Biogenic Amine Detection Systems for Intelligent Packaging Concepts: Meat and Meat Products

ABSTRACT

During meat and meat product spoilage metabolites are formed which can be detected by indicator systems to evaluate products’ deterioration, suitable to provide the consumer information about the freshness of a meat or meat product. Due to strong correlation between microbial count and biogenic amine concentration, biogenic amines were identified as suitable target metabolites for meat and meat product freshness determination in intelligent packaging concepts. The objective of this review is to provide an overview of different indication systems to detect biogenic amine formation during spoilage of meat and meat products. Different classes of bioreceptors, chromophores and fluorophores are outlined and discussed regarding their feasibility as indicator substances with a potential use for intelligent packaging concepts. Overall, this review points out that there is great potential for some of the discussed indication systems and their combination to be used as spoilage indicators for meat and meat products, though further research is necessary to overcome commercial application limitations, including precision, reliability, and low-cost producibility of the indicators.

KEYWORDS:

• Freshness determination
• spoilage indicators
• bio-receptors
• chromophores
• fluorophores

Introduction

Food safety and low food waste are generally two important challenges that the food industry is facing. In the last years, the demand of consumers for fresh, safe, and qualitative food has increased.^[1–4] A critical issue is that a large amount of packed food is discarded by retailers and consumers, when their use-by-date has been exceeded. The use-by-date is determined by the manufacturer and printed onto the foods package on the day it has been produced. It is usually assembled by the estimated products’ shelf life minus a safety margin. The products’ shelf life depends on multiple parameters including the (microbiological) quality of the used raw material, the hygiene level during the manufacturing process, storage, and transportation conditions as well as the protection properties derived from the used packaging. These diverse parameters lead to a complex determination of the estimated shelf life. In turn, this may lead to a discrepancy between the use-by-date and the actual shelf life of the product that determines the point until which the product maintains its microbiological and sensory properties as well as physical stability, when stored under given storage conditions.^[5]

One approach to fulfill consumers’ demand for fresh, safe, and qualitative food and simultaneously check the products’ shelf life in real time is the use of smart packaging concepts. Smart packaging can be divided into active and intelligent packaging and generally describes packaging that can interact with its content and/or interact with its environment and communicate with consumers for quality and safety reasons. While active packaging systems aim to increase the shelf life of food, intelligent packaging concepts provide information about the quality of food or environmental condition during storage of food products.^[6] Intelligent packaging can have several functions including detection, tracking, and communicating information regarding the products quality, storage, or packaging conditions. Intelligent packaging systems can contribute to food waste reduction, acting as active shelf life labelling device optimizing distribution control or the stock rotation system.^[7,8]

Diverse intelligent packaging systems have been developed to indicate food spoilage of highly perishable food such as fresh meat. The different concepts include indicators (freshness, integrity, time-temperature), data carriers (radiofrequency identification tags, barcodes) and sensors, which produce optical or electrical signals. While the usage of data carriers is specifically intended for storage, distribution, and traceability purposes, sensors are mostly used for analyte quantification, and indicators for food quality communication towards consumers.^[9,10]

To detect the products freshness the freshness indicator or sensor shall be placed inside the package but does not necessarily be in contact with the product. Within the European Union, requirements for intelligent packaging materials are determined in the Regulation (EC) 1935/2004. Metabolites which are formed during food spoilage, can be specifically targeted and detected by the indicator/ sensor in the package headspace through metabolite-indicator-reactions.^[7,8,11] Among those indicating substances, there are different bioreceptors, chemo responsive dyes, and fluorescent dyes.^[12–15]

Compared to the review of Kannan et al., which covers “chemical and electrochemical methodologies for the sensing of biogenic amines” in general, food, urine and blood plasma samples,^[16] this review article specifically summarizes and compares freshness indication approaches for meat and meat products with potential applicability in intelligent packaging concepts and therefore, reviewing available literature in this particular field, giving a comprehensive and most recent state-of-the-art overview.

Therefore, this review article provides a detailed description of substances, namely, bioreceptors (enzymes, molecular imprinted polymers), chemo responsive dyes (Brønsted acidic/ basic dyes, chemical pH, natural pH dyes, Lewis acid/ base dyes, conjugated polymers, colorimetric sensor arrays) and fluorophores, that are potentially applicable in intelligent packaging applications to indicate the freshness of meat and meat products. For each class of substance, the sensing mechanism is outlined, and specifically biogenic amine detection concepts are summarized and compared to provide a fundamental overview of freshness indication systems of meat and meat products using biogenic amines as target indicator substances.

Spoilage of meat and meat products

Fresh meat is a highly perishable food. Spoilage of meat is caused by three main mechanisms: microbial spoilage, lipid oxidation, and autolytic enzymatic spoilage.^[17] Thereof, according to Gram et al.^[18] microbial spoilage is considered as the major reason for meat spoilage.^[18] The microbial flora and shelf life of meat depend on several factors including water activity, initial number of psychrotrophs present on the meat surface, pH of the meat surface, storage conditions (i.e. temperature), packaging (i.e. oxygen availability), and nutritional content. In addition, meat products shelf life is also influenced by their composition, heat treatment, and handling hygiene.^[19,20]

Enterobacteriaceae, lactic acid bacteria, Brochothrix thermosphacta, Pseudomonas, and some clostridia are the main microbial groups associated with the meat microflora. Depending on the temperature and packaging atmosphere, the growth of different strains of microorganisms are favored resulting in different types of spoilage.^[21] In poultry meat for example, the dominating microorganisms responsible for aerobic spoilage are Pseudomonas, whereas the predominating microorganisms under modified atmosphere packaging (MAP) conditions are usually Brochotrix thermosphacta and lactic acid bacteria.^[22] The main microbial groups associated with the spoilage of cooked, cured meat products packed under vacuum or MAP and stored under refrigerated conditions are lactic acid bacteria.^[20] It is assumed that lactic acid bacteria, enterococci, micrococci, and yeasts predominate as spoilage microorganisms in raw salted-cured meat products such as uncooked ham or bacon, because of their resistance to curing salt.^[19]

Spoiled meat is mainly characterized by a shift in pH, slime formation, structural component degradation, off odors and visual change, causing sensorial unacceptance for consumers.^[17,18] Off-odors, perceived as a negative olfactory impact leading to customer rejection, are increasing during storage time, due to development and concentration increase of several metabolites like amines, sulfides, and volatile organic compounds (alcohols, aldehydes, ketones, organic acids).^[21,23]

Target metabolites

For the development of precise systems, able to indicate the shelf life or freshness of meat, the identification of the main quality indicating metabolites, so called target metabolites, is needed.^[8] For meat, the main quality indicating metabolites/ compounds are glucose, organic acids (lactic acid, acetic acid), ethanol, volatile nitrogen compounds, biogenic amines (tyramine, cadaverine, putrescine, histamine), carbon dioxide, and sulphuric compounds (hydrogen sulphide, methylsulphide, dimethylsulphide).^[24,25] One or several of these metabolites need to be detected by an indicator system to determine the products’ shelf life.

To clearly define a concentration threshold for an indicator compound, the indicator compound shall be initially not or in minor concentrations present in the meat product and increases in concentration during storage, being directly related to the actual shelf life. Such indicator compounds are considered as secondary compounds. Biogenic amines, sulphuric compounds, ethanol, acetate, D-lactate, CO₂, and volatile nitrogen compounds increase, whereas glucose and L-lactic acid have been reported to decrease in concentration with increasing storage time. Besides the indicator compounds, the gaseous environment of the packaging is crucial. Even though CO₂ concentration increases over storage time, the identification of microbial growth by CO₂ is shown to be quite prone to misinterpretation when used for products that are packed under modified atmosphere. This is because in MAP packaging there are high concentrations of CO₂ (≥ 20%) already present.^[24]

Overall, a concentration increase of the target metabolite should strongly correlate with microorganisms that are responsible for meat spoilage. Amongst the target metabolites mentioned above, biogenic amines (histamine, tyramine, putrescine, and cadaverine) obtain high potential to be used as target metabolite for meat spoilage and thus are focused in this review. In the following, biogenic amines and their suitability as target metabolite to indicate spoilage of meat and meat products are outlined in detail.

Biogenic amines

Biogenic amines are low molecular weight substances, which result from enzymatic amino acid decarboxylation and are usually named after their corresponding precursor amino acid.^[26–28] While tyramine derives from tyrosine, histamine from histidine and cadaverine from lysine, spermine, and spermidine derive from putrescine, which can be formed in different ways (Fig. 1). Therefore, the polyamines putrescine, spermine, and spermidine are interconvertible.^[28–30] Fig. 1 presents schematically the formation of most common biogenic amines.^[28]

Figure 1. Formation pathway of several biogenic amines (adopted and extended from^[28]).

Biogenic amines, histamine, cadaverine, putrescine and tyramine, are identified as chemical indicators for meat spoilage determination, due to their strong correlation with bacterial counts responsible for spoilage. Although, spermine and spermidine levels are reported to be naturally higher in meat products than other biogenic amines, they are not suitable as spoilage indicator because their concentration only slightly increases during storage.^[27,31] The formation of the biogenic amines depends on several factors including the availability of free amino acids, the decarboxylase enzymes, and environmental conditions. The amount of produced biogenic amines can be correlated to the presence of free amino acids and decarboxylase enzymes, which are, in turn, influenced by the raw material, processing, and microbial flora.^[28]

In high concentrations, biogenic amines are associated with food poisoning, due to their toxicological effects, potentially causing migraine, headaches, gastric and intestinal problems, and pseudo-allergic responses.^[28] However, biogenic amines concentrations in spoiled food are relatively low at an early stage of decomposition (5–8 mg/kg) and increase with increasing decomposition (17–186 mg/kg). A differentiation of biogenic amines from other amines is difficult and limited, due to their similarity and only slight difference in basicity. Therefore, recognition mechanisms should be able to detect biogenic amines at low concentrations, distinguish these nuances and utilize their unique biological structure.^[32]

Detection methods

Bioreceptors

Bioreceptors (e.g. enzymes, antibodies, nucleic acids) are biological recognition elements which can detect specific target elements (e.g. substrates, antigens, complementary DNA) based on a catalytic or binding event. Immobilized on a transducer, which converts a biochemical signal into an electrical or optical signal, they form a biosensor which can be used to analyte detection/ monitoring. In general, biorecognition elements can be classified into natural, semi-synthetic, and synthetic bioreceptors.^[15] In this section, biorecognition elements are described which have the potential to be used for meat metabolite detection.

Enzymes

Enzymes are natural bioreceptors, more precisely biocatalysts, which can specifically recognize their substrates, based on their specific three-dimensional configuration. The substrate recognition works according to the “lock and key” principle. The active site of the enzyme, suitable for a specific substrate, can catalyze the reaction by lowering the activation energy during the formation of molecular transition state between enzyme and substrate. Specific substrate recognition behavior and low detection limits enable enzymes to be suitable biorecognition elements in biosensors.^[15,33] Several broad and selective enzymes can be used for the recognition of the biogenic amines: histamine, tyramine, putrescine and cadaverine, that are outlined in Table 1.

Table 1. Suitable enzymes for the single or multiple detection of the biogenic amines: histamine, tyramine, putrescine and cadaverine, in biosensor applications.

Enzyme	Histamine	Tyramine	Putrescine	Cadaverine	References
Monoamine oxidase	x	x	x	x	^[Citation26,Citation34,Citation35]
Diamine oxidase	x	x	x	x	^[Citation26,Citation36–45]
Plasma amine oxidase	x	x	x	x	^[Citation43,Citation46]
Tyramine oxidase		x			^[Citation43,Citation47–49]
Putrescine oxidase			x	x	^[Citation12,Citation26,Citation45,Citation50–54]
Methylamine dehydrogenase	x				^[Citation55–57]
Histamine dehydrogenase	x				^[Citation50,Citation58]
Tyrosinase		x			^[Citation59–62]

In general, amine oxidases are suitable due to their reaction mechanism with biogenic amines. They catalyze, in the presence of oxygen, the oxidation of amines to the corresponding aldehyde, hydrogen peroxide, and ammonia (eq. 1)(1) $\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{e}+\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}_2{\mathrm{O}}_2$(1) . The resulting decrease in oxygen concentration or increase in hydrogen peroxide concentration can be used for the quantification of the biogenic amines.^[26]

(1) $\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{e}+\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}_2{\mathrm{O}}_2$(1)

(2) ${\mathrm{H}}_2{\mathrm{O}}_2\stackrel{\mathrm{h}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{p}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}[\mathrm{V}]}{\to }2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}+{\mathrm{O}}_2$(2)

(3) $2{\mathrm{e}}^{-}\stackrel{}{\to }\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{e}$(3)

At an electrode, the produced hydrogen peroxidase can be split into two positively charged hydrogen atoms, oxygen and two electrons, when applying a high potential (eq. 2)(2) ${\mathrm{H}}_2{\mathrm{O}}_2\stackrel{\mathrm{h}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{p}\mathrm{o}\mathrm{t}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{i}\mathrm{a}\mathrm{l}[\mathrm{V}]}{\to }2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}+{\mathrm{O}}_2$(2) . The electron flow (eq. 3)(3) $2{\mathrm{e}}^{-}\stackrel{}{\to }\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{e}$(3) is proportional to the amine concentration.^[26]

Another method to detect the concentration of biogenic amines by enzymes, is to measure the oxygen concentration (see eq. 1)(1) $\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{O}}_2+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\mathrm{o}\mathrm{x}\mathrm{i}\mathrm{d}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{e}+\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}_2{\mathrm{O}}_2$(1) . A decrease in oxygen concentration can change for instance the fluorescence intensity of an oxygen sensitive fluorophore like ruthenium complex, which can be used to visualize the decrease in oxygen concentration and in turn the formation of biogenic amines.^[48]

The dehydrogenases (methylamine- and histamine-dehydrogenase) are suitable enzymes to detect short-chain aliphatic amines (i.e. methylamine) and primary amines (i.e. histamine). The used detection mechanism is based on the oxidation of primary amines by the enzyme to its corresponding aldehyde and ammonia (eq. 4(4) $\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{e}+\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}^{+}$(4) ).^[50,55,57]

(4) $\mathrm{A}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{H}}_2\mathrm{O}\stackrel{\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{r}\mathrm{o}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }\mathrm{c}\mathrm{o}\mathrm{r}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{p}\mathrm{o}\mathrm{n}\mathrm{d}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{a}\mathrm{l}\mathrm{d}\mathrm{e}\mathrm{h}\mathrm{y}\mathrm{d}\mathrm{e}+\mathrm{N}{\mathrm{H}}_3+{\mathrm{H}}^{+}$(4)

The oxidation of a primary amine results in the reduction of the dehydrogenase. The reduced form of the dehydrogenase can be oxidized by an electrochemical mediator, where the electron flow is proportional to the amine concentration^[57] or a shift in adsorption is induced when oxidized by a chromophore.^[58]

Tyrosinase is a suitable enzyme that can be used to detect the biogenic amine tyramine and other monophenols. The reaction mechanism includes two oxidation steps, enabled by the two active sites of tyrosinase. First, tyrosinase catalyzes the oxidation of tyramine to dopamine (eq. 5)(5) $2\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{O}}_2\stackrel{\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }2\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}$(5) and second, the oxidation of dopamine to ortho-dopaquinone (eq. 6)(6) $2\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\stackrel{\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }2\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{o}-\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{n}\mathrm{e}+2{\mathrm{H}}_2\mathrm{O}$(6) . The resulting ortho-dopaquinone can be reduced at an electrode to dopamine (eq. 7)(7) $\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{o}-\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{n}\mathrm{e}+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}\stackrel{\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{e}}{\to }\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}$(7) . Dopamine in turn indicates the enzyme catalyzation and is thus proportional to tyramine concentration.^[59–62]

(5) $2\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}+{\mathrm{O}}_2\stackrel{\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }2\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}$(5)

(6) $2\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}\stackrel{\mathrm{t}\mathrm{y}\mathrm{r}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{s}\mathrm{e}}{\to }2\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{o}-\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{n}\mathrm{e}+2{\mathrm{H}}_2\mathrm{O}$(6)

(7) $\mathrm{o}\mathrm{r}\mathrm{t}\mathrm{h}\mathrm{o}-\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{q}\mathrm{u}\mathrm{i}\mathrm{n}\mathrm{o}\mathrm{n}\mathrm{e}+2{\mathrm{H}}^{+}+2{\mathrm{e}}^{-}\stackrel{\mathrm{e}\mathrm{l}\mathrm{e}\mathrm{c}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{d}\mathrm{e}}{\to }\mathrm{d}\mathrm{o}\mathrm{p}\mathrm{a}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{e}$(7)

Several studies investigated meat and meat product spoilage via biogenic amine recognition using biosensors based on enzymes (Table 2). Sensors were calibrated using defined biogenic amine solutions. The accuracy of measured biogenic amine content based on sensor responses to food samples can be controlled and compared with HPLC measurements.^{[26,43–45,52]}

Table 2. Overview of studies investigating meat and meat product spoilage via biogenic amine recognition using biosensors based on enzymes.

Meat/ Meat product	Enzyme/ enzyme combination	Targeted biogenic amines	Sensor principle	Reference
Chicken	Putrescine oxidase	Putrescine, Cadaverine, Spermidine	Current response (electrode)	^[Citation52]
Salami, Ham, Onion sausage	Monoamine oxidase + Tyramine oxidase + Diamine oxidase	Histamine + Tyramine + Putrescine	Current response (electrode)	^[Citation43]
Dry-fermented sausages	Diamine oxidase	Total amine content	Oxygen consumption (electrode)	^[Citation44]
Pork	Diamine oxidase Monoamine oxidase Putrescine oxidase	Total biogenic amine content Tyramine Putrescine	Current response (electrode)	^[Citation26]
Beef, Pork, Chicken, Turkey	Putrescine oxidase or Diamine oxidase	Putrescine	Chemiluminescence reaction based on (enzymatic) hydrogen peroxide production	^[Citation45]

The use of enzymes as biorecognition elements for biogenic amine detection as meat metabolites involves several obstacles: A general disadvantage of enzymes is their sensitivity to the substrate concentration and in part a low specificity towards the target molecules (e.g. enzymes that react with a broad range of amines). In addition, enzymes are sensitive to changes in environmental conditions that are temperature, pH, and ionic strength that influence the activity rate and thus the catalytic reaction rate. Enzymes often also lack in storage life and processing stability.^[15,63] In all listed studies (Table 2), the detection of biogenic amines by enzymes was conducted in solution, which means the food needed to be mashed and dissolved to analyze biogenic amine concentration. No literature on biogenic amine detection via a vapor recognition method using enzymes has been found during the literature search for this review article. However, an approach for a non-destructive in-package application could be based on the partition of volatiles in the package headspace and dissolution in an aqueous phase, which was conducted in a non-enzymatic approach by Heising et al.^[64]

Molecularly imprinted polymers

Molecular imprinted polymers (MIP) are synthetic biorecognition elements, which can be specifically synthesized for the detection of a target molecule, similar to the “lock and key” principle of enzymes. A target molecule or a similarly structured molecule is used as a template which is incorporated into a polymer matrix. After removing the template, resulting cavities in the polymer matrix can bind complementary structured templates. In detail, molecular imprinting includes three steps^[15,63]:

1. Pre-arrangement of the template (printed molecule) and the functional monomers (including cross-linkers and initiators) via dissolution in a solvent

2. Co-polymerization of the functional monomers by applying heat over a longer time period

3. Template removal when washing the polymers in an extraction process

Compared to other biorecognition elements, MIP are more robust and stable under a wide range of chemical and physical conditions.^[15] In addition to the use as biosensors, MIP are widely used in other fields like bioseparation, medical diagnostics, and drug delivery.^[65] The synthetization of MIP for the recognition of biogenic amines has been widely studied. Several compositions of synthesized MIP for histamine or tyramine recognition have been summarized in Table 3.

Table 3. Composition of molecularly imprinted polymers (MIP), which can detect either histamine or tyramine.

Template	Functional monomers^a				Solvent^b	Reference
	Monomer	Cross-linker	Initiator	Other
Histamine	ZnPP MAA	EGDM			DMSO	^[Citation66]
Histamine	MAA	EGDM	AIBN		DMSO	^[Citation67–70]
Histamine	MAA	EGDM			DMF	^[Citation71]
Histamine	MAA	EGDM TRIM	DABE	PETMP	ACN	^[Citation72]
Histamine	MAA	EGDM	AIBN		ACN	^[Citation73]
Histamine	MAA	EGDM		AIBN PVC THF	DMSO	^[Citation74]
Histamine	MAA	EGDM			Chloroform	^[Citation75,Citation76]
Histamine	MAA	EGDM	ABDV		ACN	^[Citation77]
Histamine	MMA	EGDM TRIM	DABE	CTA	ACN	^[Citation78]
Tyramine	TEOS TEPS				Ethanol	^[Citation79]
Tyramine	MMA	TRIM	AIBN		ACN	^[Citation80]

^aZnPP = zinc(II)–protoporphyrin, MAA = methacrylic acid, EGDM = ethylene glycol dimethacrylate, AIBM = azobisisobutyronitrile, TRIM = trimethylolpropane trimethacrylate, DABE = N,N-diethyldithiocarbamic acid benzyl ester, PETMP = pentaerythritol-tetrakis-(3-mercaptopropionate), PVC = polyvinyl chloride, THF = tetrahydrofuran, ABDV = 2,2ʹ-azobis 2,4-dimethylvaleronitrile, CTA = pentaerythritoltetrakis-(3-mercaptopropionate) TEOS = tetraethoxysilane, TEPS = triethoxyphenylsilane

^bDMSO = dimethylsulfoxide, DMF = dimethylformamide, ACN = acetonitrile

In the literature, MIP for biogenic amine recognition have been reported in sensors, based on surface enhanced Raman spectroscopy,^[73,81] impedance spectroscopy,^[67] quartz crystal microbalance,^[67,82] heat-transfer method,^[68,69] potentiometry,^[72] amperometry,^[79] impedimetry,^[70] voltammetry,^[75,80] fluorescence,^{[66,76,77,83,84]} and chromogenic adsorption.^[65,85,86] For simple visualization of the MIP sensing capability, chromogenic or fluorogenic agents can be incorporated in the MIP sensors matrix. For instance, tyramine detecting molecularly imprinted fluorescence sensors can be developed by quantum dots incorporation to achieve improved selectivity and sensitivity of the combined sensor.^[83,84]

Whereas histamine and tyramine recognition were mostly conducted in aqueous medium, Ying et al. (2018–2020)^[65,85,86] studied putrescine vapor detection. In their first study, Ying et al.^[85] developed three different imitate templates of imprinted poly(vinyl alcohol) coloring membranes, based on the chromogenic agent ninhydrin, for selectively detecting putrescine vapor by naked eye. The use of imitate templates was necessary, because of permanent covalent bond formations between putrescine and ninhydrin molecules, resulting in impossible putrescine removal after membrane polymerization. A reaction between ninhydrin and the target molecule putrescine resulted in a violet coloration spot in the membrane, which shape, and color intensity depend on putrescine adsorption concentration, polymer structure, and incorporated imitate templating molecules. When selecting an imitate template, molecular size and group configuration need to be as similar as possible to the target molecule. The imitate template should be also easy to remove and suitable for the chosen detection method (e.g. reaction inertness to chromogenic agent). The imitate templates 1,4-butylene glycol, adipic acid and succinic acid were tested for their affinity to bind putrescine, when incorporated into the ninhydrin/ poly(vinyl alcohol) membrane. 1,4-butylene glycol (1,4-butanediol) was found the best matching imitate template based on similarity in molecular size and functional groups as well as resulting imprinting efficiency and adsorption.^[85] Therefore, the authors used 1,4-butylene glycol for their following studies as the imitate template for putrescine.^[65,86]

A lower detection limit and shorter detection time were found for putrescine detection when incorporating the imitate template 1,4-butylene glycol into chromogenic electrospun fibers^[65] compared to hydrogels made of poly(vinyl alcohol) and ninhydrin as chromogenic agent.^[85] Further improvement in adsorption capacity, adsorption equilibrium time, and selectivity to putrescine were achieved in their most recent work, investigating a double-layer nanofiber membrane. The double-layer consists of a filtering layer (poly(vinyl alcohol) + 1,4-butanediol) in front of a chromogenic layer (poly(vinyl alcohol) + Ninhydrin) (Fig. 2).^[86]

Figure 2. Schematic visualization of the double-layer membrane (adapted from^[86]).

Besides the detection using a chromogenic layer, fluorophores can be also used to visualize the binding or complex formation of the MIP with a biogenic amine, which will be outlined in detail in section 5.3. Overall, the literature research showed that MIP can be specifically synthetized for the detection of biogenic amines and can be visualized by incorporation of chromogenic or fluorogenic agents. A possible detection of biogenic amines via MIP in vapor points out that MIP may be a more suitable biorecognition system for in packaging application than enzymes.

Chemo responsive dyes

Chemo responsive dyes are dyes with an interaction center that is strongly coupled to a chromophore, which changes its color dependent on the environmental conditions. In general, there are different dye classes: Chemo responsive dyes feasible for colorimetric sensor arrays can be subdivided into Brønsted acidics or basic dyes, Lewis acid/base dyes, and zwitterionic solvatochromic dyes with a large dipole.^[87]

Brønsted acidic/ basic dyes

Depending on the proton (Brønsted) acidity or basicity of the environment, pH indicators change their color according to the following equation (eq. 8(8) $\mathrm{H}\mathrm{I}\mathrm{n}\mathrm{d}+{\mathrm{H}}_2\mathrm{O}\overrightarrow{\leftarrow \phantom{{ }_{\text{vbox}to.5ex\text{vss}}}}\mathrm{I}\mathrm{n}{\mathrm{d}}^{-}+{\mathrm{H}}_3{\mathrm{O}}^{+}$(8) ), with Hind representing the acid form and Ind⁻ the conjugated base.^[88]

(8) $\mathrm{H}\mathrm{I}\mathrm{n}\mathrm{d}+{\mathrm{H}}_2\mathrm{O}\overrightarrow{\leftarrow \phantom{{ }_{\text{vbox}to.5ex\text{vss}}}}\mathrm{I}\mathrm{n}{\mathrm{d}}^{-}+{\mathrm{H}}_3{\mathrm{O}}^{+}$(8)

Increasing meat degradation metabolites, such as CO₂, hydrogen sulfide, ammonia, organic acids, and volatile amines (including biogenic amines) lead to a change in pH that can also be detected in the package headspace. Thus, a change in pH can be used as freshness indication.^[89–92] Several studies investigated pH colorimetric sensors/ indicator films as intelligent freshness indicators for spoilage determination of chicken,^[93] beef,^[94] pork,^[95] fish,^[96] shrimp,^[97] and others.^[98] These colorimetric indictors generally consist of one or multiple pH sensing dyes, which are incorporated into a carrier system. Biopolymers are suitable for carrier systems based on their good biodegradability, safety, and film-forming properties.^[95] The principle of colorimetric indicators is to generate an (irreversible) color change, based on the protonation/deprotonation of the incorporated or immobilized pH indicator dye(s). The related colorimetric response is easy to read and understand by the consumer.^[63,99] PH sensing dyes, that can be incorporated into labels and films, can be subdivided into chemical and natural dyes.

Chemical pH dyes

Plenty of studies used chemical dyes like bromothymol blue,^[100–102] bromocresol green,^[103–105] methyl red,^[92,106,107] bromocresol purple,^[108,109] methyl orange,^[110] phenol red,^[111] or mixtures of different chemical dyes^[112–115] as pH sensing component(s). Carrier systems used for these dyes were filter paper^{[102,109,115,116]} or polymer-matrices based on methylcellulose,^{[112–114,117]} cassia gum,^[100] bacterial cellulose,^[92,111] gelatin,^[104,110] cellulose acetate,^[106] agarose,^[105] and others.^[118] There are numerous studies analyzing chemical pH dyes as indicator systems measuring the headspace of packed fish,^{[101,105,107,119]} meat,^{[91,92,100,102–104,106,108,109,111,113–116]} and other food products.^{[91,101,112,117,118]} To investigate the suitability of chemical pH dyes to indicate meat spoilage, studies measuring the headspace of packed chicken,^{[92,103,104,108,114]} beef,^[116] pork,^[113] and buffalo meat^[102] at refrigerated temperature are reviewed in this section.

A chemical indicator can be based on the pH dye bromocresol purple that is immobilized onto a carrier system such as agarose gel. The light-yellow indicator stripes were attached inside polyethylene terephthalate (PET) boxes with fresh chicken meat and stored at 4°C for one week. During storage time, the indicator label changed its color to purple at the end of the 7^th day, which means that the pH changed from pH 5.78 to pH 6.34. For fresh poultry meat, the microbial limit, which is still acceptable for human consumption, is 6–7 log₁₀ cfu/g. The microbial limit of 7 log₁₀ cfu/g was found on the 7^th day (initial count: 3.4 log₁₀ cfu/g), which is in alignment with the final color change of the pH indicator turning purple.^[108]

Another study investigated the use of bromocresol green mixed with gelatin as a carrier to indicate chicken spoilage, stored in a box at 4°C for 10 days. A color change of the initially yellow indicator was observed on the 8^th day (green) and the 10^th day (blue). Correlating to the color change, pH increased during storage from 5.8 (1^st day) over 6.7 (8^th day) to 7 (10^th day). The point of spoilage was determined on the 8^th day where a microbial count of 6.6 log₁₀ cfu/g was analyzed (initial count: 3.9 log₁₀ cfu/g) and the color of the indicator turned visible from yellow to green.^[104]

A methyl red based cellulose on-package sticker sensor was investigated for the freshness determination of broiler chicken cuts that were stored at 4°C for 14 days. The initially red indicator (pH 5.61) turned orange on the 7^th day by exceeding the pH threshold (pH 6.16), potentially indicating spoilage. According to microbial and sensory analysis, chicken samples were rejected on the 8^th day with a total microbial count of 6.7 log₁₀ cfu/g corroborating with an orange/yellow indicator response. The authors concluded a correlation between the sensors response and chicken spoilage, indicating that the packed chicken has spoiled when the indicator turned yellow.^[92]

Another study, investigating the use of a pH-indicator for chicken freshness determination, used bromocresol green, incorporated into a three-layered structure. The three-layered structure consists of a clear outer layer (low density polyethylene film), color changing middle layer (bromocresol green + ethylene-vinyl acetate) and an inner gas and vapor permeable layer (porous Tyvek® sheet). Chicken breasts were stored at 4°C for 10 days. Spoilage was indicated with a color change from yellow to green, which occurred on the 7^th day of storage. Accordingly, the microbial count on the 7^th day was approximately 6 log₁₀ cfu/mL (initial count: 4.5 log₁₀ cfu/mL), corresponding to the lower human consumption limit level. The color change can be either detected by naked-eye or color-measuring instruments such as by using a smartphone that becomes a favorable device for freshness determination systems.^[103]

The spoilage of chicken breast was also studied at 4°C by using chemical dye mixtures composed of bromothymol blue and methyl red (mixture 1) as well as bromothymol blue, bromocresol green, and phenol red (mixture 2) coated onto cellulose. During the storage period of 8 days the indicator with the mixture 1 turned from green to yellow-orange and the indicator with the mixture 2 turned from violet to green. The visible color change of the indicator with the mixture 1 on the 6^th day was in alignment with lower consumption limit of 6 log₁₀ cfu/g. Therefore, the authors concluded that mixture 1 was more sensitive and faster in chicken breast spoilage detection than the mixture 2 due to an easier color change determination of mixture 1.^[114]

Kuswandi & Nurfawaidi^[116] aimed to develop an indication system based on multiple dyes because the authors stated that the onset of detection, which is correlated with microbial growth may be difficult to determine using single indicators (one colorimetric response). To enhance the visible determination of the point of spoilage, a dual sensor (two colorimetric responses) based on bromo cresol purple (inner circle) and methyl red (outer ring) immobilized onto filter paper has been developed (Fig. 3). This system was studied as an indicator system of pH changes in the headspace of packed beef. During beef spoilage, the color of the dual sensor label changed that was detected by naked eye and categorized into the following freshness states: (a) fresh, (b) ok or (c) not fresh. While the outer indicator (methyl red) turned from red over orange to yellow, the inner circle (bromo cresol purple) changed its color from yellow over grey to purple. As schematically represented in Fig. 3, the color of the dual sensor changed in alignment with the microbial analysis.^[116]

Figure 3. Schematic visualization of the dual sensor label changing its colour over the time of fresh beef stored at 4°C. Presented is a dual sensor label showing three different freshness degrees: (a) fresh, (b) ok, (c) not fresh (adapted and extended from^[116]).

To monitor freshness of lean pork, six methylcellulose-based indicators had been developed containing different dyes or dye mixtures (Table 4). Pork samples were stored in PET trays at 5°C for 8 days with the indicators attached to the lid of the tray. The authors divided the storage period of 8 days into three freshness periods with corresponding changes in indicator dyes as summarized in Table 4.^[113]

Table 4. Investigated pH dye mixtures by Chen et al. (adopted and extended from^[113]).

Indicator	Dye	pH of mixture	Fresh day 0 to 3	Medium day 4 to 5		Spoiled Day 6 to 8
1	Bromocresol purple	4.5	Yellow		Yellow		Grey
2	Bromothymol blue	5.3	Yellow		Yellow		Blue/ Green
3	Bromothymol blue: methyl red (3:2)	5.0	Red		Goldenrod		Green
4	Bromocresol purple	4.2	Yellow		Yellow		Grey
5	Bromothymol blue	4.2	Yellow		Yellow		Green
6	Bromothymol blue: methyl red (3:2)	4.4	Red		Orange		Green

A microbial analysis of the packed lean pork showed a microbial count of 6.3 log₁₀ cfu/g on the 6^th day (initial count: 3.1₁₀ log cfu/g), which was defined as the beginning of the spoiled period, exceeding the lower consumption limit of 6 log₁₀ cfu/g. The authors identified indicator 3 as the most suitable dye mixture to indicate lean pork spoilage corroborating with a clear visible color change on day 4 and 6 (Table 4).^[113]

The freshness of buffalo meat was evaluated using bromophenol blue immobilized onto filter paper. Both were placed into polystyrene boxes and stored for 9 days at 4°C. The indicator turned from yellow on the 1^st day (fresh) to yellow/green on the 3^rd day (acceptable), light blue on the 7^th day (marginally acceptable) and dark blue on the 9^th day (unacceptable). Color changes can be directly linked to the microbial counts of 4.4 log₁₀ cfu/g (1^st day), 4.6 log₁₀ cfu/g (3^rd day), 6.6 log₁₀ cfu/g (7^th day), and 7.1 log₁₀ cfu/g (9^th day), thus being directly linked to meat spoilage.^[102]

Several studies showed that there are chemical pH dyes or pH dye mixtures that are feasible to indicate meat spoilage due to the direct correlation of meat spoilage and indicator color change based on the increase of volatile metabolites.^{[92,102,104,108,113,114,116]}

However, chemical dyes have been reported to obtain disadvantages such as difficulties in handling and incorporation into the carrier system, as well as in part harmfulness for the consumer, when leaked onto the product. In contrast to chemical dyes, the use of natural pigments as pH indicators is often assessed as environmentally friendly and cost-effective alternative, because per se they do not leave chemical residues in the packaged product leading to an increased research focus within the last years.^[2,97,120]

Natural pH dyes

Natural pH dyes are substances from a natural source that change their color depending on the pH conditions and thus being suitable for a potential application as freshness indicator. Natural pH dyes are mainly represented by plant extracts, containing pigments like anthocyanins.^{[97,98,120,121]} In addition to pH, the color expression of natural pigments, such as anthocyanins, is also strongly influenced by its structure, co-pigmentation, temperature, and UV-radiation. Anthocyanins are water-soluble pigments, that can be extracted from leaves, roots, and caudexes of high plants. For quality indicating purposes, the literature shows that anthocyanins or anthocyanin rich extracts are mainly extracted from sweet potatoes,^[98] grapes or grape skins,^{[95,122–125]} red cabbage,^{[93,126–128]} black carrot,^[129,130] black rice bran,^[97] black chokeberry pomace,^[131,132] blueberry,^[132,133] curcumin,^[96,134,135] and many more.^[136–138] In the literature, carrier systems investigated for natural pH dyes are mainly biopolymer films based on chitosan,^{[125,131,132]} starch,^{[124,129,133]} cellulose,^[98,130] as well as other biopolymers or mixtures of biopolymers.^[93,122,123] Even though a lot of studies developed natural and bio-based pH-indicators, only a fraction of those studies measured the headspace of packed meat at refrigerated temperature, investigating the dyes suitability as freshness monitoring systems.^{[93,94,120,124,139,140]}

For instance, red cabbage extract incorporated into a pectin-based film was investigated to detect spoilage of fresh perishable food, including chicken filet and beef. Samples and indicators were placed inside petri dishes and stored at 4°C for 72 to 96 h. Total volatile basic nitrogen (TVBN) levels were analyzed to determine the point of spoilage, which was defined as 20 ± 2 mg/100 g TVBN content. This threshold was reached by chicken samples after 72 h along with a decrease of absorbance at 553 nm and visual color change, thus indicating spoilage. After 72 h the TVBN-levels of beef samples were below 5 mg/100 g, indicating that the beef sample was not spoiled after 3 days and probably won’t be after 4 days, which however was not measured by the authors.^[93]

In another study, spoilage of fresh beef was investigated using litmus paper as the indicator. During the storage period of 14 days at 4°C the red litmus paper (fresh) changed its color to purple on the 3^rd day (medium fresh) and to blue on the 7^th day (not fresh). The color change on the 7^th day corresponded with an increase in pH (from 5.67 on 1^st day to 6.02 on the 7^th day), exceeding pH threshold of 6.0, at which spoilage of meat has been reported to occur. Furthermore, sensory evaluation showed meat rejection on the 7^th day and the analyzed microbial count of 6.7 log₁₀ cfu/g on the 7^th being in alignment with the acceptable consumption limit of 6–7 log₁₀ cfu/g.^[140]

There are also studies indicating that natural indicator responses can in part not be directly linked to meat spoilage via microbial count. For instance, one study immobilized anthocyanin rich Jamun fruit (Syzgium cumini) skin extract onto filter paper strips, to monitor the freshness of chicken patties, stored at 4°C for 21 days. During the storage period, the indicator stripes had an initial violet color (1^st day) which faded on the 18^th day and turned to yellow on the 21^st day. PH significantly decreased from 6.22 to 6.04, TVBN-levels significantly increased from approximately 16.1 mg/100 g to 20.4 mg/100 g, and total viable count increased from 1.7 log₁₀ cfu/g to 5.1 log₁₀ cfu/g from day 1 to day 21^st, respectively. Based on measured TVBN- levels, which exceeded the recommended threshold of 20 mg/100 g, decreasing pH, and decreasing sensory acceptance, the authors concluded spoilage after 21 days, being in alignment with results from similarly conducted studies and the indicators color change (yellow).^[120]

To monitor minced beef, Ezati et al.^[94] developed a pH indicator based on the natural dye alizarin, incorporated into a cellulose-chitosan film. During a storage period of 6 days at 4°C, the indicator turned from an initial brown color into a purple color on the 4^th day, which intensified towards the 6^th day. The color change on the 4^th day correlated with increasing pH, total viable count, and TVBN-values. The TVBN- value of the minced beef exceeded 17 mg/100 g, which was defined as the acceptable threshold after 4 days and therefore indicating meat spoilage. The total viable count increased over the storage period from approximately 5.6 log₁₀ cfu/g (initial value) to 6.3 log₁₀ cfu/g (2^nd day), 8.2 log₁₀ cfu/g (4^th day) and 9.3 log₁₀ cfu/g (6^th day). Due to the two-day measurement rhythm, the indicator must have reached the acceptability limit of 7 log₁₀ cfu/g on the 3^rd day, which might have not been visualized by the developed pH indicator system by naked eye. The measured visual color change occurred after the thresholds of 7 log₁₀ cfu/g (total viable count) and 17 mg/100 g (TVBN-level) were exceeded. The TVBN threshold of 20 mg/100 g, which was used in other studies,^[93,120] was reached on the 4^th day and would therefore be in alignment with the indicators color change from brown to light purple.^[94]

Golasz et al.^[124] investigated pork spoilage using grape anthocyanins immobilized onto cassava starch-based films as a potential indicator. Samples were stored at 4°C for two weeks. According to the psychrotrophic aerobic count, pork meat spoiled after 10 days exceeding the microbial count of 6–7 log₁₀ cfu/g. However, a significant increase in pH was observed after the 10^th day and indicator color only changed up to the 7^th day. That means that the point of spoilage could not be indicated with a change in color of the anthocyanin containing indicator film.^[124]

In another study the spoilage of fresh pork meat was analyzed by using a bilayer indicator film composed of an agar-anthocyanin layer functioned as a sensing layer and gellan gum-TiO₂ layer as a light barrier and conducting layer. Meat samples were stored inside PET boxes and the indicator film was attached either on the inside of the lid or its outside covering a 15 mm hole. The indicator film inside the packaging showed a leaching of the anthocyanins and thus could not be used as spoilage indicator. Only the indicator attached to the outside of the lid was used for further analysis. The change in color of the indicator film were described by the G (green) value divided by the sum of R- (red), G-, B (blue) values. Accordingly, the film changed its color from 31.45% to 33.39% over the storage of 14 days at 4°C. Using TVBN contend for spoilage evaluation the TVBN-value exceeded the rejection limit of 15 mg/100 g, which is regulated in the Chinese standard GB 2707–2016, on the 8^th day. A color value of 32.55% was analyzed on the 8^th day. As a result, authors suggested that the pork samples were spoiled and should not be consumed when the color value exceeds 32.55%. The authors concluded that the indicator showed visible color changes during pork storage, which, however, could not be directly seen in the published pictures.^[139]

After analyzing the studies investigating meat spoilage under refrigerated conditions by using natural pH dyes, two out of six studies were able to visibly indicate the point of spoilage via change in coloration based on microbial counts^[93,140] and one out of six studies based on TVBN-levels.^[120] In two out of six studies the onset of the indicator (change in color) was generated before^[124] or after^[94] the investigated meat sample has reached or slightly exceeded the microbial consumption limit of 6–7 log₁₀ cfu/g.^[94,124] However, the alignment of indicator response and meat or meat product point of spoilage highly depended on the used parameters (total viable count, TVBN-levels, pH) for spoilage determination and the used thresholds, evaluating the point of spoilage.

Compared to chemical pH dyes, natural pH dyes are more environmentally friendly but in part detect meat spoilage less precisely.^[94,124] One reason for this could be that natural plant extracts are composed of several different anthocyanins, which can react in different manners to different volatile compounds and thus differently indicate changes in pH. Their response, that is the change in color, may be non-linearly correlated with the volatiles, which may result in a high threshold and therefore late response. However, according to Dudnyk et al. this non-linear response can also be considered advantageous enabling to differentiate between different volatiles such as amines.^[93]

Lewis acid/ base dyes

While Lewis acids are described as electron acceptors, Lewis bases “donate” their free electron pair to form a covalent bond with the Lewis acid. Due to amino group(s) of biogenic amines, they can be regarded as Lewis bases which can covalently bind to an electron acceptor. A dye, which shows changes in electron delocalization and therefore adsorption upon reaction with a biogenic amine, can be used as an indicator towards the biogenic amine.^[14,88,141]

A chromophore which can be used to detect biogenic amines in vapor is 4-(dioctylamino)-4ʹ-(trifluoroacetyl)-azobenzene.^[142] With the trifluoroacetyl group being an electron acceptor and the dioctylamino group being an electron donor, electron delocalization across the 4-(dioctylamino)-4ʹ-(trifluoroacetyl)-azobenzene molecule occurs. The trifluoroacetyl groups reaction with amines results in the formation of hemiaminals (Fig. 4), that lead to a blue shift of the optical absorbance peak upon decreasing acceptor strength.^[14,142] Compared to ammonia and ethylamine vapor (monoamines), the exposure of 4-(dioctylamino)-4ʹ-(trifluoroacetyl)-azobenzene to putrescine and cadaverine vapor (diamines) resulted in a seemingly irreversible reaction (see Fig. 4). This could be based on the presence of two amine groups of the analyte forming hemiaminals with two trifluoracetyl groups of the indicator dye molecules.^[142]

Figure 4. Bonding reaction of an amine with 4-(dioctylamino)-4ʹ-(trifluoroacetyl)azobenzene (adapted and extended from^[142]).

The azobenzene dyes 4-N,N-dioctylamino-4ʹ-dicyanovinylazobenzene (CR-528) and 4-N,N-dioctylamino-2ʹ-nitro-4ʹ-dicyanovinylazobenzene (CR-555) have been immobilized in ethylene-vinyl acetate copolymer (EVA) to form vaporous biogenic amine selective indicators. Indicator films have been exposed to different biogenic amine vapors (dimethylamine, trimethylamine, spermidine, ethanolamine, isopentylamine, and cadaverine). A change in color of both indicators (CR-528/EVA and CR-555/EVA) have been observed only by reaction with isopentylamine. A color change from purple to orange-yellow and maximum absorbance change from 455 nm to 470 nm was also analyzed after the exposure of CR-555/EVA indicator to cadaverine vapor.^[143]

Conjugated polymers

Conjugated polymers are widely used as chemical sensors, due to their optical/electrical response to various analytes. When exposed to biogenic amine vapor, such as putrescine and cadaverine, the conjugated polymer nitrated polythiophene likely forms an intermolecular charge transfer complex with the analyte. As a result, a naked to the eye visible hyperchromic shift can be observed (darkening in color).^[144]

Colorimetric sensor arrays

To specify the detection of target molecules, multiple chemo responsive dyes, each enabled to detect a variety of metabolites, can be immobilized in a solid support system to create a unique odor depended colored pattern (Fig. 5).^[145]

Figure 5. Schematic visualization of a colorimetric sensor array (own illustration based on^[145]).

The color of each color spot can be described by three values, according to the RGB-system. A colorimetric sensor array with 12 dyes, as shown in Fig. 5, can therefore provide 36 variables (12 dyes x 3 color components). The difference map (Fig. 5c) can be obtained by subtracting the initial image (before exposure to biogenic amines, Fig. 5a) from the final image (after exposure to biogenic amines, Fig. 5b). The color change profile of the sensor array can be used for volatile compounds and thus freshness quantification/ determination.^[146] Immobilization of the chemo responsive dyes onto the carrier system was reviewed to be usually carried out by adsorption, entrapment, ion exchange, or covalent bonding techniques.^[88] In a sensor array, there are several requirements for chemo responsive dyes and solid support systems, which are presented in Table 5.^[88]

Table 5. Requirements for chemo responsive dyes and solid support systems for colorimetric sensor array application.^[88].

Chemo responsive dyes:	Solid support system:
Strong interaction between the analytes and the dye Corresponding color change after binding/reaction with the target molecule Compabillity of diverse dyes with cross-responsive properties Reproducible and reliable color change	No interaction with the chemo responsive dyes Suitable surface properties to enable a uniform color spread Uniform white background Hydrophobic properties to reduce the interference of ambient humidity (preferable) Accessible microstructure and high surface area for a high analyte diffusion and binding

Chemo responsive dye classes, fulfilling the listed requirements in Table 4, are Brønsted acidic or basic dyes (i.e. pH indicator dyes), Lewis acid/base dyes (i.e. metal ion containing dyes) and zwitterionic solvatochromic dyes with large permanent dipoles. Thereof, porphyrins, metalloporphyrins, and pH indicator dyes are typically used in colorimetric sensor arrays.^[88,145,146] Porphyrins and metalloporphyrins have been used to identify different volatile organic and inorganic compounds including alcohols, amines, ethers, and ketones,^[147] making them a suitable sensor array component for meat freshness sensing. Several studies investigated the use of sensor arrays using different chemo responsive dyes for meat (i.e. chicken^{[146,148,149]}) or meat products (i.e. fresh pork sausage^[150]) as freshness indicator system. An accuracy of 90% or more of developed colorimetric sensor arrays for optical food odor detection was analyzed in several studies, of which data analysis and sensor array composition were reviewed by Xiao-wei et al.^[88]

Fluorophores

Another approach to detect neutral analytes that can be used as freshness indicator substances of meat product spoilage, like biogenic amines, is the reaction between fluorophores and the analyte. The analyte recognition can be based on the formation of a covalent bond, changing the indicator dyes fluorescence intensity and absorbance.^[14,63]

In the literature, an induced emission of the fluorophore (in their non-fluorescent form) upon interaction with the analyte (fluorescent form) is referred to as “turn-on” fluorescence. Turn-on sensing of fluorophores with aggregation induced emission can be also induced through non-covalent interactions, including hydrogen bonds and van der Waals interactions as described elsewhere.^[151] Dyes with a fluorescent and colorimetric signal are advantageous towards single signal dyes because they recognize the analyte and simultaneously change their color. In this section, several turn-on fluorescent dyes with mostly simultaneous color changing behavior feasible for the detection of biogenic amines are described.

To detect volatile amines such as the biogenic amines putrescine and cadaverine, 1,2-dihydroquinoxaline derivatives, with aggregation induced emission characteristics, were loaded onto filter papers. In their protonated form, 1,2-dihydroquinoxaline derivates are non-emissive and red colored. Deprotonation occurs as soon as amines are available leading to a molecular change of the derivates into their emissive yellow-colored form. In a food spoilage test with different fish, stored at room temperature, a faster turn-on fluorescence response (after 18 and 24 h) than colorimetric change (after 48 h) of the indicator paper was observed which revealed that the fluorescent response of the indicator was more sensitive to biogenic amines then its colorimetric response.^[152]

Another “turn-on” fluorescent sensor based on aggregation with biogenic amines induced emission has been developed using carboxylic acid modified tetraphenylethenes. The sensor is based on the binding affinity of the carboxylic acid with the amine resulting in aggregation which activates fluorescence emission (Fig. 6). A higher fluorescent response has been observed for amines with higher chain lengths (spermidine >cadaverine >histamine).^[13]

Figure 6. Schematic drawing of the “turn-on” fluorescence response of carboxylic acid modified tetraphenylethenes towards amines based on their binding affinity, where amine 1 represents a long chain diamine (e.g. cadaverine) and amine 2 represents an aromatic amine (e.g. histamine) (AIE: aggregation induced emission, TPE: tetraphenylethene) (adapted and extended from^[13]).

Colorimetric and fluorescent dual signals have been investigated for pyrrolopyrrole aza-BODIPY (PPAB) probes after exposure to gaseous biogenic amines.^[32] With increasing amount of putrescine or cadaverine molecules in the vapor, emission intensity gradually decreased from dark to bright yellow and the color changed visibly from green to yellow. The authors assume that the change in conjugation resulted from a split of the large PPAB molecules into smaller molecules by biogenic amines attacking the imine and B-N (boron – nitrogen) bonds of the large PPAB molecules. PPAB was analyzed to be specific towards di-, poly-, and aliphatic amines as no significant response towards other amines such as secondary-, tertiary-, and aromatic amines were detected. With their synchronous colorimetric and fluorescent turn-on process and naked eye visibility, the authors concluded a potential use as an intelligent package.^[32]

Another fluorophore feasible for biogenic amine detection during meat spoilage are pyrilium dyes. Pyrylium dyes react with amino groups by exchange of oxygen with the amidic nitrogen, thereby changing from a blue and virtually non-fluorescent dye into a red fluorescent conjugate (pyridinium derivatives). For instance, the biogenic amines putrescine, histamine and tyramine induce a fluorescent signal when complexing with the chameleon dye, namely 2,6-dimethyl-4-[(E)-2-(2,3,6,7-tetrahydro-1 H,5 H-pyrido [3,2,1-ij] quinolin-9-yl)-vinyl]-pyranylium. A color change from blue (620 nm) over green to red (502 nm) as well as a change in fluorescence emission intensity (quantum yield) from under 0.01 up to 0.5 was exclusively observed for primary amines by Azab et al.^[153] In another study, a mixture of pyrylium dyes and methylpyridinium derivates were studied to detect biogenic amines. The results showed a chromatic shift from blue (650 nm) to red (521 nm) after reaction with histamine, putrescine, and cadaverine.^[154] As a representative biogenic amine, histamine was also detected by a 2,6-dimethyl-4-[(E)-2-(2,3,6,7-tetrahydro-1 H,5 H-pyrido[3,2,1-ij]quinolin-9-yl)-vinyl]-pyranylium tetrafluoroborate-based sensor. The initial non-fluorescent blue sensor also formed a red fluorescent derivate with histamine, showing similar absorption and emission^[155] to those analyzed in other studies demonstrated.^[153]

Lee et al.^[156] investigated the detection of biogenic amines in buffered aqueous solution by different coumarin derivates. They showed micromolecular detection of important biogenic amines via UV-Vis or fluorescence spectroscopy. Although the study focused on buffered aqueous solutions, a possible application for sensing gaseous amines by the coumarin derivate 7-(N,N-dimethylamino)-4-hydroxycoumarin has been examined as well. Behaving like a chemodosimeter, the coumarin dye showed an irreversible and cumulative visible color respond to gaseous putrescine. The 7-(N,N-dimethylamino)-4-hydroxycoumarin drop coated poly(methyl methacrylate) layer showed fading of its yellow color, demonstrating a possible application for optical detection.^[156]

A ratiometric fluorescent gaseous amine indicator was developed by immobilizing isothiocyanate (indicator) and protoporphyrin IX (reference) onto cellulose acetate. Different indicator forms including printing ink, coating, flexible film, and nanofibrous membrane were tested. Upon increasing concentration of different amines, including putrescine and histamine, the indicator fluorescence emission changed from red over orange/yellow to green. Electrospun nanofibrous membranes were identified as the most cost-efficient indicator form and therefore additionally used for monitoring seafood freshness. Conducted freshness trials of shrimps and crabs at different storage temperatures showed possible usage of the indicator as a freshness indicator.^[157]

A chemiresistive sensor detecting volatile aromatic biogenic amines, including histamine and tyramine, was developed by Liu et al. using semiconducting naphthyl end-capped terthiophene derivate films. The sensor showed a good selectivity, due to the sensors little response to aliphatic amines including cadaverine, putrescine spermidine, and spermine. In addition, an increasing sensor response has been observed during pork, chicken, and fish storage at refrigerated and room temperature.^[158]

Different chemodosimetric detection assays have been investigated by Chow et al. In their previous trial, gaseous biogenic amines, including putrescine, were detected by a Ru(II)-Eu(III) heterobimetallic complex. Using different heterobimetallic

Ru(II)−Ln(III) complexes in a following study, putrescine and histamine vapors were detected in a colorimetric and luminescent manner. For sensor verification, spiked putrescine samples of fish were used and a linear spectrofluorimetric response upon increasing putrescine concentration has been observed. Furthermore, no cross-interference was observed towards other biogenic volatile compounds.^[159,160]

Another approach to detect biogenic amines was conducted using asymmetric perylene diimide molecules in a fluorescent nanotube system. Measuring the fluorescence response cadaverine, ammonia, and putrescine showed the highest response intensity and lowest detection limit among several different vapors. Under refrigerated conditions, fluorescence quenching also increased over four days of storage period of shrimp, fish, chicken, and pork indicating an increase in volatile compounds and progressing spoilage.^[161]

Conclusion

This review provides an overview of different biogenic amine detection approaches as freshness indicator systems for meat and meat products, describing and discussing the use of bioreceptors, chromophores, and fluorophores.

Among bioreceptors, enzymes are well studied and well-known in the food sector, however, so far, enzymatic detection of biogenic amines seems only applicable in aqueous-based system, thus not (yet) suitable for in-packaging biogenic amine vapor recognition. Molecularly imprinted polymers, mimicking the recognition mechanism of enzymes, reveal more potential as biogenic amine detectors in vapor, when combined with a chromophore or fluorophore for visual indication. However, to elucidate the in-packaging application of molecularly imprinted polymers further research is needed.

Biogenic amine selective chromo- and fluorophores reveal high potential based on the high precision to detect specific analytes.

In general, the commercial application of freshness indication systems is still limited, because there are many requirements. Besides others, the indicator shall be easy to read and understand by consumers, the system needs to be precise, the indicator substances need to be implemented into the packaging material, and the indicator substances need to be harmless. Based on the cited references, two trends have been identified, one is research about natural indicator substances e.g. natural pH dyes that are more environmentally friendly but so far less precise, and systems that can be detected by naked eye or by using smartphones to interpret the freshness indication systems response. Recent and future challenges of the intelligent packaging sector are the production of reliable indicator responses and the low-cost production of the indicator, meeting the requirements of a packaging application feasible to meet the consumers demand for fresh, safe, and qualitative food.

Disclosure statement

The author(s) declare no conflict of interests.

Additional information

Funding

This study was carried out as part of the SmartMaterial research project. The SmartMaterial Project is funded by the Carl Zeiss Foundation;Carl-Zeiss-Stiftung;