1 INTRODUCTION

Seafood products are rich in high-quality proteins, unsaturated fatty acids and biologically available trace elements, which were not readily found in terrestrial food.^{1, 2} In addition, 17% of global edible meat production is seafood, so it played a vital role in people's healthy diet.³ As consumers demand for seafood products, more and more fresh seafood is being shipped across the country. During transportation, the uncontrolled storage environment and transportation temperature can lead to microbial growth and multiplication in seafood products, producing a large number of spoilage and pathogenic bacteria, which can lead to diarrhoea, vomiting, and even death, and other food-borne diseases.⁴ Food safety widely concerned consumers.

Traditional methods of monitoring food freshness include gas chromatography,⁵ liquid chromatography,⁶ electronic tongue,⁷ electronic nose⁸ and capillary electrophoresis.⁹ However, these methods had limitations, relying on expensive instruments and complex operations, time consuming and destructive to samples. The ‘shelf life’ on food package can no longer accurately show the remaining consumption time of food.¹⁰ In recent years, people began to pay attention to the research of intelligent packaging, hoping to achieve real-time monitoring of packaging through internal changes in product quality, and provide consumers with information about product status.¹¹ It is urgent to develop fast, simple, cheap, biodegradable and accurate sensors for real-time detection of shrimp freshness.

Microbiological and chemical spoilage of seafood products can lead to the release of different compounds such as amines, ammonia, hydrogen sulfide (H₂S), carbon dioxide and organic acids.¹² H₂S is a volatile gas produced during meat deterioration, which is mainly produced in the degradation of sulfur-containing amino acids.¹³ The accumulation of H₂S is one of the indicators of seafood spoilage.¹⁴ Wang et al. prepared an aggregation-induced emission (AIE)-active fluorescent probe with aggregated state transition for sensing H₂S in a ratiometric manner for the detection of freshness in shrimp and beef.¹⁵ The colour of the strips changed from green to red under UV light as the meat changed from fresh to rancid with increasing storage time. Lin et al. synthesized a mimetic enzyme based on Cu²⁺-modified boron nitride nanosheets-supported gold nanoparticles (AuNPs/Cu²⁺-BNNS), which can be used to catalyse the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB).¹⁶ The H₂S gas can inhibit the activity of AuNPs/Cu²⁺-BNNS toward catalytic oxidation of TMB. The AuNPs/Cu²⁺-BNNS sensors were successfully applied to detect H₂S produced by pork spoilage with satisfying results. Therefore, H₂S is considered to be a characteristic compound for assessing meat deterioration. Lead(II) acetate basic (LAB) is a well-known dye that reacted with H₂S to form dark brown precipitates of lead sulfide (PbS) (Pb (CH₃COOH)₂ + H₂S=PbS↓ + 2CH₃COOH).¹⁷ Poly-L-lactic acid (PLLA) is a new type of biodegradable material, mainly derived from the starch extracted from natural crops such as wheat, sugar cane, corn and renewable plant resources, fermented into lactic acid and then synthesized PLLA of a certain molecular weight through chemical synthesis.¹⁸ PLLA has good bio-compatibility and mechanical properties. PLLA is one of the most promising compost plastics.¹⁹ It could be fully degraded into carbon dioxide and water in nature, which is an excellent polymer base.

Accordingly, in this study, we developed a colour indicator membrane for monitoring the freshness of shrimp at 25°C. The membrane is based on the correlation between the release of H₂S and the freshness of shrimp during spoilage of seafood products. The colour indicator membrane is prepared by electrostatic spinning of LAB and PLLA solutions. LAB could be evenly distributed in the PLLA solution. The colour indicator membrane prepared by electrostatic spinning technology increased the specific surface area of the membrane to facilitate adequate contact between LAB and H₂S for monitoring the freshness of shrimp. The results confirmed that the prepared colour indicator membrane can accurately and quickly respond to the corresponding colour of H₂S released from shrimp. Therefore, it can be used as a non-contact naked eye visual colour indicator membrane for seafood freshness monitoring.

2 MATERIALS AND METHODS

3 RESULTS AND DISCUSSION

3.1 Characteristics of colour indicator membrane

3.1.1 Morphology and diameter distribution

Figure 1 showed the microstructure of the colour indicator membrane at different concentrations. The PLLA membrane structure had uniform fibres, a smooth surface, and a diameter of 799 mm (Figure 1A,D). After adding LAB to the PLLA solution, the fibre of the obtained fibrous membrane remained uniform and surface was smooth (Figure 1B,C). Moreover, LAB was observed to aggregate in the fibrous membrane. Then, with the increase of LAB concentration, the fibre diameter increased from 1.10 to 1.54 μm (Figure 1E,F). This phenomenon may be due to the increase of solution concentration leading to the increase of viscosity with the increase of LAB concentration.²⁰ As the solubility of the solution increased, the viscoelasticity of the polymer jet increased, making it more difficult to stretch the fibre, so the fibre diameter increased. The fibrous shape was beneficial to the increase of the specific surface area of the colour indicator membrane, and the lead acetate dye was more likely to be exposed to gas. In summary, the colour indicator membrane had a larger specific surface area, thus improving the sensitivity of freshness detection.

3.1.2 FTIR spectra

The chemical interaction between the PLLA and LAB was evaluated using FTIR, and the consequent FTIR was shown in Figure 2. In the spectra of PLLA, PLLA 0.3 LAB and PLLA 0.6 LAB, the absorption peaks observed around 1753 and 1456 cm⁻¹ were attributed to the bending vibrations of the -C=O and -CH₃.^{21, 22} In addition, the absorption peaks at 1183 to 1087 cm⁻¹, which was due to the multimodal distribution of -C-O-C- bonds of different groups and atoms.²³ The peak value around 1361 cm⁻¹ was bending the vibration of -C-H.²⁴ In the spectrum of LAB, PLLA 0.3 LAB, and PLLA 0.6 LAB, two arm peaks at 1509 and 1419 cm⁻¹ could be attributed to the symmetric stretching vibration of -C=O and the absorption vibration of the asymmetric stretching vibration.²⁵ No new chemical bonds were generated in PLLA 0.3 LAB and PLLA 0.6 LAB, and only physical changes occurred in PLLA/LAB colour indicator membrane. Thus, FTIR spectra revealed that LAB was successfully incorporated in the PLLA.

3.1.3 WCA

The WCA was an important indicator of the wettability of a material's surface, with WCA θ = 90° being the boundary between hydrophobicity and hydrophilicity.^{26, 27} When WCA θ > 90°, it indicated that the colour indicator membrane was hydrophobic and not easily wet by liquids, and the larger the contact angle, the better its hydrophobicity. When WCA θ < 90°, it indicated that the colour indicator membrane was hydrophilic and easily wetted by liquids, and the smaller the contact angle, the stronger the hydrophilic ability. As shown in Figure 3A, the WCA of the PLLA membrane was 116.45°, indicating that PLLA was hydrophobic due to the large number of hydrophobic groups contained in PLLA, such as methyl groups and so forth.^{28, 29} As shown in Figure 3B,C, the WCA of the colour indicator membrane was 117.85° when adding LAB at a concentration of 0.3%, and 119.95° as the concentration of LAB increased to 0.6%. Therefore, the colour indicator membrane with the LAB concentration of 0.6% was the best for hydrophobicity. The WCA concentration of the added LAB was greater than 90°, indicating that the colour indicator membrane is hydrophobic and not susceptible to water molecules, which creates beneficial conditions for subsequent experiments.³⁰

3.2 Stability of PLLA/0.6 LAB colour indicator membrane

3.2.1 Humidity stability

During spoilage, the proteins of seafood products was decomposed under the action of micro-organisms and protein enzymes, leading to an increase in free water.^31-33 The humidity stability test was designed to investigate the corresponding colour stability of the colour indicator membrane when the moisture content changed in the packaging environment. This experiment investigated the colour change of the colour indicator membrane at 20% and 80% humidity. As shown in Figure 4, the colour indicator membrane indicated the colour change by L*, a* and b* values. In Figure 4, there was a slight change of L* values in the colour indicator membrane at 20% relative humidity (RH) and 80% RH, while a* and b* values were almost the same. The results showed that humidity had no effect on the colour of the colour indicator membrane. The experiment showed that the PLLA/0.6 LAB membrane had good humidity sensitivity. It can be applied to shrimp freshness monitoring.

3.2.2 Temperature stability

Temperature stability could be assessed as the reliability of the colour indicator membrane in practical application. In order to simulate the actual storage environment of shrimps, 4°C and 25°C were used as experimental temperatures to determine the colour stability of the colour indicator membrane. In Figure 5, the colour of the colour indicator membrane was more stable in 2–8, 10–14 and 16–18 days at 4°C, and colour changed more in 8–10 and 14–16 days. The colour of the colour indicator membrane changed little at 25°C. Although there were fluctuations in the colour change of the colour indicator membrane, ΔE < 3.³⁴ With ∆E > 3, human eyes could identify colour changes. In general, the colour indicator membrane exposed to air for a long time would be oxidized. It was because LAB was not readily decomposed.³⁵ As a result, the PLLA/0.6 LAB colour indicator membrane had excellent colour stability and could be used for shrimp freshness monitoring.

3.3 H₂S responsivity

H₂S produced during spoilage of seafood is an important indicator of quality. Therefore, to determine the ability of the colour indicator membrane to detect H₂S, H₂S gas with different concentrations was used to simulate the environmental changes caused by the seafood denaturation process. As shown in Figure 6A, the colour of the colour indicator membrane changed from white to dark brown as the H₂S concentration increased, indicating that the membrane was as sensitive as expected. Moreover, it can be seen in Figure 6B that the colour change of the colour indicator membrane was mainly due to the change of L value caused by the generation of black–brown PbS. In addition, as shown in Figure 6C, the detection limit of the colour indicator membrane for H₂S was calculated to be 0.45 ppm, which verified the ability of the membrane to detect H₂S.³⁶ The colour indicator membrane was prepared simply. Some H₂S response labels today require the preparation of metal nanoparticles as an indicator material.^{15, 16} The colour indicator membrane provided a rapid colour response to different concentrations of H₂S.

3.4 Application of colour indicator membrane in shrimp freshness monitoring

Shrimps are rich in nutrients such as proteins, minerals, and vitamins.^{37, 38} Therefore, it can provide a rich source of nutrients for microbial growth.^{39, 40} The growth and multiplication of microorganisms is one of the main factors in shrimp spoilage and is one of the most important indicators of shrimp freshness.⁴¹ According to Koutsoumanis,⁴² a TVC count exceeding 7.0 Log (CFU/g) was considered to be spoiled and unfit for consumption. As shown in Figure 7A, the initial colony count of shrimp at 0 h was 2.48 Log (CFU/g). With the increase of storage time, the total colony count of shrimps at 36 h was 7.65 Log (CFU/g), exceeding 7.0 Log (CFU/g). Therefore, at 36 h, the shrimp had deteriorated and could not be eaten. The colour change of the colour indicator membrane and the corresponding shrimp freshness were shown in Figure 7A,B. It was observed that ∆E in Figure 7A increased with the increase of storage time. This result was due to the accumulation of PbS. In Figure 7B, the PLLA/0.6LAB colour indicator membrane was white at 0 h and gradually darkened with the increase of storage time. At 12–24 h, the TVC content was close to the spoilage limit, and the shrimp was in a sub-fresh state. At this time, the colour indicator membrane was grey. The shrimp deteriorated at 36 h, and the colour of the colour indicator membrane became dark brown. The experimental result of ΔE was consistent with the results of the shrimp TVC. Compared with pH-responsive freshness detection labels responding to biogenic amines produced by meat during spoilage,^{43, 44} the colour indicator membrane responds only to H₂S and detects the substance more uniquely. The results showed that colour indicator membranes could accurately detect the freshness of shrimp.

4 CONCLUSIONS

The PLLA/LAB colour indicator membrane was developed for shrimp freshness monitoring. The LAB was dispersed into PLLA-based fibres using electrostatic spinning technology to give the colour indicator membrane a larger specific surface area. Through H₂S responsivity, we found that the colour indicator membrane had high colour responsiveness to H₂S. When applied to the packaging of shrimps with a TVC value above the spoilage threshold, the colour indicator membrane displays a visually distinguishable colour, from white to dark brown. The experiments on shrimp samples showed that the PLLA/LAB colour indicator membrane had considerable potential in monitoring food freshness.