Effect of Antigonon leptopus extract in corn starch - glycerol based film as colourimetric indicator film for monitoring fish freshness

A colourimetric indicator can be used to test the freshness of fish in a rapid, straightforward and non - destructive manner. Anthocyanin, a natural dye found in Antigonon leptopus flowers, has a sensitive colour reactivity to a wide range of pH levels and can be made into colourimetric indicator films on a laboratory scale, making it a viable replacement for artificial dyes. The study's purpose was to see how the concentration of A. leptopus extract affected the colourimetric assessment of fish freshness. Ethanol was used to extract anthocyanin from A. leptopus . FTIR and pH - respond spectroscopies were used to characterise the extracts. Colourimetric indicator films were developed to assess fish freshness by incorporating A. leptopus extract into a corn starch - glycerol matrix via the solution casting method, with concentrations of 10%, 30% and 50%, respectively. The effect of A. leptopus extract on visual aspects, thickness, morphology, FTIR spectra, UV - Vis spectra and colour responses was analysed. Microbial analysis, TVB - N contents, firmness, and pH of the fish samples were analysed after storage. The colour of the original films became darker as the extracted content increased. There was no significant (p>0.05) effect of A. leptopus extract on film thickness. SEM micrographs revealed that the composite films had homogeneous and whitish granules on the surface and that increasing the A. leptopus extract concentration caused the films to become rougher. FTIR and UV - Vis spectroscopies showed successful binding between A. leptopus extracts and corn starch - glycerol film. With increasing pH, the colour change of the films exposed to pH = 3 - 11 solutions was no significant difference due to improper storage. An increase in the microbial population, TVB - N content and pH was observed over the storage period as a result of fish deterioration. Colour changes were also identified in the film which became darker. Overall, colourimetric indicator film with 50% A. leptopus extract was found to be the optimal formulation since it had the highest values in ∆ E* during progressive spoilage of fish.


Introduction
Intelligent packaging films are packaging systems that can track the condition of the packaged food when it is stored or delivered in order to improve food safety and quality (Fang et al., 2017), which attached a colourimetric indicator film.When the pH of the food inside the package changes, the colourimetric indicator changes colour so that consumers can immediately discern between fresh and damaged food without having to open the packaging, potentially improving food quality and reducing food waste (Sani et al., 2021).Many researchers have used pH-sensitive dyes such as bromothymol blue, dimethylphenol blue, bromocresol green, bromocresol violet, phenol red, methyl red, and others in food packing materials to check the freshness of food.Anthocyanins have a colour reaction that is sensitive to a wide pH range (Choi et al., 2017).The sensitivity of anthocyanins differs depending on the plant source (Rawdkuen et al., 2020).For example, in their RESEARCH PAPER analysis, anthocyanin derived from butterfly pea had the strongest pH sensitivity whereas red dragon fruit and roselle had low pH sensitivity.In this study, the anthocyanins derived from a new source, A. leptopus were used to develop colourimetric indicator films to monitor the freshness of fish.Antigonon leptopus is native to Mexico, it is widely naturalized and cultivated as ornamental in warm, tropical climates around the world, such as Africa, India, Australia, North, Central and South America and numerous islands in the Pacific Ocean (Burke and Ditommaso, 2011).Antigonon leptopus is used to make honey as a nectar source (Burke and DiTommaso, 2011).In the West Indies and Central America, it's also used in traditional medicine.Antigonon leptopus contains anthocyanin, which can be extracted in 15 mg by solvent extraction (Youssef et al., 2021).However, less information about anthocyanin from A. leptopus was found in the literature.
Monitoring fish freshness is of great importance for consumers, retailers, and industries.There are many common methods used to investigate the freshness of fish (Huang et al., 2019).However, most of these methods generally require long analysis time and professional operators.Normal food packaging is unable to monitor the state of the packaged food or the food environment inside the package, as well as give the user accurate information (Merz et al., 2020).The use of intelligent food packaging, such as colourimetric indicator films, to check the freshness of fish has just been launched, although this is not a variable study.Chemical dyes are commonly used in new concepts of intelligent food packaging, such as pH-sensitive colourimetric indicators, but they are potentially toxic and harmful to humans.Natural colours are non-toxic, they should be utilised to substitute chemical dyes.Anthocyanin from A. leptopus could be used to replace artificial dyes because it is a natural dye with a colour reaction that is sensitive to a wide pH range (Pereira et al., 2015).Furthermore, employing anthocyanin from A. leptopus can increase its value in the food packaging industry.The purpose of the study was to determine the effect of A. leptopus extract concentration as a colourimetric indicator in assessing fish freshness.

Materials
Antigonon leptopus flowers were obtained from Seberang Takir, Kuala Nerus, Terengganu in August 2021.All samples were harvested and the chosen parts, which were the proper flowers, stored at -18 o C. Rastrelliger kanagurta were purchased in August 2021 at Jeti Pulau Kambing, Kuala Terengganu, Terengganu, kept in ice (0°C) during transportation to the laboratory, and stored in -18°C.

Extraction and quantification of Antigonon leptopus extract
Antigonon leptopus flowers were dried at 50°C for 24 hrs (Memmert drying cabinet, United Kingdom).Forty grams of dried A. leptopus were blended with ethanol:1 M HCl mixture solution (85:15, v/v) at a solid: liquid ratio of 1:30 (Wu et al., 2019).The mixture was then stirred continuously for 24 hrs in the dark.The mixture was then filtered, and centrifuged at 4000 rpm for 20 mins.The supernatant was neutralised to pH 7.0 with NaOH (1 M), then concentrated at 40°C in the dark in a rotary evaporator.The concentrated solution was tested for its absorbance at the maximum wavelength (λmax) using a spectrophotometer (IRTracer-100 Spectrophotometer, Shimadzu, Japan) in the range of 400-4000cm -1 .The colour changes of the extract were analysed after mixing in different pH solutions (pH 3-10).

Preparation of the colourimetric indicator films
Approximately 4 g of commercial corn starch powder and 1.5 g of glycerol were dissolved in distilled water to form corn starch-glycerol solution (Prietto et al., 2017).The solution was heated to 80°C and stirred at 8 rpm for 2 hrs.After 2 hrs, the solution was cooled until it reached 40°C.A certain content of extract (0.1 g, 0.3 g, or 0.5 g) was then added into the corn starch-glycerol solution with continuous stirring for 30 min to obtain extract/corn starch-glycerol mixtures with extract concentrations of 10%, 30%, and 50% (w/w, based on corn starch-glycerol), respectively.The mixture was homogenized at 8000 rpm for 20 mins.The mixture was then poured on a clean mould and dried for 48 hrs.

Thickness and morphology of colourimetric indicator films
The thickness of the films was measured with a digimatic calliper (0.001mm, Mitutoyo), and the average of five different point positions measured in each film was calculated (Merz et al., 2020).A scanning electron microscope (SEM) (Hitachi tabletop TM1000, Hitachi High-Technologies Corp., Tokyo, Japan) was used to examine the surface section of films.The photos were taken at a 10kV acceleration voltage with magnifications varying from 300 to 1000× (Loo and Sarbon, 2020).

Application of the colourimetric indicator films for monitoring fish freshness
The fish samples were placed inside a container with a colourimetric indicator film attached to the bottom.The containers were stored at 4°C for 14 days.The change in film colour was visually monitored, and the colour was measured using a chromameter.

Firmness measurement
Texture Analyzer TA-XT plus (Stable Micro Systems Ltd., UK) equipped with a 36-mm diameter cylindrical probe (P/36R) and controlled by Texture Exponent 32 software was used to measure whole fish firmness by compression on the left side, approximately 2 cm from the operculum and over the lateral line.All samples were thawed at room temperature for 3 hrs prior to the texture analysis.For the determinations, the mackerel fish were compressed by 30% of their height (1 mm/s crosshead speed, 30 kg load cell) and the maximum force (N) was recorded (Wan Mohamad Din et al., 2020).

Microbial analysis of fish samples
Approximately 10 g of fish sample were taken under aseptic conditions and mixed with 90 mL of peptone water in stomacher bags to prepare a 10 to 1 dilution, then homogenised for 30 s at 25°C in a stomacher (BagMixer 400, Interscience, France).A 10 -2 , 10 -3 , 10 -4 and 10 -5 dilution series was prepared.One millilitre of 10 -3 , 10 -4 , and 10 -5 dilution were pipetted onto agar.An Lspreader was used to spread the inoculum in an aseptic manner.After 24 hrs of incubation at 37°C, colony numbers were counted and reported as log CFU/g (Mohd Zin et al., 2021).

pH measurement
pH measurements of fish samples were performed using a pH meter.pH values were obtained using 5 g of minced fish samples with 5 mL of distilled water.The pH meter was calibrated using pH buffer solutions at pH values of 10.00±0.01,7.00±0.01,and 4.01±0.01at 25°C (Chong et al., 2020).

Determination of functional groups
The functional groups of A. leptopus flowers were determined using FTIR Transmission (Nicolet iS10, Thermo Scientific, US) in which 4 mg of A. leptopus flowers powder was mixed with 200 mg of KBr (1:50).The mixture was homogenized using agate mortar and pestle and then it was pressed into pellet (1-2 mm thick films) with a 15-ton hydraulic press.The FTIR spectra were obtained from wave numbers of 600 to 4000 cm -1 during 64 scans with 2 cm -1 resolution.The resulting spectrum represented the molecular absorption and transmission which then created a molecular fingerprint of the sample (Chew et al., 2020).

Total volatile basic nitrogen contents
Total volatile basic nitrogen (TVB-N) contents were determined by steam distillation based on the method described by Berizi et al. (2018) with a slight modification.Approximately 10 g of minced fish was blended with 50 mL distilled water and impregnated further for 30 mins with shaking every 10 mins.After that, the mixture was filtered.Approximately 5 mL of filtrate was mixed with 5 mL magnesium oxide (0.1% w/ v).Steam distillation was performed using a Kjeldahl distillation unit (Vapodest 30, Gerhardt, France).The volatile base was distilled into 10 mL of 20 g/L boric acid containing methyl red and bromocresol green indicators.Then, the collected distillate was titrated with 0.01 mol/L HCl.The results are expressed as mg/100 g.The content of TVB-N was calculated using the following equation: Where V1 is the titration volume for the tested sample (ml), V2 is the titration 145 volume of blank (ml), and c is the actual concentration of HCl (mol/l), m is the 146 weight of minced pork sample (g).

Statistical analysis
All data were analysed by Minitab software and were described as the mean ± standard deviation (SD).The results were computed using one-way analysis of variance (ANOVA) and Fischer's Least Significant Difference (LSD) test, with p < 0.05 considered significant.

FTIR spectra of extract solutions from Antigonon leptopus
Figure 2 depicts the FTIR spectra of A. leptopus extract solutions.The functional groups were separated based on their bonding site, and the presence of a diverse range of functional groups of bioactive compounds was confirmed by the peaks obtained when the extract was passed through the FTIR region (Table 1).Skoog et al. (2017) reported that the different functional groups will absorb radiation of a different characteristic frequency of the infrared spectrum.Ghassempour (2008) reported that the wave number that lies between 3700 cm -1 to 3100 cm -1 indicates the vibration characteristics of hydroxyl groups so the broad peak at 3348.42 cm -1 indicated the hydroxyl group (O-H) and the peak and wide band that corresponded to the vibration amplitude of an O-H group is highly intense.Figure 2 also shows absorption bands for saturated hydrocarbon groups, 2924.09cm -1 for the methyl group (C-H 3 ) and 2854.65 cm -1 for the methylene group (C-H 2 ) (Vasincu et al., 2014).Moreover, the bands at 1643.35 cm -1 can be assigned to the stretching vibration of C=O groups (Favaro et al., 2018) or benzene skeleton vibration (Fei et al., 2020).Other significant bands at 1450.47 cm -1 and 1273.02cm -1 can be referred to as C-N vibration (Favaro et al., 2018) and the C=C bond that arises from the pyran ring (Musa et al., 2019), respectively.

Visual aspects, thickness and morphology
Figure 3 displays the visual appearance of colourimetric indicator films containing 10%, 30% and 50% extract, respectively.The brown colour of the films is due to the addition of extract to the film formulation.The visual colour difference was confirmed in the image demonstrated in Figure 3.A similar observation of an increase in concentration of anthocyanin, the films becoming darker was also reported by Li et al. (2021), who extracted anthocyanin from purple tomato to create a colourimetric indicator for application in intelligent packaging.The results of colour parameters, as presented in Table 2, also verified this observation.Table 2 illustrates the colourimetric parameters (L*, a*, b* and ∆E) of colourimetric indicator films containing 10%, 30% and 50% extract.The amount of extract concentration placed into the colourimetric indicator films determined the degree of colour shift.The L* and ∆E* values fell with the addition of extract, however, the a* and b* values increased, showing a propensity toward redness and blueness.The increase in extract level causing a reduction in the L* value of colourimetric indicator films is assigned to the higher indicator dye content in the films.Nogueira et al. (2019), also noticed a reduction in the brightness of arrowroot starch films with blackberry powder.Moreover, Lee and Coates (2003), reported that the ∆E* had more than 2 proves that there will be a visually perceptible difference to the  ).This could be explained by the addition of A. leptopus extract into the film's formulation which will change the colour appearance.
The thickness of the developed films varied between 0.096 mm (10%) and 0.108 mm (50%) (Table 3).The effect of the A. leptopus extract concentration on the film's thickness was not obvious.There is no significant difference observed between the thickness of colourimetric indicator films when analysed using oneway ANOVA between the percentage of A. leptopus extract addition.Similar results are reported by Prietto et al. (2017), who reported that the difference between various concentrations of anthocyanin did not affect the thickness of the indicator film in black bean seed coat and red cabbage.However, Nogueira et al. (2019) had different results, they found that the thickness of arrowroot starch films increased as the blackberry powder concentrations increased.Farias et al. (2012) also discovered that the thickness of cassava starch films increases when the plasticizer and acerola pulp concentrations rise.This difference was produced by a higher concentration of extract-integrated solids in the same mass of film-forming solution per unit area in the carrier plate.
The surface morphology of samples with 10%, 30% and 50% anthocyanin and the images are presented in Figure 4.The surface morphology of the corn starchglycerol-A.leptopus extract films appeared almost homogeneous and had whitish granules on the surface.This study displays the distinct patterns that correspond to withering ghost starch granules (Medina-Jaramillo et al., 2017).Corn starch-glycerol with 50% anthocyanin shows a rougher surface structure compared to 10% and 30% anthocyanin.It has been shown that various concentrations of anthocyanin alter the film's morphology.A similar result was observed by Hamzah et al. (2021), anthocyanin concentration increased the sago starch films becoming rougher surfaces.Mary et al. (2020) reported that the rough film surface indicated the anthocyanins were mixed homogeneously and a strong reaction between starch and anthocyanin existed.

Functional groups analysis
Figure 5 presents the FTIR spectrum for corn starchglycerol films with (a) 10% A. leptopus extract (b) 30% A. leptopus extract (c) 50% A. leptopus extract.The 10%, 30% and 50% A. leptopus extract films showed similar results, which were depicted at 3300 cm -1 , 2924.09 cm -1 , 1643.35 cm -1 , 1242.16 cm -1 and 1010.70 cm -1 .The data shows that increasing A. leptopus extract concentration from 10% to 50% resulted in the decrease of the peak of O-H group in films from 3294.42 cm -1 to 3286.70 cm -1 .The slight reduction in wavenumber was probably due to an increase in the concentration of A. leptopus extract, which increases the interaction between anthocyanin polyphenols and corn starch hydroxyl groups (Merz et al., 2020).Corn starch was the major component in the films.The glycerol contained in the films consisted of C-H stretching vibration that shows at 2924.09 cm -1 in both colourimetric indicator films (Bilanovic et al., 2016).Liu et al. (2017) reported that the C=O from the amylose and amylopectin contribute to the film to withstand force and support the structure of the films.The addition of A. leptopus extract in the film mixture induced chemical and physical interactions with corn starch and glycerol, which involved the addition of a pyran ring detected at 1242.16 cm -1 and shifted to a  RESEARCH PAPER higher wavenumber.The results of the current study were in agreement with an FTIR analysis by Musa et al. (2019) on corn starch-glycerol and anthocyanin from Hibiscus sabdariffa.The strong bands were found at 3378.11 cm -1 (O-H stretching), 2937.11cm -1 (stretching vibration of C-H), 1648.33 cm -1 (stretching vibration of C=O), and 1417.87 cm -1 (CH-CH 2 bending vibration).
Figure 6 shows the maximum absorption for both colourimeter indicator films at 300 nm and 550 nm in the UV and Vis region.Those absorptions described the wavelength of maximum anthocyanidin detection.Qin et al. (2010) reported that 2 benzene rings will create a conjugated system in anthocyanin molecules in the UV-Vis region at maximum absorption around 280 nm and 500-550 nm respectively.In addition, Musa et al. (2019) also reported that the maximum absorbance at 520 nm indicated the presence of C=C conjugation in the anthocyanin structure.The presence of anthocyanin in colourimeter indicator film enabled the film to change colour in different pH values.However, finding shows that the use of these films should be limited to avoid direct contact with hydrophilic chemicals, as the colour pigments may migrate to the product, which would be undesirable.
Furthermore, from this result, the absorbance increased as the concentration of A. leptopus extract was increased.This is because one of the factors that affect a sample's absorbance is its concentration.As the concentration is higher, more radiation is absorbed leading to the absorbance increase (Luong et al., 2011).As a result, the concentration is directly proportional to the absorbance.

Colour responses
Figure 7 illustrates corn starch-glycerol films containing 50% A. leptopus extract in solutions with pH values ranging from 3 to 10.When the pH was changed from 3 to 10, the colour change of the films was not obvious.This observation was proved by the finding of colour parameters, shown in Table 4. Table 4 displays the colourimetric parameters of corn starch-glycerol films containing 10%, 30% and 50% A. leptopus extract after soaking the films in several pH buffer solutions.There is no significant difference observed between the L*, a* and b* of colourimetric indicator films when analysed using one way ANOVA between the pH.However, previous studies reported by Luchese et al. (2017) had different results, who confirmed that the colourimetric indicator films are visually perceptible differences to the human eye between the samples (corn starch films containing blueberry powder).Besides that, Li et al. (2021) reported that the change in colour of the film with anthocyanin was from pink (pH = 3) to light  The reason why the colour change of films was not obvious may be due to the degradation of anthocyanin that is present in the A. leptopus extract.The stability of anthocyanin is influenced by a number of factors such as pH, light, temperature, co-pigmentation, sulfites, ascorbic acid, oxygen and enzymes.This degradation of anthocyanin that is present in the A. leptopus extract may be caused by improper storage temperature of A. leptopus extract.Kırca et al. (2006) reported that the degradation of anthocyanins was clearly affected by storage temperature.The breakdown of anthocyanins was substantially faster at 37°C than it was at 4°C when stored refrigerated.Moreover, light also accelerates the degradation of anthocyanins present in A. leptopus extract.Anthocyanins are affected by light in 2 ways.Anthocyanin production is aided by light, but it also accelerates their destruction.Enaru et al. (2021), report that the greatest substantial loss of anthocyanins occurs when pigments are exposed to fluorescent light.To prevent the harmful effects of light on anthocyanins, A. leptopus extract should be stored in a container composed of materials that can block light from the visible spectrum, particularly the ultraviolet field of the spectrum, producing a protective barrier.

Application of the colourimetric indicator films in monitoring fish spoilage
To validate the colourimetric indicator films, this study was done using cornstarch-glycerol films with three different concentrations (10%, 30% and 50%) of A. leptopus extract in detecting fish deterioration.Figure 8 shows that the 50% A. leptopus extract level of colourimeter indicator film exhibits the highest colour change rate, followed by 30% and then 10% of A. leptopus extract.Among the colourimeter indicator films, the ∆E* value of the 50% A. leptopus extract of colourimeter indicator film expresses the highest difference between before and after storage.The ∆E* value of colourimeter indicator films with 50% A. leptopus extract concentration increased from 74.63 to 77.19 (Table 5).Previous studies have reported different results; in some, the colourimeter indicator film with 30% anthocyanin had the highest rate of colour change, followed by 50% anthocyanin and no variation in 10% of anthocyanin (Wu et al., 2019).Zhai et al. (2017) reported that a higher colour variation rate occurred in RESEARCH PAPER the colourimetric films with fewer anthocyanin and this was due to the fact that the discoloured anthocyanin took higher proportions of the total anthocyanins within the same reaction time.Colourimeter indicator films' colour fluctuation mechanism responds quickly to TVB-N in the container this can indicate fish freshness in the early phases of storage.TVB-N hydrolysed to produce hydroxyl ions after they diffused into the films, causing the environment of the film to become alkaline, and the latter induced the colour change of anthocyanins (Zhai et al., 2017).TVB-N value, pH value, firmness and microbial content of Indian mackerel samples were also determined.
Table 6 shows the changes in fish freshness after 14 days.The initial TVB-N value of the Indian mackerel sample was 7.60 mg/100 g then it increased with storage time and reached 179.2 g after 14 days.The Chinese standard for fish (GB 2733(GB -2015) ) that the of should be less than mg/100 Standards of People's Republic of China, 2015).European Union Commission Regulation (EC) no.2074/2005 (Annex II) reported that the consumption limit of TVB-N value ranges from 25 to 35 mg/100 g, which indicates that the quality of Indian mackerel has declined and is not suitable for consumption (European Union, 2005).TVB-N value is one of the most widely used methods to determine the freshness and spoilage of fish.TVB-N is an off-flavour substance that is produced by enzymes or microbial degradation of protein-rich foods which include ammonia, dimethylamine, trimethylamine and so on.The pH of the sealed package may change due to the formation of TVB-N (Zhai et al., 2017).Kyrana et al. (1997) reported that the high value of TVB-N is attributed to the high degree of protein degradation.Low molecular weight nitrogenous compounds were broken down and volatile base nitrogen was produced.The formation of volatile nitrogenous compounds causes the formation of an alkaline environment on the films (Ma et al., 2018).Moreover, microorganisms also cause biogenic amines and result in a rise in pH value.The pH of Indian mackerel samples was 5.67 at the start of storage and increased to 7.53 at the end.This is related to the development of alkaline substances such as ammonia compounds and TMA, which are mostly formed from microbial action at refrigeration (Chudasama et al., 2018).These alkaline compounds have a high pH, which causes anthocyanins to generate carbinol bases, resulting in film colour shifts.
The total aerobic mesophilic count on the spoiled fish sample was 1.835×10 6 CFU/g.The standard count method relies on bacteria growing a colony on a nutrient medium (Kuley et al., 2017).The nutrient agar that is used in this research for standard plate count is Plate Count Agar (PCA).PCA is not a selective medium.Bacterial growth nutrients are provided by peptone, yeast extract and glucose.Yeast extracts are the main source of B-complex vitamins.Glucose is a carbohydrate that provides energy.These nutrients, together with the nutrient factors present in the products to be evaluated will support the growth of most organisms found in the product.In a solid sample, the number of bacteria is measured in colony forming units per gramme (CFU/g).

RESEARCH PAPER
In addition, some parasites can cause tissue breakdown, leading to undesirable texture defects in fish (Batt and Patel, 2014).Changes in texture have a direct impact on seafood freshness.The firmness of Indian mackerel changes from 1872.38 N to 1490.21N. Prabhakar et al. (2020) also reported that one of the factors that affect the textural quality changes of fish is due to autolysis that produces hypoxanthine and formaldehyde.

Conclusion
Colourimetric indicator films containing 10%, 30%, and 50% A. leptopus extract were found to be compatible with corn starch and glycerol in this study.These colourimetric indicator films containing A. leptopus extract can be used to monitor the freshness of fish.The 50% A. leptopus extract film showed the most significant colour change after fish preservation.These findings imply that A. leptopus is a good source of and that colourimetric indicator films containing 50% A. leptopus extract can be used to determine the freshness of marine commodities like However, due improper storage of A. leptopus extract, the colourimetric indicator films were not responsive to colour change, and this was not noted throughout the investigation.

Figure 1
Figure 1 illustrates the colour variation of extract solution from A. leptopus in different pH solutions.The colour of the solution changed from reddish brown to yellow when the pH was raised from 3 to 10. Anthocyanins may have been present in the extract solution when the colour changed.Since anthocyanin's chemical features make it unstable and cause it to decay quickly.Aliaño-González et al. (2020) claimed that the

Figure 1 .
Figure 1.Colour variation of extract solution from A. leptopus.

Figure 8 .
Figure 8. Colour change of colourimetric indicator films after 14 days.

Table 1 .
Functional group assignment of A. leptopus extract.

Table 2 .
Colourimetric parameters (CIELab*) of colourimetric indicator films containing 10%, 30% and 50% of A. leptopus extract.Values are presented as mean±SD.Values with different superscripts within the same column are statistically significantly different between sample by Fischer's Least Significant Difference (LSD) test, with p < 0.05 considered significant.

Table 5 .
Colourimetric parameters (CIELab*) of colourimetric indicator films containing 10%, 30% and 50% A. leptopus extract after 14 days.Values are presented as mean±SD.Values with different superscripts within the same column are statistically significantly different between sample by Fischer's Least Significant Difference (LSD) test, with p < 0.05 considered significant.

Table 6 .
The change in fish freshness after storage.Values are presented as mean±SD.Values with different superscripts within the same column are statistically significantly different between sample by Fischer's Least Significant Difference (LSD) test, with p < 0.05 considered significant.