Shelf life of Penaeus vannamei coated with gelatin extracted from Oreochromis niloticus scales

doi:10.21203/rs.3.rs-4137197/v1

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Shelf life of Penaeus vannamei coated with gelatin extracted from Oreochromis niloticus scales

https://doi.org/10.21203/rs.3.rs-4137197/v1

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Fish products have high nutritional value but are very perishable. Fish gelatine coatings are a renewable technology that offers numerous benefits for preserving highly perishable fishery products with high nutritional value. A recent study evaluated the shelf life of Pacific white shrimp (Penaeus vannamei) coated with gelatine extracted from Nile tilapia (Oreochromis niloticus) scales and glycerol. The coated shrimp were stored frozen at -18°C for 180 days. The researchers applied the gelatine coating at a concentration of 1.5%, with 0.2% glycerol, to peeled and headless shrimp (“treatment G”) and compared them with uncoated shrimp (control – “treatment C”). They evaluated bacterial growth, pH, formation of volatile bases (TVB-N and TMA-N), and lipid oxidation (TBARS) every 30 days (day T0, T30, T60, T90, 120, 150 and 180). The study found that the gelatine extraction yield obtained was 24.64%. The total bacterial count (TBC) range for treatment C was 0.00 to 2.32 log CFU/g, while for treatment G it was only 0.00 to 1.48 log CFU/g. The shelf-life assessment showed that the gelatine and glycerol-based coating solution was effective in preserving P. vannamei in terms of microbiological parameters, pH, TVB-N, TMA-N and TBARS. The study concluded that the use of this coating combined with frozen storage could be a good alternative for maintaining the quality of Pacific white shrimp for an extended period.

fish waste

Nile tilapia

gelatin coating

TVB-N

TMA-N

TBARS

Gelatine is a protein derived from the denaturation of collagen. It can be obtained from the by-products and waste generated in the poultry, beef, and fishing industries (Rigueto et al. 2022). However, due to concerns related to religious beliefs (halal and kosher) and diseases such as foot-and-mouth disease and bovine spongiform encephalopathy, gelatine extracted from fish collagen is preferred (Senarathna & Marapana, 2021).

Fish is the most widely produced meat worldwide, with global production reaching 178 million tons in 2020 (FAO, 2022). However, due to the sheer amount produced and the significant waste generated by the fishing industry, which can account for up to 85% of the processed weight (Liu et al. 2022), the disposal of such waste has become a major environmental concern (Aneesh et al. 2023).

Typically, fish waste comprises heads (9–12%), viscera (12–18%), skin (1–3%), bones (9–15%), and scales (5%) (Boronat et al. 2023). Unfortunately, this waste is often discarded, leading to environmental pollution (Sousa et al., 2022). Instead of wastefully disposing of this material, it is possible to extract valuable biocomposites (Alcântara et al., 2022) such as gelatin (Nitsuwat et al. 2021). This approach can significantly reduce waste generation and promote sustainable fish production, aligning with Sustainable Development Goal 12: responsible consumption and production (United Nations 2023).

Gelatin is a versatile material with diverse applications due to its excellent biocompatibility and biodegradability, which is why it is widely used in the food, cosmetic, and pharmaceutical industries (Liao et al. 2021). In the food industry, gelatine can improve the food's viscosity, texture, and stability (Zhang et al. 2020). It also provides a unique melt-in-the-mouth effect after consumption, as it is a thermo-reversible protein gelling agent (Nitsuwat et al. 2021).

Recently, gelatine has been used for the production of edible films and coatings that can retard the proliferation of spoilage and pathogenic microorganisms, thereby improving the quality and shelf life of packaged foods (Abdelhedi et al., 2019; Mirzapour-Kouhdasht & Moosavi-Nasab, 2020).

Studies show the potential value of applying edible films and coatings to extend the shelf life of fresh fishery products (Liu et al., 2023). Although fish products are very rich in protein, polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), essential vitamins and minerals (Giannakourou et al. 2023), they also contain high levels of non-protein nitrogen compounds, endogenous bacteria and autolytic enzymes that cause rapid postmortem deterioration (Jia et al., 2019).

In shrimp, anaerobic glycolysis begins soon after death, using the ATP reserves in the muscle to cause the muscle tissue to harden, known as rigor mortis, and to produce lactic acid, which lowers the pH of the muscle. As the days pass, the process of autolysis begins through the action of the shrimp's natural enzymes, producing low molecular weight compounds such as trimethylamine, which causes the pH of the muscle tissue to rise (Ge et al., 2020; Das et al., 2023). These post-mortem changes result in a distinctly unpleasant taste and require efficient and sustainable preservation technologies to maintain product quality (Pan et al., 2019).

Various methods such as low-temperature storage, high-pressure processing, modified atmosphere packaging, irradiation, biochemical preservation, and ozone preservation have been developed to maintain the freshness of aquatic products (Nagarajan et al. 2021). Freezing is the most common preservation method for fish products, but freeze-thaw cycles and temperature fluctuations during transport, storage, and distribution adversely affect their quality (Zuanazzi et al. 2020).

Low temperature alone may not completely inhibit microbial growth, lipid oxidation, and protein degradation in fish (Ge et al. 2020). And the shelf life for frozen uncooked shrimp (-18°C) is a long 180 days (FAO 1981). Against this background, researchers have been studying sustainable packaging, such as coatings and edible films, for the proper preservation of fresh aquatic products, capable of increasing their quality and shelf life (Alcântara et al. 2022).

However, the use of coatings to extend the shelf-life of frozen shrimp is a recent innovation in this field, with little literature available; and a thin layer of non-toxic coating can be an excellent choice for preserving shrimp by reducing the total microbial load, protein oxidation, melanosis, lipid oxidation and controlling the pH of the flesh (Das et al. 2023).

In view of this, the aim of this research was to extract gelatin from Nile tilapia (Oreochromis niloticus) waste (scales), apply the coating solution from this to peeled, headless, and frozen Pacific white shrimp (Penaeus vannamei), and evaluate the effectiveness of the coating on the shelf life of this product.

The material used in this study was registered in the National System for the Management of Genetic Heritage and Associated Traditional Knowledge (SisGen) under the number registered on the SISGEN platform with the code A058F53.

2.1 Thermal extraction of gelatin and yield calculation

Nile tilapia scales were purchased fresh from a fishmonger in Fortaleza, Ceará, Brazil, and taken to the Laboratory of Fishing Technology (LATEPE) of the Federal University of Ceará (UFC). The extraction of gelatin from the scales was performed according to the method reported by Martins et al. (2018), with minor modifications in the thermal extraction. The demineralized and hydrolyzed scales were immersed in distilled water at a ratio of 1:4 (m/v) for 2 h in a water bath at 60°C, with stirring, in a reactor equipped with a condenser (model 521-5D, Ethik Technology, Brazil) and a mechanical stirrer (model 713D, Fisatom, Brazil).

The mixture was then filtered. The liquid part was left to cool and the scales were returned to the reactor for a second extraction, now at a 1:2 ratio (m/v), for another hour. A new filtration was then carried out. The scales were discarded and the second liquid mixture was added to the first, then placed in aluminum containers, frozen in an ultra-low temperature freezer (model MDF-U33V, Sanyo, Japan), and lyophilized (model LP510, Liotop, Brazil) to obtain dry gelatin, and then ground in an analytical mill (model LS-06MB-N, Skymsen, Brazil) to obtain powdered gelatin. The extraction yield was calculated from the ratio between the dry mass of gelatin and the mass of scales on a dry basis (Senarathna & Marapana, 2021).

2.2 Shrimp procurement and preparation

Twelve kilograms (kg) of Penaeus vannamei [before Litopenaeus vannamei (Boone, 1931)], fresh and without preservatives, with an average weight of 11 ± 0.7 g, were purchased from a shrimp farm in Russas, Ceará, Brazil, packed in isothermal boxes with ice at a ratio of 1:1 (w/w) and transported to LATEPE/UFC within 3 hours. At the laboratory they were peeled, headed, filleted, washed with cold drinking water, weighed, packed, and frozen in a freezer at -18°C for coating the following day.

2.3 Preparation of coating solution, coating, and storage

The solution of 1.5% w/v gelatine and 0.2% glycerol 99.5% PA ACS (VETEC, Brazil) was prepared by dissolving 15 g gelatine in 1000 ml distilled water and adding 2 ml glycerol, stirring for 20 min at 30°C, as suggested by Alcântara et al. (2022). The solution was then sterilized by exposure to ultraviolet light at 25°C for 30 min.

For coating, eight kilograms of peeled, headless and frozen shrimp were separated into two treatments: the control (C) which were not coated, and the coated with gelatin (G). The shrimp from G were placed on a flat grid at room temperature and sprinkled (spray) with the aid of a sprayer with the coating solution on one side, letting it drain and dry for 2 min to form the film, turned and sprayed on the other side, letting it drain and dry for another 2 min. Then all samples, coated (G) and uncoated (C), were packed in polypropylene and sterile Styrofoam trays, sealed, identified and frozen in a freezer at -18°C.

2.4 Shelf life study

To study shelf life, microbiological analyses (psychrotrophic bacteria counts) and physicochemical analyses (pH, TVB-N, TMA-N and TBARS) were carried out in duplicate in nine replicates on peeled and headless shrimp C and G treatments on the first day of the experiment and then every 30 days (T0, T30, T60, T90, T120, T150 and T180 days).

2.4.1 Microbiological analysis

The psychrotrophic bacteria counts (PBC) were determined according to the recommendations of the American Public Health Association (APHA 2015) by preparing decimal dilutions of homogenized shrimp in 0.85% NaCl saline and inoculating on PCA agar plate media (Merck, Germany) using the pour plate method and incubating the plates at 7°C for 8 days in a BOD incubator (model Q315M, Quimis, Brazil). After the incubation period, the number of colony-forming units (CFU) was determined. To calculate the standard plate count (SPC), plates with growth between 25 and 250 CFU were selected and calculated using Eq. 1 (APHA, 2015). The results were transformed into logarithms of the number of colony-forming units (log CFU/g), and samples that did not grow within the specified range were estimated (est.).

\(\text{S}\text{P}\text{C} = \text{C}\text{F}\text{U} \times \text{i}\text{n}\text{v}\text{e}\text{r}\text{s}\text{e} \text{o}\text{f} \text{t}\text{h}\text{e} \text{d}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r}\) (Eq. 1)

2.4.2 Physicochemical analysis

The pH of shrimp samples from groups C and G was measured using a bench pH meter (Kasvi, Brazil) by homogenizing 5.0 g of the macerated sample with 50 mL of distilled water (APHA 2015).

Total volatile basic nitrogen (TVB-N) and trimethylamine nitrogen (TMA-N) were measured using the steam distillation method, as described by (Malle and Tao 1987).

Lipid oxidation was analyzed using thiobarbituric acid reactive substances (TBARS) according to Vyncke (1970). TBARS value in mg malondialdehyde equivalent per kilogram of fish (mg MAD eq/kg muscle) was determined using the standard curve of 1,1,3,3-tetraethoxypropane (TEP) based on Eq. 2:

\(\text{T}\text{B}\text{A}\text{R}\text{S} = (\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e} – \text{0,032}) / 0.0789\) (Eq. 2)

Excel was used to create graphs of the results of the shelf life study.

2.4.3 Statistical analysis

Gelatine yield was subjected to descriptive statistics and the result was expressed as mean ± standard deviation, using Excel software. The analyses of the physicochemical shelf-life parameters for the two treatments were carried out using Jamovi(R) software, version 2.4.6. First, the Shapiro-Wilk test was applied to check the normality of the values obtained, while the Levene test was used to check the homogeneity of the data. The values were then subjected to the Student's t-test to check for significant differences (p < 0.05) between treatments.

3.1 Gelatin extraction yield

The gelatine yield from Nile tilapia scales was 24.64%. This value was higher than other yields also obtained from Nile tilapia scales such as 14.32% gelatine from Nile tilapia scales extracted in a water bath at 60°C and ultrasound for 3 h (Senarathna and Marapana 2021); and 12.10% gelatine from Nile tilapia scales by acid-alkaline-acid extraction and water bath at 60°C for 1 h (Martins et al. 2018).

It was also higher than the gelatine yield obtained from other species, such as 24% from Labeo rohita scales (Das et al. 2017); 11.88%, in the extraction of gelatin from black tilapia (Oreochromis mossambicus) scales, in a water bath at 65°C for 8 h (Sockalingam and Abdullah 2015); 8. 63 and 9.27% of gelatin from spotted golden goatfish scales with 6 and 12 h extraction, respectively (Chuaychan et al. 2017); and 9.55% from sea bream scales in a water bath at 60°C for 12 h (Akagündüz et al. 2014).

However, the yield obtained in this study was lower than some fish skin extraction values reported in the literature. Liao et al. (2021) achieved yields of 35.24, 41.91, 31.66, and 36.27 percent of tilapia skin gelatin extracted at 60 and 75°C at pH 3 and at 60 and 75°C at pH 5, respectively. Sinthusamran et al. (2018) achieved yields of 44.83 to 71.5 percent of giant barramundi (Lates calcarifer) skin gelatine, extracted at temperatures of 45 to 75°C. This is because fish scales and bones generally contain less collagen than skin and collagen can be lost during processing steps such as washing, pre-treatment, and extraction (Sockalingam and Abdullah 2015).

Extraction yields depend on many factors such as species, age, tissue type, and process conditions such as pH, ionic strength, pretreatment and extraction time, temperature, and acid type (Martins et al. 2018; Tkaczewska et al. 2018). The high yield of gelatine from fish scales correlates with the high amino acid (proline) content of these structures (Das et al. 2017). In tilapia, it may be correlated with the high collagen content, as well as the effective pretreatment and extraction methods used (Sockalingam and Abdullah 2015), especially the extraction time and high extraction temperature, which significantly increase the gelatine yield with increasing temperature (Tan et al. 2019).

Here, the high yield values were probably due to the extraction time. Thus, the process used to extract gelatin from Nile tilapia scales was found to have yields that are compatible with literature values.

3.2 Microbiological analysis

Microbial spoilage of fish is mainly due to the growth of large numbers of bacteria and the products of their metabolism, which cause sensory changes such as discoloration, physical changes, changes in texture, slime or gas formation, or unpleasant odors and flavors (Jia et al. 2019).

Psychrotrophic bacteria have the ability to grow and multiply at low refrigeration temperatures and perform proteolytic and lipolytic activities and include species of Acinetobacter, Aeromonas, Alcaligenes, Arthrobacter, Bacillus, Brochothrix, Carnobacterium, Chromobacterium, Citrobacter, Clostridium, Corynebacterium, Enterobacter, Escherichia, Flavobacterium, Klebsiella, Lactobacillus, Leuconostoc, Listeria, Microbacterium, Micrococcus, Moraxella, Pseudomonas, Psychrobacter, Serratia, Shewanella, Streptococcus, Weissella, Alteromonas (formerly Pseudomonas putrefaciens), Photobacterium, and Vibrio (APHA, 2015).

The results of the average decimal logarithm of the colony forming units (CFU) of psychrotrophic bacteria detected in shrimp (P. vannamei) muscle are shown in Fig. 1.

Figure 1 - Quantification of psychrotrophic bacteria (log CFU/g) in samples of Peneaus vannamei frozen for 180 days from the control and gelatine treatments.

From Fig. 1, the total bacterial count (TBC) of treatment C was almost always higher than that of treatment G, except two moments when both had the same count: at T30, where the values were equal to zero, and at T120 with 0.70 log UFC/g/test. In addition, the TBC range for treatment C was 0.00 to 2.32, whereas for treatment G it was only 0.00 to 1.48/est. This may suggest that the combined gelatin and glycerol coating used in this study appears to have slowed microbial growth in frozen shrimp. Also, the reason why psychrotrophic bacteria were not seen in the coated samples at days 90 and 180 is that the antimicrobial effect of the gelatine (Das et al., 2023; Nagarajan et al., 2021) probably inhibited their growth in the G treatment samples.

The International Commission on Microbiological Specifications for Food (Stewart 1987) sets a maximum limit of 107 CFU/g (equivalent to 7.0 log CFU/g) for the population of these bacteria in fish intended for human consumption. Considering this limit, all samples from treatments C and G were within the standards for psychrotrophic TBC in shrimp muscle throughout the storage period.

Farajzadeh et al. (2016) found that the TBC of the control group increased significantly (p < 0.05) faster than the shrimp group (P. vannamei) coated with chitosan and commercial bovine gelatine, both stored at 4 ºC, from an initial 2. 5 log CFU/g to the maximum allowable limit of 7.0 log CFU/g according to the International Commission on Microbiological Specifications for Food (ICMSF) in only 8 days (control), while the coated group reached this limit after 14 days of storage.

Mirzapour-Kouhdasht & Moosavi-Nasab (2020) also observed that the TBC of the control group increased much more and faster (p < 0.05) than the samples of Penaeus merguiensis shrimp coated with gelatine extracted from Scomberomorus commerson skin, after 12 days of storage at 4°C. Jiang et al. (2011) found that coatings of gelatin solution (5% w/w) from catfish (Ictalurus punctatus) skin, glycerol plus potassium sorbate and/or sodium tripolyphosphate were effective against bacterial growth and extended the shelf life of fresh white shrimp (Penaeus vannamei) by up to 10 days when stored on the ice at 0°C for 31 days.

Changes in microbial populations provide useful information for understanding population changes associated with spoilage related to food storage conditions (Jia et al. 2019).

3.3 Physicochemical analysis

3.3.1 pH

Shortly after the death of the fish, some substances in the muscle begin to be converted into acidic substances, raising the pH of the muscle. Proteins are then broken down into alkaline substances that cause the pH to rise, indicating the process of deterioration of fish products (Zuanazzi et al. 2020). pH values below 4.0 indicate that bacterial growth is suppressed and yeasts and molds grow abundantly; above pH 5, proteolytic bacteria can become active (GMIA 2019). The pH of the muscles of frozen L. vannamei shrimp samples from treatments C and G are shown in Fig. 2.

Figure 2 - Graph of pH of P. vannamei muscle frozen for 180 days in the control and gelatine treatments.

Legend: * - indicates that there was a statistically significant difference (p < 0.05) between treatments using Student's t-test.

On day one (T0), the initial pH of treatment C was 6.55 ± 0.09, while the average pH of treatment G was higher (p < 0.05), reaching 6.66 ± 0.02. At 30 days (T30), there was a decrease in pH values: 6.45 ± 0.05 for C and 6.40 ± 0.10 for G, with no significant differences (p > 0.05). Then, at T60, there was an increase in the pH of the two treatments, with a significant difference (p < 0.05) between them (6.57 ± 0.10 in C and 6.69 ± 0.07 in G).

This behavior was to be expected, as anaerobic glycolysis and lactic acid formation begin soon after the shrimp die, causing the pH of the muscle to drop. Days later, autolysis begins through the action of the shrimp's natural enzymes, producing low molecular weight compounds such as trimethylamine, which causes the pH of the muscle tissue to rise (Ge et al., 2020; Das et al., 2023). Thus, the times between T0-T30 and T30-T60 correspond to these two times after the shrimp died under freezing.

The fact that treatment G had higher values than C may be due to the sampling factor, since although they are individuals from the same batch, each individual may have different degradation states. However, as the days passed, the effect of the coating reversed this trend and was able to significantly (p < 0.05) reduce the pH of the shrimp muscle in treatment G after 150 days of storage.

Analyzing only the initial (T0) and final (T180) values, treatment G hardly changed its pH (6.66 ± 0.02 and 6.67 ± 0.08, respectively), while treatment C increased by 0.14 (6.55 ± 0.09 and 6.69 ± 0.09, respectively) on a logarithmic pH scale, indicating that the shrimp had entered the autolysis and protein degradation phase. Nevertheless, on the last day of analysis, there were no significant differences (p > 0.05) between treatments C and G, and throughout the experiment, the pH of the shrimp flesh remained well below the limit of 7.85 recommended for consumption of crustaceans by current Brazilian legislation (Brazil, 2020).

Looking at treatment C in Figs. 1 and 2, there is a similarity in the behavior of the microbiological analysis and the pH curves, indicating that the higher the TBC, the more alkaline the pH of the samples and consequently the greater the action of enzymes and/or bacteria. It can therefore be seen that the use of coatings can be a good practice to inhibit the degradation of fish.

The values obtained in this research were in line with other literature. Ge et al. (2020), working with swordfish (Parapenaeopsis hardwickii), found that the pH of shrimp from the control treatment was higher (p < 0.05) from the 4th day of storage at -5 ºC until the last day of analysis (23rd day) compared to shrimp treated with acid-chlorogenic gelatine. Farajzadeh et al. (2016) found that the pH of the control samples (between 6.32 and 7.91) reached significantly higher values (p < 0.05) than the group of shrimps (P. vannamei) coated in gelatine with chitosan (between 6.22 and 6.44), both stored under refrigeration at 4 ºC.

3.3.2 TVB-N

Total volatile basic nitrogen (TVB-N) is a widely used index to evaluate the deterioration of fish due to the action of endogenous animal enzymes and/or bacterial action that degrade nitrogen compounds in muscle proteins such as peptides, amino acids, and nucleotides (Pan et al. 2019). The TVB-N value is derived from trimethylamine oxide (TMAO), ammonia, dimethylamine (DMA), trimethylamine (TMA), and other volatile basic nitrogen compounds in fish muscle during storage that cause loss of freshness (Ge et al. 2020). Figure 3 shows the TVB-N values of treatments C and G during 180 days of frozen storage.

Figure 3 - Graph of TVB-N (mg N/100 g fish) in the muscle of Penaeus vannamei frozen for 180 days in the control and gelatine treatments.

Key: * - indicates that there was a statistically significant difference (p < 0.05) between the treatments using Student's t-test.

Figure 3 shows that treatments C and G showed no significant differences (p > 0.05) in the analyses at 0, 30 and 60 days. However, at 90 days, treatment C showed the highest value of TVB-N recorded during the 180 days of storage (26.88 ± 0.94 mg N/100 g fish), with a significant difference (p < 0.05) between it and treatment G (22.68 ± 0.42 mg N/100 g fish), and where the highest value of TBC was also recorded (Fig. 1), showing a correlation between the microbiological results and those of fish deterioration.

The G samples showed the highest TVB-N values at 30 days of the experiment (25.39 ± 0.70 mg N/100 g fish) and the lowest at 150 days of the experiment (20.35 ± 1.24 mg N/100 g fish), with a significant difference (p < 0.05) between C and G treatments at T150. On the last day of the experiment (T180), TVB-N values were 21.47 ± 0.85 mg N/100 g for C and 20.63 ± 0.95 mg N/100 g for G, with no significant differences (p > 0.05) between C and G treatments. These results may be due to the storage time, showing that the chemical quality of the samples was maintained and that the formation of TVB-N was inhibited.

Lannelongue et al. (2006) present the following TVB-N acceptance scales for raw shrimp < 12 mg N/100 g for fresh; 12–20 for edible but slightly decomposed; 20–25 for borderline; and > 25 mg N/100 g for inedible and decomposed. According to current Brazilian legislation (Brazil 2020), the total volatile base value must be less than 30 mg nitrogen/100 g muscle tissue. Thus, shrimp from both treatments kept their TVB-N levels below the limits recommended in the literature. The low TVB-N values found in this study may be due to the good initial freshness of the shrimp samples analyzed, as well as the low storage temperature (-18°C) throughout the storage period.

The results of this study were also consistent with other studies described in the literature. Mirzapour-Kouhdasht & Moosavi-Nasab (2020) found that Penaeus merguiensis shrimp coated with gelatin extracted from the skin of Scomberomorus Commerson had lower (p < 0.05) TVB-N levels than the control treatment over 12 days at 4°C, with the control exceeding the limit of 30 mg N/100 g muscle on 9 days of the experiment. Huang et al. (2016) reported that the formation of TVB-N in uncoated P. vannamei increased significantly from 4.2 mg/100 g (fresh) to a final value of 49.0 mg/100 mg after 24 h of storage at 25°C. The upward trend of TVB-N was effectively limited when the temperature was lowered to 4ºC, reaching 52.8 mg/100 g after 14 days of storage at 4ºC.

Farajzadeh et al. (2016) observed that the initial TVB-N content of the control group increased exponentially (p < 0.05) until the 8th day of storage under refrigeration at 4 ºC (10.48 to 33.58 mg N/100 g), while shrimp coated with gelatine and chitosan took 14 days to reach the same level (10.43 to 33.27 mg N/100 g). According to these authors, low-temperature storage combined with edible coating technologies showed a significant reduction in the formation of TVB-N. The current study confirmed this.

3.3.3 TMA-N

The accumulation of nitrogenous compounds such as TVB-N, trimethylamine (TMA), and biogenic amines in fish products is caused by the metabolism of spoilage bacteria and enzymes, affecting the sensory quality, flavor, nutritional value, and safety of the products (Li et al. 2018). TMA is one of the main substances responsible for fish odor, and some biogenic amines (such as histamine, cadaverine, and putrescine) are potentially toxic to humans (Yu et al. 2018). To measure only TMA, formaldehyde can be used to block primary and secondary amines (Huang et al. 2016). This was the method used in this study. Figure 4 shows the evolution of TMA-N present in the muscles of P. vannamei shrimp samples from treatments C and G over the course of 180 days of frozen storage.

Figure 4 - Graph of TMA-N (mg N/100 g fish) in the muscle of P. vannamei shrimps frozen for 180 days in the control and gelatine treatments.

Legend: * - indicates that there was a statistically significant difference (p < 0.05) between treatments using Student's t-test.

Figure 4 shows that the treatments started with no significant difference (p < 0.05) in the TMA-N content of the muscle of frozen shrimps. However, after 60, 90, 120 and 180 days of storage, the samples with the edible Nile tilapia scale gelatin coating (treatment G) showed lower values (p < 0.05) compared to treatment C. Nevertheless, the highest value for treatment C was 7.93 ± 0.61 mg N/100 g of fish and, according to Connell (1995), the maximum level of TMA recommended for human consumption is 10 to 15 mg N/100 g. Thus, none of the samples exceeded this limit.

The changes in TVB-N and TMA-N appear to be somewhat consistent with changes in the pH of the shrimp muscle resulting from the accumulation, however small, of basic compounds induced by bacterial or enzymatic activity. The increase in pH, especially in group C, reflected the production of alkaline bacterial metabolites in shrimp muscle.

The results obtained in this research were lower than those of Tsironi et al. (2009), who found values of up to 25 and 14 mg N/100 g TVB-N and TMA-N, respectively, after 8 months in samples of uncoated whole shrimp frozen and stored between − 5 and − 15°C. Huang et al. (2016) reported a 50% lower increase in TMA content after 14 days of storage of P. vannamei at 4°C compared to 25°C, both without coating. According to the authors, TMA content is widely used as a good indicator of the bacterial contamination level of seafood.

According to (Jia et al. 2019), the microbiota present in shrimp produces various compounds associated with deterioration, such as TMA, hydrogen sulfide, ammonia, and acetic acid, which cause shrimp tissue to lose elasticity and produce unpleasant odors and flavors. In this research, gelatine combined with low temperature slowed down the production of volatile compounds.

3.3.4 TBARS

In seafood, lipid oxidation leads to discoloration, rancid flavors, potentially toxic compounds, reduced protein functionality, and nutritional loss of some amino acids (Yu et al. 2018). The thiobarbituric acid reactive substances (TBARS) test is used to determine the state of lipid oxidation in fish. Thiobarbituric acid (TBA), the main reagent used in this method, reacts with the tissues to produce a pink color as a result of the formation of a complex between TBA and oxidized lipid compounds, mainly malonaldehyde (MA) and malondialdehyde (MDA) (Vyncke 1970). The quantification of the levels of malonaldehyde, in mg equivalent/kg fish muscle, present in the muscles of P. vannamei samples from treatments C and G is shown in Fig. 5.

Figure 5 - Graph of TBARS (mg MAD eq./kg fish) in Peneaus vannamei muscle frozen for 180 days in the control and gelatine treatments.

Figure 5 shows that the TBARS value of treatment C was 0.349 ± 0.29 mg MAD eq./kg fish at T0 and then showed a decreasing trend until its values reached zero after 120 days of storage, except at T90 when there was an increase in TBARS. At this time, the samples from the control treatment may have been more exposed to the environment and handling during the processing stages of the analyses, which would also justify the peak in total bacterial count (TBC) and TVB-N of the control treatment at 90 days.

The gelatine treatment, on the other hand, showed a zero TBARS value throughout the 180 days of frozen storage, demonstrating that the coating prevented lipid oxidation of the shrimp flesh. According to Connell (1995), TBA levels between 1.0 and 2.0 mg MDA/kg in fish muscle are associated with unpleasant taste and odor. Therefore, based on these parameters, the samples from both treatments were below this limit.

The results of this analysis are similar to others in the literature. Bono et al. (2012) evaluated the TVB-N levels of frozen marine shrimps during storage at -18°C for one year and found that the lipid oxidation, also measured by malonaldehyde content, of all their samples (control, modified atmosphere, sulfite treatment and vacuum packaging) remained below the limit proposed by Connell (1995) throughout the 12 months of the experiment; however, the control samples deteriorated more rapidly in the second half of the year than the other samples.

Das et al. (2023) observed that TBARS levels in uncoated shrimp (control) were significantly higher than those in shrimp coated with commercial gelatine and peppermint oil at the beginning and end of the storage period. However, all samples remained below the upper limit of 1–2 mg MAD/kg shrimp. Yu et al. (2018) found that edible coatings effectively slowed the lipid oxidation of seafood products during storage, mainly by acting as a barrier to oxygen and through their antioxidant properties.

Coatings help to improve food safety and shelf life by slowing down lipid oxidation, preventing weight loss (moisture) and loss of protein functionality, and reducing unpleasant odors and discoloration (Farajzadeh et al. 2016). Gelatine is a promising coating material due to its film-forming or gelling ability, as well as its resistance to drying, light, and oxygen (Feng et al. 2017). which can efficiently extend the shelf life and ensure the acceptability of frozen fish products (Zhang et al. 2020).

Gelatine derived from fish waste is a good alternative to reduce waste and environmental impact and add value to the product (Martins et al. 2018). According to FAO (2022), Oreochromis niloticus, is the third most produced species in the world and 4.4 million tons (live weight) of this fish will be produced in 2020. Considering this production, the residual scale of about 5% (Boronat et al. 2023) and the yield obtained in this study (24.64%), a total of 54,200 tons of Nile tilapia scale gelatine could be produced annually, an edible, non-toxic, biodegradable gelatine that is biocompatible with fish products (Abdelhedi et al. 2019). It could be used at low temperatures to significantly inhibit the degradation of fish and effectively extend the shelf life and ensure the acceptability of frozen fish products (Liu et al. 2023b), specially Penaeus vannamei, a very noble but highly perishable product (Alcântara et al. 2022).

1. This research confirmed the suitability of Nile tilapia scales as a good source of gelatin and demonstrated the efficiency of adapting the methodology used to extract gelatin, guaranteeing a high yield of material.

2. The study showed that many of the deterioration analyses (growth of psychrotrophic bacteria, pH, TVB-N, TMA-N, and TBARS) of the peeled, headless and frozen shrimp samples coated with a solution of Nile tilapia scale gelatin and glycerol showed lower values than the control samples, suggesting that coating is a good technique to inhibit deterioration and preserve the quality of Penaeus vannamei for longer.

Author Contribution

S.M.S.N.: Conceptualization, Methodology, Investigation, Validation, Formal analysis, Writing - original draft, Writing - review & editing, Data Curation, Visualization. J.M.L., D.A.C. and C.C.S. O.: Investigation. C.B.V.: Methodology, Investigation, Formal analysis. A.G.L.M.: Formal analysis. A.L.A.M.: Methodology, Investigation. M.S.M.S.F.: Conceptualization, Methodology, Supervision, Writing - review & editing, Resources. And B.W.S.S.: Conceptualization, Methodology, Validation, Resources Writing - review & editing, Supervision, Project administration.

Abdelhedi O, Jridi M, Nasri R, Mora L, Toldrá F, Nasri M (2019) ‘Rheological and structural properties of Hemiramphus far skin gelatin: Potential use as an active fish coating agent’ Food Hydrocolloids 87, 331–341. doi:10.1016/j.foodhyd.2018.08.005
Akagündüz Y, Mosquera M, Giménez B, Alemán A, Montero P, Gómez-Guillén MC (2014) ‘Sea bream bones and scales as a source of gelatin and ACE inhibitory peptides’ LWT 55, 579–585. doi:10.1016/j.lwt.2013.10.026
Alcântara LO, de Sousa JR, Andrade FK, Teixeira EH, Cerqueira MÂ, da Silva ALC, Souza Filho M de sá M, de Souza BWS (2023) ‘Extraction and characterization of hyaluronic acid from the eyeball of Nile Tilapia (Oreochromis niloticus)’ International Journal of Biological Macromolecules 226, 172–183. doi:10.1016/j.ijbiomac.2022.12.016
Alcântara L, Sousa J, Martins ME, Silva AL, Souza Filho M de sá, Souza B (2022) ‘Evaluation of Surface Properties of Chitosan and Scale Gelatin Coatings on Shrimp Fillets (Litopenaeus vannamei)’ Journal of Aquatic Food Product Technology 31, 1115–1127. doi:10.1080/10498850.2022.2133581
Aneesh PA, Anandan R, Kumar LRG, Ajeeshkumar KK, Kumar KA, Mathew S (2023) ‘A step to shell biorefinery—Extraction of astaxanthin-rich oil, protein, chitin, and chitosan from shrimp processing waste’ Biomass Conversion and Biorefinery 13, 205–214. doi:10.1007/s13399-020-01074-5
APHA (2015) ‘Compendium of Methods for the Microbiological Examination of Foods.’ (Yvonne Salfinger and Mary Lou Tortorello, Eds.). doi:https://doi.org/10.2105/MBEF.0222
Bono G, Badalucco CV, Cusumano S, Palmegiano GB (2012) ‘Toward shrimp without chemical additives: A combined freezing-MAP approach’ LWT 46, 274–279. doi:10.1016/j.lwt.2011.09.020
Boronat Ò, Sintes P, Celis F, Díez M, Ortiz J, Aguiló-Aguayo I, Martin-Gómez H (2023) ‘Development of added-value culinary ingredients from fish waste: Fish bones and fish scales’ International Journal of Gastronomy and Food Science 31,. doi:10.1016/j.ijgfs.2022.100657
Brasil (2020) ‘Decreto N^o 10.468, de 18 de agosto de 2020, dispõe sobre o Regulamento da Inspeção Industrial e Sanitária de Produtos de Origem Animal (RIISPOA). Brasília, 18 de agosto de 2020.’ (Brasília)
Brazil (2017) ‘Decreto N. 9.013 de 29 março de 2017.’ (Brazil)
Chuaychan S, Benjakul S, Kishimura H (2017) ‘Characteristics and Gelling Property of Gelatin from Scale of Spotted Golden Goatfish (Parupeneus heptacanthus)’ Journal of Food Processing and Preservation 41,. doi:10.1111/jfpp.13139
Connell JJ (1995) ‘Control of Fish Quality.’ (Wiley-Blackwell: Nova Jersey, EUA) Available at https://www.wiley.com/en-us/Control+of+Fish+Quality%2C+4th+Edition-p-9780852382264
Das MP, R. SP, Prasad K, Jv V, M R (2017) ‘Extraction and characterization of gelatin: a functional biopolymer’ International Journal of Pharmacy and Pharmaceutical Sciences 9, 239. doi:10.22159/ijpps.2017v9i9.17618
Das SK, Vishakha K, Das S, Ganguli A (2023) ‘Study of Gelatin and Peppermint Oil Nanoemulsion Coating Against Food-Borne Pathogens, as Well as Its Effect on Shrimp Quality and Preservation’ Journal of Aquatic Food Product Technology 32, 142–157. doi:10.1080/10498850.2023.2174392
FAO (1981) Standard for quick frozen shrimps or prawns. In ‘Codex Alimentarius’. pp. 1–6
FAO (2022) ‘The State of World Fisheries and Aquaculture 2022.’ (FAO) doi:10.4060/cc0461en
Farajzadeh F, Motamedzadegan A, Shahidi SA, Hamzeh S (2016) ‘The effect of chitosan-gelatin coating on the quality of shrimp (Litopenaeus vannamei) under refrigerated condition’ Food Control 67, 163–170. doi:10.1016/j.foodcont.2016.02.040
Feng X, Ng VK, Mikš-Krajnik M, Yang H (2017) ‘Effects of Fish Gelatin and Tea Polyphenol Coating on the Spoilage and Degradation of Myofibril in Fish Fillet During Cold Storage’ Food and Bioprocess Technology 10, 89–102. doi:10.1007/s11947-016-1798-7
Ge Y, Li Y, Wu T, Bai Y, Yuan C, Chen S, Gakushi I, Hu Y (2020) ‘The preservation effect of CGA-Gel combined with partial freezing on sword prawn (Parapenaeopsis hardwickii)’ Food Chemistry 313,. doi:10.1016/j.foodchem.2019.126078
Giannakourou MC, Stavropoulou N, Tsironi T, Lougovois V, Kyrana V, Konteles SJ, Sinanoglou VJ (2023) ‘Application of hurdle technology for the shelf life extension of European eel (Anguilla anguilla) fillets’ Aquaculture and Fisheries 8, 393–402. doi:10.1016/j.aaf.2020.10.003
GMIA (2019) Gelatin Handbook. USA. Available at http://www.gelita.comhttp://www.nitta-gelatin.comhttp://www.pbleiner.comhttp://www.rousselot.comhttp://www.weishardt.com
Huang YR, Zelaya MFG, Shiau CY (2016) ‘Changes in Biochemical Compositions and Quality of White Shrimp (Litopenaeus vannamei) During Storage’ Journal of Aquatic Food Product Technology 25, 35–45. doi:10.1080/10498850.2013.824944
Jia S, Liu Y, Zhuang S, Sun X, Li Y, Hong H, Lv Y, Luo Y (2019) ‘Effect of ε-polylysine and ice storage on microbiota composition and quality of Pacific white shrimp (Litopenaeus vannamei)stored at 0 °C’ Food Microbiology 83, 27–35. doi:10.1016/j.fm.2019.04.007
Jiang M, Liu S, Wang Y (2011) ‘Effects of antimicrobial coating from catfish skin gelatin on quality and shelf life of fresh white shrimp (Penaeus vannamei)’ Journal of Food Science 76,. doi:10.1111/j.1750-3841.2011.02056.x
Lannelongue M, Finne G, Hanna M, Nickelson R, Vanderzant G (2006) ‘Storage Characteristics of Brown Shrimp (Penaeus aztecus) Stored in Retail Packages Containing CO2‐Enriched Atmospheres’ Journal of Food Science 47, 911–913. doi:10.1111/j.1365-2621.1982.tb12743.x
Li E, Xu C, Wang X, Wang S, Zhao Q, Zhang M, Qin JG, Chen L (2018) ‘Gut Microbiota and its Modulation for Healthy Farming of Pacific White Shrimp Litopenaeus vannamei’ Reviews in Fisheries Science and Aquaculture 26, 381–399. doi:10.1080/23308249.2018.1440530
Liao W, Zhu Y, Lu Y, Wang Y, Dong X, Xia G, Shen X (2021) ‘Effect of extraction variables on the physical and functional properties of tilapia gelatin’ LWT 146,. doi:10.1016/j.lwt.2021.111514
Liu S, Lau CS, Liang K, Wen F, Teoh SH (2022) ‘Marine collagen scaffolds in tissue engineering’ Current Opinion in Biotechnology 74, 92–103. doi:10.1016/J.COPBIO.2021.10.011
Liu Z, Yang W, Wei H, Deng S, Yu X, Huang T (2023a) ‘The mechanisms and applications of cryoprotectants in aquatic products: An overview’ Food Chem 408,. doi:10.1016/j.foodchem.2022.135202
Liu Z, Yang W, Wei H, Deng S, Yu X, Huang T (2023b) ‘The mechanisms and applications of cryoprotectants in aquatic products: An overview’ Food Chem 408,. doi:10.1016/j.foodchem.2022.135202
Malle P, Tao SH (1987) Rapid Quantitative Determination of Trimethylamine using Steam Distillation.
Martins MEO, Sousa JR, Claudino RL, Lino SCO, Vale DA do, Silva ALC, Morais JPS, De Souza Filho M de SM, De Souza BWS (2018) ‘Thermal and Chemical Properties of Gelatin from Tilapia (Oreochromis niloticus) Scale’ Journal of Aquatic Food Product Technology 27, 1120–1133. doi:10.1080/10498850.2018.1535530
Mirzapour-Kouhdasht A, Moosavi-Nasab M (2020) ‘Shelf-life extension of whole shrimp using an active coating containing fish skin gelatin hydrolysates produced by a natural protease’ Food Science and Nutrition 8, 214–223. doi:10.1002/fsn3.1293
Nagarajan M, Rajasekaran B, Benjakul S, Venkatachalam K (2021) ‘Influence of chitosan-gelatin edible coating incorporated with longkong pericarp extract on refrigerated black tiger Shrimp (Penaeus monodon)’ Current Research in Food Science 4, 345–353. doi:10.1016/j.crfs.2021.05.003
Nitsuwat S, Zhang P, Ng K, Fang Z (2021) ‘Fish gelatin as an alternative to mammalian gelatin for food industry: A meta-analysis’ LWT 141, 110899. doi:10.1016/J.LWT.2021.110899
Pan C, Chen S, Hao S, Yang X (2019) ‘Effect of low-temperature preservation on quality changes in Pacific white shrimp, Litopenaeus vannamei: a review’ J Sci Food Agric 99, 6121–6128. doi:10.1002/jsfa.9905
Rigueto CVT, Rosseto M, Alessandretti I, de Oliveira R, Wohlmuth DAR, Ferreira Menezes J, Loss RA, Dettmer A, Pizzutti IR (2022) ‘Gelatin films from wastes: A review of production, characterization, and application trends in food preservation and agriculture’ Food Research International 162, 112114. doi:10.1016/J.FOODRES.2022.112114
Senarathna PDS, Marapana RAUJ (2021) ‘Comparative Analysis of the Effect of Ultrasound-Assisted and Conventional Water Bath Extraction Methods on the Physicochemical Characteristics of Tilapia Scales Gelatin’ Journal of Aquatic Food Product Technology 30, 893–906. doi:10.1080/10498850.2021.1950252
Sinthusamran S, Benjakul S, Hemar Y, Kishimura H (2018) ‘Characteristics and Properties of Gelatin from Seabass (Lates calcarifer) Swim Bladder : Impact of Extraction Temperatures’ Waste and Biomass Valorization 9, 315–325. doi:10.1007/s12649-016-9817-5
Sockalingam K, Abdullah HZ (2015) Extraction and characterization of gelatin biopolymer from black tilapia (Oreochromis mossambicus) scales. In ‘AIP Conf Proc’,(American Institute of Physics Inc.) doi:10.1063/1.4919191
Sousa EM de, Melo EF de, Ribeiro HL, Andrade Feitosa JP de, Moreira M de S, Melo MM de, Castro-Silva II (2022) ‘Biocompatibility and biodegradation analysis of Nile Tilapia gelatin and apatite membranes’ Revista Ciencia Agronomica 53,. doi:10.5935/1806-6690.20220035
Stewart GS (1987) ‘Micro-organisms in food-2. Sampling for microbiological analysis: principles and specific applications’ Meat Science 19, 1–310. doi:10.1016/0309-1740(87)90078-7
Tan CC, Karim AA, Uthumporn U, Ghazali FC (2019) ‘Effect of extraction temperature on the physicochemical properties of gelatine from the skin of black tilapia (Oreochromis mossambicus)’ Journal of Physical Science 30, 1–21. doi:10.21315/jps2019.30.s1.1
Tkaczewska J, Morawska M, Kulawik P, Zając M (2018) ‘Characterization of carp (Cyprinus carpio) skin gelatin extracted using different pretreatments method’ Food Hydrocolloids 81, 169–179. doi:10.1016/j.foodhyd.2018.02.048
Tsironi T, Dermesonlouoglou E, Giannakourou M, Taoukis P (2009) ‘Shelf life modelling of frozen shrimp at variable temperature conditions’ LWT 42, 664–671. doi:10.1016/j.lwt.2008.07.010
United Nations (2023) ‘Political Declaration of the Sustainable Development Goals Summit.’
Vyncke W (1970) ‘Direct Determination of the Thiobarbituric Acid Value in Trichloracetic Acid Extracts of Fish as a Measure of Oxidative Rancidity’ Fette, Seifen, Anstrichmittel 72, 1084–1087. doi:https://doi.org/10.1002/lipi.19700721218
Yu D, Xu Y, Regenstein JM, Xia W, Yang F, Jiang Q, Wang B (2018) ‘The effects of edible chitosan-based coatings on flavor quality of raw grass carp (Ctenopharyngodon idellus) fillets during refrigerated storage’ Food Chemistry 242, 412–420. doi:10.1016/J.FOODCHEM.2017.09.037
Zhang T, Xu J, Zhang Y, Wang X, Lorenzo JM, Zhong J (2020) ‘Gelatins as emulsifiers for oil-in-water emulsions: Extraction, chemical composition, molecular structure, and molecular modification’ Trends Food Sci Technol 106, 113–131. doi:10.1016/j.tifs.2020.10.005
Zuanazzi JSG, Goes ES dos R, Almeida FLA, Goes MD, Lara JAF, Ribeiro RP (2020) ‘Effect of multiple freeze-thaw cycles on the quality of chicken breast meat’ Food Science and Technology 40, 300–304. doi:https://doi.org/10.1590/fst.11119

No competing interests reported.

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Shelf life of Penaeus vannamei coated with gelatin extracted from Oreochromis niloticus scales

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Abstract

Figures

1. Introduction

2. Material and methods

2.1 Thermal extraction of gelatin and yield calculation

2.2 Shrimp procurement and preparation

2.3 Preparation of coating solution, coating, and storage

2.4 Shelf life study

2.4.1 Microbiological analysis

2.4.2 Physicochemical analysis

2.4.3 Statistical analysis

3. Results and discussion

3.1 Gelatin extraction yield

3.2 Microbiological analysis

3.3 Physicochemical analysis

3.3.1 pH

3.3.2 TVB-N

3.3.3 TMA-N

3.3.4 TBARS

4. Conclusions

Declarations

Author Contribution

References

Additional Declarations

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