Coating based on cassava starch and chitosan in maintenance of vacuum packaged shrimp quality

Shrimp is a product with good nutritional characteristics and strong market appeal, however, highly susceptible to microbiological contamination. For this reason, the use of appropriate preservation methods is essential to guarantee the quality of this food throughout its marketing chain. Conventional methods of shrimp preservation make use of non-biodegradable plastic packaging associated with refrigeration and vacuum. As an alternative to these plastic packages, coatings based on biopolymers such as polysaccharides and proteins can be used. In this work, the quality of the shrimp covered with films from cassava starch and chitosan was evaluated. Fresh shrimp were collected, peeled, and dipped in filmogenic solutions of cassava starch, chitosan, and mixed cassava starch with chitosan (1:1), a negative control was performed. The samples were stored in vacuum packages for six days, and on days 3 and 6, three repetitions of mesophilic bacteria count, pH, and humidity analysis were performed. The groups with coatings presented lower bacterial counts than the control group, especially the groups containing chitosan.  The pH and humidity changed during the days of experiment only in the group covered with cassava starch and control group, being pH variation associated with the increase of mesophilic bacteria count in these groups. The influence of the application of the coating on the conservation of the shrimp, in addition to creating a barrier around it, shows antimicrobial properties when considering chitosan, and may be a promising method for the conservation of minimally processed food.


Introduction
Food quality is a topic of worldwide relevance and involves several sensory, safety, and conservation aspects (Yang, Wei, & Pei, 2019). An example of alternative technology is the use of edible coatings in food, whose contribution to the preservation of the integrity of the food has been verified by several authors, in foods of plant origin (Coelho et al., 2017;Nunes et al., 2017;Rodrigues et al., 2019) and animal origin (Angioletti et al., 2018;Nunes et al., 2017;Pereira et al., 2018).
Some animal source foods, such as shrimp, are highly susceptible to deterioration, as they present characteristics like chemical composition favorable to the metabolism of microorganisms, mucus production, and richness of nutrients. (Furuya et al., 2008). Thus, despite the numerous benefits attributed to shrimp, such as richness of proteins of great biological value, those types of food are highly prone to microbial and chemical changes, being of great importance the research and application of conservation methods to prolong its useful life (Queiroga et al., 2014).
The temperature and time are extrinsic factors that affect this type of seafood, then soon after its arrest the shrimp must be cooled (Furuya et al., 2008). Cooling is very important because it prevents the action of microorganisms and enzymes on the shrimp. Adequate conservation methods can minimize losses in the processing, storage, and marketing of the shrimp, also add value to the product (Carlos & Neto, 2002).
Edible coatings and films are thin layer of material used to coat or pack food and extend their shelf life. For film composition, chemical components have advantages and limitations, polysaccharides, proteins, and lipids are the most used ones. Polysaccharides have a great advantage for being an economical material, in their molecular composition there is a Research, Society and Development, v. 9, n. 8, e275985714, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5714 5 large number of hydroxyls and polar groups (Dehghani et al., 2018).
The raw materials can have different origins for formation of the coatings, for example the proteins can be from animal sources or plant-based. The initial stage of the formation process is the denaturation of proteins caused by heat, pH, or use of solvents followed by a peptide association caused by molecular interactions (Azeredo, 2012).
Polymer blend is a mixture of a least two polymers with several advantages, including the power to combine several component properties in order to obtain good thermal, mechanical, and chemical conditions with low economic value. To obtain a polymer blend, it is necessary to understand the chemical and physical characteristics and their applications (Fernandes et al., 2012). Cassava starch is an example of polymer that has several advantages for the formulation of these coverings, such as the low cost and the ability to form resistant, transparent, and efficient barriers against gas passage . Another prominent polymer is chitosan, a product derived from chitin that has good gel point, in addition to high biocompatibility and biodegradability (Gonçalves et al., 2019).
Chitosan is the second most abundant polymer in nature, it is non-toxic and has the ability to be easily converted into gel (Chevalier et al., 2016), is considered a promising polymer in the composition of food coatings mainly due to its antimicrobial potential (Mejía, Martínez & Lara, 2018).
Vacuum packaging is a very simple method with a low investment cost. It consists of removing air from the package, until the amount of oxygen available at the end of the process is less than 1%, in which there must still be a minimum permeability of gases such as water vapor. Due to the absence of large amounts of oxygen, the growth of aerobic microorganisms and oxidation reactions are inhibited. It also has the advantages of retaining compounds responsible for the aroma and preventing the occurrence of injuries caused by cold in food (Iglesias, Cabezas, & Luis, 2006).
A sustainable and innovative packaging has the purpose of minimizing food waste and to preserve the microbiological and organoleptic quality of the food, in addition to contributing to reduce the use of petroleum-derived plastics.
Thus, considering the nutritional value of the shrimp and aiming to produce biodegradable coating in order to reduce the use of plastics and preserve the sensory and organoleptic characteristics of the crustacean, the present study aimed to evaluate the effect of the application of edible cover based on chitosan and cassava starch in vacuum packaged shrimp stored under refrigeration conditions in qualitative parameters. Research, Society and Development, v. 9, n. 8, e275985714, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5714 6 2. Material and methods

Study object
Samples of shrimp of the genus Litopenaeus vennamei were purchased in the region of Mossoró in Rio Grande do Norte under cooling conditions, transported immediately to the Food Technology Laboratory at UFERSA and packed in a thermal insulation box. The shrimps were minimally processed by removing the head and shell followed by washing in sterile distilled water.
The study consisted of a microbiological and physical-chemical evaluation of minimally processed shrimp covered with chitosan, cassava starch, polymer blend, and an uncoated group (control). All samples were placed in vacuum and refrigerated packaging at 7 ° C ± 1. The coverings applied on the shrimp were composed of polysaccharides (chitosan and / or cassava starch) and a plasticizer (glycerol).

Study design
After processing, the shrimp samples were separated into four groups: I C (No Cover), II CC (edible Chitosan Coating) III SC (edible Cassava Starch Coating) and IV CSC (Polymeric blend edible coating). After processing and vacuum packaging, the samples were stored in refrigeration for six days and subjected to evaluation of microbiological parameters (total count of aerobic mesophilic bacteria) and physicochemical parameters (potential hydrogen and moisture content).

Control (C)
The control group consisted of shrimp samples that underwent minimal processing operations, but received no coating. This group served as a basis for comparing and analyzing the microbiological and physical chemical data obtained with the other coated samples.

Cassava Starch Coating production (SC)
The production of the cassava starch coating was performed as proposed by Nunes et al. (2017). 30g/L of cassava starch and 6 g/L of glycerol were added to sterile distilled water.
The mixture was heated at 70 ° C for 15 minutes under stirring.

Chitosan and Cassava Starch Coating production (CSC)
For the production of this coating, a polymer blend was made using 20g/L of chitosan and 30g/L of cassava starch. The plasticizer was added at a concentration of 6g/L of the total solution. The solution was kept under stirring for 24 h at room temperature.
The CSC coating was produced by mixing the aforementioned coatings in a 1: 1 ratio.

Methodology for analysis
The aerobic mesophilic bacteria count was performed according to Freire et al (2016). Serial dilutions were produced by weighing 1 g of the sample in 9 mL of 0.1% peptone salt solution (D-1), the dilutions used were: 10 ^ -2 and 10 ^ -3, based on the principle that the food could be contaminated and just one dilution would not be enough to count microorganisms, the other dilutions were obtained from the first. The dilutions were spread on a standard hardened agar surface for microorganisms counting, the plates were inverted and incubated in a bacteriological culture oven at 37 ± 1 ° C. The plates were analyzed after 48 hours of incubation. The values of colony forming units (CFU) obtained in the counts were expressed in log CFU / g, the counts were performed in duplicates, where at the end of the counting, it was calculated an average value for each group.
The physical chemical parameters pH and moisture content were performed, with adaptations, according to Lima et al. (2019). The potential of hydrogen (pH) was measured by macerating 5 g of the sample in 10 mL of distilled water and measuring it in a properly calibrated digital potentiometer. The moisture content was measured by consecutive weighing of sample aliquots before and after 24 hours of drying in a stove at 105°C, the results were expressed as water loss percentage. Development, v. 9, n. 8, e275985714, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5714 8

Data analysis
All analyzes were performed with three repetitions per treatment, the data obtained were statistically evaluated by one-way analysis of variance (ANOVA) and the means of all groups were compared by Tukey test at the 5% level of significance. Figure 1 shows the general appearance of shrimp coated with cassava starch, chitosan, and both polymers.

Aerobic mesophilic count
The data obtained in the aerobic mesophilic count is shown in Table 1. Analyzing the efficiency of the coating on day 6, microorganism counts were made on the plates. According to the obtained data, it was observed the efficiency of the coats regarding the development of these bacteria, where all edible coatings presented a lower value of microorganisms UFC / g when compared to the control group. The coating with the lowest count was the one formulated with chitosan. This is a biopolymer with antimicrobial potential (Alencar et al., 2018) and prevents the growth of this type of contaminant, which can cause deterioration in Development, v. 9, n. 8, e275985714, 2020 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v9i8.5714 9 the organoleptic and sensory properties of the food. By reproducing an encapsulating effect between the coat and the food, an efficient barrier is created preventing direct contact between the environment and the food (Dias et al., 2018).
SC group presented a larger number of mesophilic bacteria compared to the CSC group, making it evident, according to Table 1, that the association of cassava starch and chitosan is more efficient to prevent the development of aerobic mesophilic bacteria. Table 1 shows the microbiological data of the shrimps treated over 6 days. Equal lowercase letters on the same line indicate statistical similarity between the groups, equal uppercase letters in the same column indicate statistical similarity between the days. C (negative control); SC (cassava starch coated shrimp); CC (chitosan coated shrimp); CSC (cassava starch and chitosan coated shrimp). P <0.05 was considered statistically significant at 95% confidence. Source: The author (2019).

pH
The pH increases during the experiment. It is a parameter closely linked to microbial contamination (Fernandes et al., 2016), the increase in the number of microorganisms generates an increase in pH due to the probable accumulation of volatile bases (Freire et al., 2016). For this parameter, the coating with chitosan and cassava starch was the most efficient, preventing the development of volatile bases in the shrimp. In this regard, the association of starch with chitosan was more efficient than the use of the components alone.
As opposed to the microbiological question, the starch coating obtained better results than chitosan, proven by the value of the pH being lower in the SC group than in the CC group, which shows the importance of this type of study, since a polymer that prevents the development of microorganisms may not be as efficient to avoid the influence of extrinsic factors, making an association necessary to obtain greater efficiency of the coating.

Moisture
Still in line with the importance of this type of study, we see the highest humidity value on day 6 for the CC group, even higher than the control group. It is noticed that the chitosan coating directly affects the humidity, this value increased during the experiment while the other groups had a reduction in humidity. Shrimps coated with polymeric blend obtained a reduction in moisture content, proving the benefits of the association between chitosan and cassava starch. Again, the starch obtained better results than chitosan, because it is considered an effective barrier to gas exchange, thus preventing the passage of O2 into the food, however, it has a high permeability to water vapor, being an inefficient barrier against moisture (Hassan et al., 2018).

Conclusion
The application of edible coatings based on chitosan and cassava starch, associated with vacuum packaging, can contribute to a better conservation of the coated shrimps based on the physical, chemical, and microbiological aspects analyzed. This group showed the best results in this experiment. However, further studies should be carried out analyzing other experimental conditions and other types of analysis of the coating's effectiveness.