Studying the microbial, chemical, and sensory characteristics of shrimp coated with alginate sodium nanoparticles containing Zataria multiflora and Cuminum cyminum essential oils

Abstract Retardation of quality loss of seafood has been a new concept in recent years. This study's main objective was to evaluate the microbial, chemical, and sensory attributes of shrimp coated with alginate sodium nanoparticles containing Zataria multiflora and Cuminum cyminum essential oils (EOs) during refrigerated storage. At the end of storage time (15 days storage at 4°C), the pH, thiobarbituric acid reactive substances (TBARS), and total volatile basic nitrogen (TVBN) amounts in shrimps coated with the alginate nanoparticles were 7.62, 1.14 mg MDA/kg, and 117 mg/100 g which were significantly (p < .05) lower than the control groups. The count of all bacteria groups was also lower in this treatment, which was 2–2.74 Log CFU/mL on day 15 of cold storage. This combined treatment also obtained the highest sensory scores (around 7) and the lowest melanosis score (2.67) due to the effective delaying microbial and oxidation activities. Therefore, this edible coating could substantially retard microbial and chemical changes and improve the organoleptic properties of shrimp under refrigerated storage.

and using some artificial or chemical preservatives are the best preservation method to increase the shelf life of shrimp. However, repeated freezing and thawing reduce the quality of shrimp. Moreover, given the toxic fate of some chemical additives and consumers' preference to buy foods containing natural additives, there is a need to use safer preservation methods (Alparslan et al., 2016;Arancibia et al., 2015;Zhang et al., 2015).
Zataria multiflora Boiss (Lamiaceae family), known as thyme-e-Shirazy, is an aromatic herb that grows in several parts of Asia, such as southern Iran. This shrub has been applied as an antiseptic, antispasmodic, and anti-inflammatory agent in traditional medication.
Zataria multiflora is also commonly consumed as a flavoring and antimicrobial agent in home applications and the food industry (Hashemi et al., 2022). Cuminum cyminum L. (Apiaceae family), known as cumin, is also a small annual herbaceous plant native to Egypt and southeastern Iran. It has an intense and distinct taste due to having compounds like cuminaldehyde (Homayonpour et al., 2021). However, the straight addition of EOs into the food is mostly restricted due to several limiting factors, including the creation of strong taste, low solubility in water, high volatility, and probable reactions with other food ingredients (Zhang et al., 2021).
Incorporating EOs into edible films and coating (lipids, proteins, or polysaccharides) is a promising approach to solving these problems. Therefore, they remain around the product for a long time to have the best antimicrobial and antioxidant effectiveness during storage (Zarei et al., 2015). Alginate is a linear polyanionic biopolymer composed of βd-mannuronic acid and αl-guluronic acid parts (Yousefi, Azizi, et al., 2018;. It is naturally derived from brown seaweed (Phaeophyceae family). Besides the impermeability of alginate with oils and its poor water vapor barrier properties, alginate shows potentials like forming gels, spheres, and micro-and nanoparticles (Gheorghita Puscaselu et al., 2020).
Natural food packaging materials containing nanoparticles possessing active and intelligent traits have the ability to improve some of the food sector concerns. These materials could extend the shelf life of foods, develop food safety, and decline the amount of food waste resulted from deterioration. There is no regulatory construction for nanoparticle usage in packaging materials. However, using the metallic nanoparticles in active and intelligent packaging in the European Union (EU) is disallowed (except titanium nitride in plastic bottles). Nevertheless, nanoparticles could also be fabricated from nonmetals like organic materials such as proteins and polysaccharides as well as plant extracts (like EOs). They are food grade nanoparticles existing naturally or have been made in the nano size from food-compatible materials. There is a higher public acceptance concerning these nanoparticles in comparison with metal nanoparticles because of their natural fate and no toxicity. The application of these nanoparticles is a solution to the intrinsic toxicity of metallic ones. There are numerous different food and plant extracts that possess antimicrobial characteristics and have the potential to be incorporated into food packaging (Hannon et al., 2015).
Nowadays, biopolymer nanoparticles have attracted attention due to their inherent biological properties and size-dependent features such as higher area and improvement effectiveness (Kraśniewska et al., 2020).
Many studies have been conducted on the effects of edible coatings and films combined with different EOs on several food products to increase their shelf life (Abdollahzadeh et al., 2021;Mojaddar Langroodi et al., 2021;Yousefi et al., 2022). However, to the best of our knowledge, there is no study investigating the combined incorporation of Z. multiflora and C. cyminum to alginate nanoparticles for the active packaging of shrimp. The main aim of the present study was thus to investigate the microbial, chemical, and sensory characteristics of shrimp coated with sodium alginate nanoparticles containing Z. multiflora and C. cyminum EOs during 15 days of storage at 4°C.

| Materials
The freshly caught shrimp were obtained from a local market in Fasa (Fars province, Iran) and brought to the laboratory with ice. After properly washing shrimps, they were kept at −18°C until testing. All culture media, including plate count agar (PCA), cetrimide fucidin cephaloridine agar (CFC), de Man-Rogosa-Sharpe agar (MRS), and violet red bile glucose (VRBG), as well as peptone water (PW), Tween 20 and Tween 80 were provided by Merck Company (Merck, Darmstadt, Germany). Sodium alginate and CaCl 2 were also obtained from Sigma Company (Sigma-Aldrich, Germany). Zataria multiflora and C.
cyminum EOs were purchased from Zardband Pharmaceuticals and Tabibdaru companies (Iran), respectively. Distilled water was used in all tests.

| GC-MS analysis
The gas chromatography device used was Agilent 6890 with column length, inner diameter, and layer thickness of 0.25 mm, 0.25 mm, and 0.25 μm, respectively (type BPX5). The process was described in our previous study. Briefly, to recognize the components of the EOs of each plant (Z. multiflora and C. cyminum), 1 μL of the sample diluted by n-hexane was injected into the GC/MS system. The temperature program of the column was set as follows: the primary temperature of the oven was 50°C and stopped at this temperature for 5 min. Then the temperature increased to 300°C, and 3 min was held at this temperature. Thus, the response time was 75 min. The temperature of the injection part was 250°C as a 1:35 split. Helium was used as carrier gas with a course rate of 0.5 mL/min. The mass spectrometer applied was the Agilent 5973 model with ionization voltage, ionization method, and ionization source temperature of 70 electron volts, EI, and 220°C, respectively. The scan range of mass spectra was adjusted from 40 to 500. The spectra were identified using their inhibition index, the standard compounds' mass spectra, and the evidence in the apparatus library (Mojaddar Langroodi et al., 2021).

| Characterization of nanoparticles
A DLS device (SZ-100 series, HORIBA Scientific, Japan) was used to check the size, polydispersity index (PDI), and zeta potential of the prepared nanoparticles. ATR-FTIR was also used to confirm the loading of EOs inside the nanoparticles. The spectra of Z. multiflora EO, C. cyminum EO, Alg-free, and Alg-NPs (from 400 to 4000 cm −1 ) were recorded. DPPH test was used to check the antioxidant properties of nanoparticles. A solution of 0.3 mM DPPH in ethanol was first prepared. Then, 150 μL of DPPH solution and 50 μL of serial dilutions were poured into each well of the plate . The plate was then incubated for 30 min in a dark environment, and the optical density (OD) was read at 517 nm. The antioxidant property was calculated according to Equation (1):

| pH
According to Liu et al. (2020), 10 g of minced shrimp was mixed with 90 mL of distilled water for 30 s, and then the pH of the obtained solution was measured using a pH meter (3510 pH meter, GENWAY) at room temperature (Liu et al., 2020).

| Thiobarbituric acid reactive substances (TBARS)
The TBARS test was conducted regarding Pikul et al. (1989) method with slight modification (Pikul et al., 1989). This test is usually performed to determine secondary oxidation products, that is, Malondialdehyde (MDA) (Mojaddar Langroodi et al., 2021). First, 200 mg of minced shrimp sample was blended with a little amount of 1-butanol, and the volume of the obtained mixture was increased to 25 mL by the same solvent. Then, 10 mL of trichloroacetic acid (0.2%) reagent was added to 5 mL of the previously obtained mixture and kept in a hot water bath (95°C, 2 h). After cooling the resulting solution at ambient temperature, its absorbance was measured at 532 nm. TBARS value was calculated, as mg MDA/kg shrimp, using Equation (2): where A, B, and m are the absorbance of the sample solution, absorbance of the control solution, and weight of minced shrimp (mg).

| Total volatile basic nitrogen (TVBN)
To measure the TVBN amount, 10 g of minced shrimp was mixed with 2 g of magnesium oxide (MgO) and 250 mL of distilled water and a droplet of silicone (as an antifoam agent). Then, the obtained solution was poured into a tube containing 20 mL of boric acid (3% aqueous solution) in the presence of methyl red and methylene blue (as the indicator). After that, the solution was titrated with hydrochloric acid (HCl 0.01 N). Finally, the TVBN amount was calculated by the volume of consumed HCl and was stated as mg/100 g shrimp (Goulas & Kontominas, 2005).

| Microbiological analysis
The count of lactic acid bacteria (LAB), coliform, Pseudomonas, and aerobic mesophilic was investigated during refrigerated storage. For this purpose, 1 g of minced shrimp was aseptically homogenized with 9 mL of peptone water (0.1%) using a stomacher (Labtron, UK) for 2 min. In the case of all microbial assays, decimal serial dilutions were made in peptone water (1.0%) of the homogenized prepared solutions.

| Sensory evaluation
The sensory properties of shrimp samples were examined by seven trained panelists who do not have an allergy to shrimp and other seafood. The assessors did not know about the experimental investigation, and the shrimp samples were provided with a numerical code (with no special meaning). The assessment of texture, odor, color, and overall acceptability was performed using a 9 points hedonic test (9 = very favorable and 1 = very unfavorable). In addition, the assessors' satisfaction with being involved has been considered (Liu et al., 2020).

| Melanosis assessment
Shrimp samples' melanosis (black spots) was visually investigated by the same 7 trained panelists based on a scoring system during refrigerated storage (Alparslan et al., 2016).

| Statistical analysis
In the present study, all tests were carried out in triplicate, and all data are expressed as the average (mean) ± standard deviation (SD).
All obtained data were evaluated using a two-sample mean comparison using STATA software (version 11). p < .05 was reflected as a significance cutoff.

| Chemical composition of Zataria multiflora and Cuminum cyminum EOs
Active chemical components in Z. multiflora and C.

| Characterization of nanoparticles
DLS analysis and potential zeta profile of nanoparticles are depicted in Figure 1. The size, PDI, and zeta potential of nanoparticles were 195 ± 7 nm, 0.109, and −29 ± 2 mV, respectively. The wavelength of the light is much longer than the size of the nanoscale particles, so it does not contain any light angle and makes the nanoparticles appear clear or slightly hazy (Abbasi et al., 2021). In the study by Masoumi et al. (2016), the average particle size of the Z. multiflora EOs' nanoemulsion was 66.5 nm, which was lower than the results of this study (Masoumi et al., 2016). Conversely, Gahruie et al. (2017) reported that the particle size of nanoemulsion (oil in water) of Z. multiflora EOs (210.5 nm) was higher than the results of the present study. This difference could be attributed to the differences in the ultrasound intensity and preparation time (Gahruie et al., 2017).
Alginate, a natural anionic polymer derived from brown seaweed, has been widely used because of its biocompatibility, low toxicity, reasonably low price, and slight gelation using divalent cations like Ca 2+ (Lee & Mooney, 2012). The first alginate particles for encapsulation were developed in 1980 (Lim & Sun, 1980). Advances in nanotechnology provide many synthetic protocols to produce polymer nanoparticles with dimensions below 100 nm. Polymeric nanoparticles have attracted great consideration because of the diversity of their characteristics in many applications . The great surface-to-volume proportion of nanoparticles is related to significant physicochemical characteristics compared with their macroscale counterparts (Geetha et al., 2016). Sodium alginate has wide applications in the food industry, including beverages, dairy products, frozen products, salads, canned products, sauces, dessert gels, and puddings.
It is also applied as an edible film and coating in many food products (Gheorghita Puscaselu et al., 2020). The possible reason for using alginate nanoparticles could be because alginate nanoparticles can contain many types of natural antimicrobial and antioxidant compounds, including EOs, enzymes, drugs, and other agents (Paques et al., 2014).
To the best of our knowledge, there is no research regarding the alginate nanoparticles containing both Z. multiflora and C. cyminum EOs.

| Attenuated Total Reflectance-Fourier Transform InfraRed Spectroscopy (ATR-FTIR)
ATR-FTIR analysis was used to confirm the loading of EO inside the nanoparticles. Figure 2 shows ATR-FTIR spectra of C. cyminum and F I G U R E 1 DLS analysis, 195 ± 7 nm (a) and Zeta potential, −29 ± 2 mV (b) of the alginate nanoparticles containing Zataria multiflora and Cuminum cyminum EOs. showed the C=C vibration of the aromatic compounds. The peak at 1074 cm −1 is attributed to C-O stretching vibration. The band at 948 cm −1 is related to C-H bending absorption, and the strong peak at 827 cm −1 is assigned to benzene rings' C-H vibration absorption.
The peak at 687 cm −1 corresponds to the vibration absorption of alkenes.
Regarding the ATR-FTIR spectrum of Z. multiflora EO, the broadband at 3200-3500 cm −1 can be attributed to the stretching vibra-

| DPPH free-radical scavenging activity
The antioxidant potency of the mixture of nanoparticles is shown in Figure 3. According to the obtained results, the concentration of and Alg-NPs) were lower than the acceptable level, even during the emergence of undesirable sensory characteristics in the sample, it could be said that the pH index is not a correct quality value .

| Assessment of TBARS changes
The ported that TBARS values less than 5 mg MDA/kg represent highquality food (Kilinc et al., 2009). All calculated TBARS contents in the present study were lower than this limit.

| Microbiological analysis
Food-borne microorganisms are one of the most sources of deterioration in foodstuff (Hashemi et al., 2021). are mostly related to that thymol, carvacrol, and cuminic aldehyde probably caused the breakdown of bacteria membrane, resulting in the cytoplasmatic seepage, cell lysis, and finally its damage (Hashemi et al., 2021;Li et al., 2020). Our results revealed that the alginate nanoparticles cannot decline bacterial growth efficiently while applied alone (Alg-Free sample), but it limited bacterial contamination when the combination of Z. multiflora and C. cyminum EOs were added (Alg-NPs sample). It has been revealed that nanoemulsified EOs has a higher antimicrobial effect in comparison with their typical emulsions due to the reduction in EOs particle size and higher diffusion of the active agents into the microorganisms (Dini et al., 2020) which confirm the present result. Similar trends were reported in previous studies, such as EOs nanoemulsion reducing microbial growth in food products (Dini et al., 2020;Li et al., 2020;Zhang et al., 2019Zhang et al., , 2020. Several reports also have shown synergistic and/or additive antimicrobial effects between different EOs (Abbasi et al., 2021;Lambert et al., 2001;Mojaddar Langroodi et al., 2021;Zarei et al., 2015). For example, Gibriel et al. (2013) reported that using a mixture of EOs (C. cyminum, Z. multiflora, and rosemary) showed a synergistic impact; so lower amounts of each EOs can be incorporated (Gibriel et al., 2013).
As depicted in Figure 7a, the LAB count of all samples increased F I G U R E 6 TVBN changes of shrimp samples during refrigerated storage (differences were significant at all times between all samples [p < .05], however, asterisks are not shown due to figure crowding).

F I G U R E 7
Changes in LAB (a), coliform (b), Pseudomonas (c), and aerobic mesophilic (d) counts of shrimp samples during refrigerated storage (in c and d, the differences were significant at all times between all samples [p < .05], however, asterisks are not shown due to figure crowding).
A similar trend was perceived for coliform growing during storage ( Figure 7b). Initial coliforms ranged from 1.24 to 1.67 Log CFU/g. Fresh shrimp is spoiled during cold storage mostly by Gramnegative psychrotrophic bacteria (especially Pseudomonas species) (Dehghani, Hosseini, Golmakani, et al., 2018;Dehghani, Hosseini, & Regenstein, 2018). Pseudomonas species are one of the most important microflora of meat. They are recognized as particularly aerobic bacteria which could not survive in situations with inadequate oxygen (Behbahani et al., 2020;Chamanara et al., 2013). It is reported that the spoilage of meat products kept under aerobic-refrigerated storage is mostly related to the growth and metabolic activity of Pseudomonas species, which are capable to decompose amino acids and glucose under cold storage (Behbahani et al., 2020;Chamanara et al., 2013). The proteolytic activity of this bacteria is probably responsible for meat spoilage and the following slime generation (Majdinasab et al., 2020). It is reported that when Pseudomonas's number is between 7 and 8 Log CFU/g, they cause spoilage in raw meat (Mehdizadeh & Langroodi, 2019). The Pseudomonas's growth in shrimp samples during cold storage is depicted in Figure 7c. These results are also consistent with Farshidi et al. (2018) study investigating the influence of whey protein coating containing lactoperoxidase on shrimp quality and Pseudomonas's growth inhibition . Moreover, in the study done by Mehdizadeh and Langroodi (2019), the number of Pseudomonas in chicken breast meat samples also increased during cold storage, and the samples coated with chitosan-containing propolis extract and Z. multiflora EO displayed considerably lower counts, undeniably due to the antimicrobial effect of EOs during the storage period (Mehdizadeh & Langroodi, 2019). It is worth noting that the edible coating of alginate nanoparticles containing EOs was effective in limiting the oxygen distribution amount and prevented the development of this bacteria group which is the main psicrotrophic bacteria (Behbahani et al., 2020;Chamanara et al., 2013). These results are in good accordance with a previous study investigating the effect of the edible film based on chitosan-containing nanoemulsion EOs on beef loins, lamb meat, and pork (Dini et al., 2020;Pabast et al., 2018;Zhang et al., 2019Zhang et al., , 2020. The result verifies the intense antimicrobial activity of EOs due to the high concentration of thymol, carvacrol, and cuminic aldehyde (Majdinasab et al., 2020). Figure 7d shows the count of aerobic mesophilic in all shrimp samples during 15 days stored in the refrigerator. Microbial contamination of fresh raw meat products including shrimp during cold storage is inevitable. As a result, the counting of mesophilic bacteria is mandatory in several safety regulations (Dehghani, Hosseini, Golmakani, et al., 2018;Dehghani, Hosseini, & Regenstein, 2018;Majdinasab et al., 2020). As presented in this figure, the number of this bacterial group also increased significantly (p < .05) with storage period in all treatments, especially in the control samples, which increased from 1.97 (on day 0) to 3.52 Log CFU/g (at the end of storage  properties. In the case of Alg-NPs, the results showed that the score of all parameters maintained higher than 7 until the 15th day, except for the color, which was lower than 7 after the 15th day of storage.

| Sensory evaluation
However, the texture and odor of the control sample were taken to unacceptable scores after 9 days (5.33 and 5.33, respectively), while the color and overall acceptability were unacceptable after 12 days

| CON CLUS ION
This study determined that coating shrimp with alginate nanoparticles containing Z. multiflora and C. cyminum EOs was greatly efficient against developing several types of spoilage bacteria vis.
LAB, coliform, pseudomonas, and aerobic mesophilic. This process also delayed lipid oxidation and sustained sensory characteristics, consequently increasing the fresh shrimp's shelf life during storage in the refrigerator (4°C). So alginate nanoparticles incorporated with Z. multiflora and C. cyminum EOs could be considered effective new active packaging for preserving fresh seafood under cold storage.

ACK N OWLED G M ENTS
The authors would like to thank the Fasa University of Medical Sciences for supporting this study (IR.FUMS.REC.1400.055).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors have no conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.

F I G U R E 9
Melanosis changes of shrimp samples during refrigerated storage. *p < .05.