Preserving quality and enhancing sensory attributes of chicken lunch meat: free and microencapsulated shrimp shell extract

doi:10.21203/rs.3.rs-5230366/v1

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Preserving quality and enhancing sensory attributes of chicken lunch meat: free and microencapsulated shrimp shell extract

https://doi.org/10.21203/rs.3.rs-5230366/v1

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This study aimed to investigate the effects of free (FAST) and microencapsulated forms (MFAST, and MSAST) of astaxanthin (AST) on the quality, antioxidant and sensory properties of chicken lunch meat (CLM) over a 45-day period at 4°C, and were compared along with synthetic sodium nitrate and a control sample. The results demonstrated that AST levels in CLMs decreased during storage; however, microencapsulation significantly preserved approximately twice as much AST compared to CLMs containing free AST. Samples containing AST showed significantly higher antioxidant activity compared to both the control and sodium nitrate samples. Microencapsulated forms exhibited stronger radical scavenging activity, surpassing the free forms by more than 10 units, particularly in CLMs supplemented with freeze-dried AST (approximately 44% higher). Microbial growth rate increased over time, ranging from 2.82 ± 0.03 log₁₀^(CFU/g) on the first day to over 5.35 ± 0.04 log₁₀^(CFU/g) on day 45, while still remaining within acceptable microbial limits. In terms of sensory attributes, the FAST treatment at different concentrations resulted in moderately acceptable levels, however, the MFAST and MSAST treatments at higher concentrations received excellent scores. These findings highlight the potential of microencapsulation as a valuable technique for preserving AST and developing high-quality meat products with extended shelf life and enhanced sensory characteristics.

Microencapsulation

Astaxanthin

Chicken lunch meat

Antioxidant activity

Microbial growth

Sensory attributes

Chicken meat, known for its high protein content and low- calorie count, ranks as the second most consumed meat worldwide (Sobral et al., 2020). Heat-processed meats, such as sausages and chicken lunch meats, enjoy popularity due to their high production efficiency, low cost, and substantial nutritional value (Muchekeza et al., 2021). However, microbial contamination and oxidation contribute to the spoilage of chicken meat, with oxidation being the primary factor affecting meat color, texture, nutrition, and shelf life. Therefore, extending the shelf life of meat products by preventing microbial spoilage and oxidation is a key focus in the meat industry (Song et al., 2022).

Using antioxidants is a common strategy to inhibit protein oxidation and extend the shelf life of meat products (Ben Akacha et al., 2023). Both synthetic and natural antioxidants can prevent protein oxidation (Jia et al., 2023). Nitrite and nitrate salts, such as sodium and potassium salts, are typical preservatives used in meat processing (Ferysiuk & Wójciak, 2020). They inhibit microbial growth, delay rancidity, enhance cured meat flavor, and stabilize the red color of meat (Andrade et al., 2024; Ferysiuk & Wójciak, 2020). The effectiveness of nitrites and nitrates makes them essential additives for meat products, particularly cured meats (Ferysiuk et al., 2020). However, nitroso compounds have been linked to carcinogenesis and DNA damage (Gassara et al., 2016), limiting their use and increasing the demand for natural preservatives (Bonifacie et al., 2021; Flores & Toldrá, 2021).

Shrimp, a globally prized seafood, generates considerable waste during processing (Sae-Leaw & Benjakul, 2019). Research has focused on extracting a lipid-rich extract containing α-tocopherol, polyunsaturated fatty acids (PUFAs), and astaxanthin (AST) from this waste (Gómez-Guillén et al., 2018; Montero et al., 2016). AST (3,3'-dihydroxy-β,β-carotene-4,4'-dione), a carotenoid pigment responsible for the red color of various aquatic organisms, holds promise as a natural food colorant with potent antioxidant properties (Montero et al., 2016; Yuan et al., 2011). While synthetic production exists, AST derived from biological sources shows superior chemical stability and antioxidant capacity due to its natural esterification with fatty acids (Vakarelova et al., 2017; Yang et al., 2023). As an antioxidant, AST scavenges free radicals and oxidants, protecting cell membranes from peroxidation through its polar ionic rings and non-polar conjugated carbon-carbon bonds (Hwang et al., 2024; Sun et al., 2023). Its antioxidant activity surpasses other carotenoids like zeaxanthin, lutein, canthaxanthin, and β-carotene by tenfold, potentially offering significant health benefits (Chen et al., 2022; Santos-Sánchez et al., 2020). However, AST's highly unsaturated structure makes it vulnerable to degradation by heat, light, and oxygen (Qiao, Yang, Gu, et al., 2021; Qiao, Yang, Hu, et al., 2021; Yang et al., 2021). Studies indicate that AST dispersed in sunflower oil has a half-life of only seven days at room temperature (Vakarelova et al., 2017), highlighting the need for methods to ensure its long-term stability and preserve its valuable properties.

Microencapsulation, which involves encasing small particles or droplets within a biopolymer to form microparticles ranging from 1 to 1000 micrometers, offers a promising solution. This technique involves coating oil droplets containing AST with proteins and carbohydrates for protection (Ahmed et al., 2015; Burgos-Díaz et al., 2020; Fang et al., 2022). Spray drying and freeze drying are particularly favored for preserving delicate and expensive bioactive compounds like AST (Guo et al., 2020). Sharayei et al. (2021) identified an optimal wall material composition for encapsulating AST, consisting of 18.40% maltodextrin with 7 free chain ends (MD7), 41.78% modified starch (Hi-Cap 100), and 39.81% maltodextrin with 20 free chain ends (MD20).

Several studies emphasize the importance of global strategies for sustainable agriculture and reducing environmental food waste. However, limited information exists on using microencapsulated AST from shrimp shell waste as an antioxidant in chicken lunch meat (CLM) compared to synthetic antioxidants. This research aimed to compare the effects of free AST (ASN), spray-dried AST (MSAST), and freeze-dried AST (MFAST), with commonly used preservatives, such as sodium nitrate, on the quality, microbial, and sensory properties of CLM.

The Fig. 1 presents a comprehensive experimental procedure for extracting and microencapsulating AST from shrimp shells and using it in CLM. The materials used included shrimp shells, chicken breast, and various chemicals. The methods involved ultrasound-assisted extraction of AST, microencapsulation, preparation of CLM, measuring AST content, antioxidant testing, microbial analysis, and sensory evaluation.

2.1. Materials

2.1.1. Raw materials

Frozen chicken breast was sourced from the local market in Mashhad, Iran. Green tiger shrimp (Penaeus semisulcatus) shells were obtained from shrimp processing plants in Bushehr province, Iran. To ensure freshness during transportation to the laboratory, the shells were packed in an insulated container with ice. Upon arrival, the shells were cleaned of any remaining appendages, packaged in polyethylene bags, and stored at -20°C until further processing. Before extraction, the shells were finely ground using a laboratory grinder (Mulinex mill, Depose-Brevete S.G.C.G., France) and passed through a 425 µm mesh sieve.

2.1.2. Chemicals, reagents, and bacterial culture media

Various chemicals were procured from reputable suppliers, including Sigma-Aldrich Co. (St. Louis, MO, USA) and Merck Co. (Darmstadt, Germany). These included ethanol (96%), petroleum ether, acetone, hexane, hydrochloric acid, sodium chloride, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), potassium chloride, aluminum chloride, sodium hydroxide, ferric chloride, potassium carbonate, sodium acetate, methanol, maltodextrin (degree of hydrolysis 7 and 20), and Tween 80. Hi-Cap 100®, a chemically modified octenyl succinic anhydride (OSA) starch derived from waxy maize, was obtained from the National Starch Company (England). Microbial media such as Plate Count Agar (PCA), Polymyxin-Sulfadiazine agar, Violet Red Bile Agar (VRB Agar), Dichloran Rose-Bengal Chloramphenicol Agar, Baird Parker Agar, Peptone Water Agar, Tetrathionate Broth Agar, Rappaport-Vassiliadis Salmonella Enrichment Broth, Salmonella Shigella Agar, and Brilliant Green Agar were sourced from Oxoid (Lancashire, UK).

2.2. Methods

2.2.1. Ultrasound-assisted extraction of AST

50 g of powdered shrimp shells were placed in a cartridge containing 200 ml of solvent (mixture of petroleum ether, acetone, and water in a 15:75:10 ratio), and transferred to the ultrasound chamber. Ultrasound waves were applied at 25% intensity for 13.90 min at 27°C, based on optimal conditions designed using the Box-Behnken model. An ultrasound device (Heilscher, Germany – UP400S) with a 400W titanium probe (7 mm diameter, and 100 mm length) was used. The extraction process was continued with Soxhlet extraction, including sonication, for 6 h, and the solution was concentrated using a vacuum rotary evaporator (Laborota 4000 Efficient, Eco, Germany) at 45°C to complete dehydration (Sharayei, et al., 2021).

2.2.2. Microencapsulation of AST

2.2.2.1. Preparation of Microcapsule Wall Materials

According to Sharayei et al. (2021) the solution containing different wall material (40% MD7, 41.78% modified starch, and 39.81% MD20 for spray- drying and the solution containing different wall material 29.39% MD7, 34.07% modified starch, and 36.55% MD20 for freeze- drying) were prepared and mixed in distilled water at room temperature (25 ± 1°C) using a magnetic stirrer for 30 min. The mixture was then refrigerated at 4°C for 24 h to ensure complete hydration. Finally, an aqueous solution with a total solids concentration of 10% (w/w) was then prepared.

2.2.2.2. Spray-drying and freeze-drying of AST extract

Tween 80, serving as a stabilizer, and the shrimp shell extract were added to the aqueous solution at a weight ratio of 2.5%. The mixture was homogenized using an ultraturrax homogenizer (Model 50T, IKA, Germany) at 15000 rpm for 10 min. Further homogenization was achieved with an ultrasonic generator (Heilscher, Germany, UP400S model) with a power output of 400W and a frequency of 20 kHz for 3 min at room temperature.

The prepared solutions, containing both wall and core materials, were spray dried using a spray dryer (Model 190 B, BUCHI, Switzerland) with an inlet air temperature of 180°C, an outlet air temperature of 71°C, an airflow rate of 600 L/h, and an air humidity of 65%. The resulting dried powders were stored in the dark at -18°C until further analysis.

For freeze-drying, the prepared solutions, containing both wall and core materials was stored at -70°C for 19 h, then freeze-dried using an Operon FDB-550 model (Korea) for 48 h at -55°C and a pressure of 0.15 mmHg. The dried samples were stored in the dark at -18°C for further analysis (Sharayei, et al., 2021).

2.2.3. Preparation of Chicken Lunch Meat (CLM) and Formulation with AST

Chicken meat was cut into strips and ground into minced meat with a meat grinder. The chicken lunch formula was prepared according to Table 1. The CLM dough was then directly treated with AST extract (50 and 100 ppm), and indirectly with AST freeze-dried and spray-dried microencapsulated forms added to CLM at two concentration levels, 200 and 400 ppm. For comparison, CLM was also treated with sodium nitrate (1000 ppm). CLM with no additives served as the control. After soft homogenized dough was achieved, the mixture was used to form chicken lunches with a diameter of 2 cm and length of 10–12 cm in polyamide casings. The CLMs were tied with string at 13 cm intervals, steamed (69–72°C for 2.5 h), and stored at 4°C for 45 days.

Table 1

Formulation of Chicken Lunch Meat (CLM) with a Total Weight of 3500 g
Ingredient	Quantity (g)	Ingredient	Quantity (g)
Chicken breast	2220	Starch	55.5
Ice	518	Gluten	37
Vegetable oil	370	Soy flour	185
Sodium nitrate	3.5	Casein	83
Polyphosphate	7.4	Ascorbic acid	3.7
Salt	59.2	Spices	120

2.4. Measurement of AST content

The concentration of AST in the free and microcapsulate forms was determined by dissolving a sample in 3 ml of hexane and measuring the absorbance at 470 nm. The AST concentration (µg/g) was calculated using the equation below:

$$\:AST\left(\frac{\mu\:g}{g}\right)=\frac{A\times\:D\times\:{10}^{6}}{100\times\:G\times\:d\times\:{E}_{1cm}^{1\%}}$$

Where AST, AST concentration in µg/g; A, Absorbance; D, Extract volume in hexane (ml); $\:{10}^{6}$, Dilution factor; G, Sample weight in grams; d, Cuvette width (mm); E, Extinction coefficient, which is 2100 (Sharayei et al., 2021).

2.5. DPPH radical scavenging assay

The DPPH radical scavenging activity of CLMs supplemented with free and microencapsulated AST, compared with samples containing sodium nitrate, as well as control samples was evaluated with slight modifications to the method by Sabeghi et al. (2024). Initially, 500 mg of each CLM sample was stirred in 5 ml of distilled water for 30 min. Subsequently, 1.0 ml of the AST and CLM extract was mixed with 4.0 ml of DPPH solution (0.12 mM). The mixture was kept in the dark at 25°C for 25 min, and its absorbance was measured at 517 nm. The DPPH scavenging effect (%) was calculated using the following formula:

$$\:\:\text{D}\text{P}\text{P}\text{H}\:\text{s}\text{c}\text{a}\text{v}\text{e}\text{n}\text{g}\text{i}\text{n}\text{g}\:\text{e}\text{f}\text{f}\text{e}\text{c}\text{t}\:\:\left(\text{%}\right)=100\times\:\left(\frac{{A}_{blank}-{A}_{sample}}{{A}_{blank}}\right)$$

Where, $\:{A}_{blank}$ and $\:{A}_{sample}$ represent the absorbance of the control and the sample, respectively.

2.6. Microbial analysis of CLM

To determine the microbial counts, the surface of the CLM was disinfected using a 70% ethanol solution. Then, it was cut with a sterile cutter. The first dilution was prepared by mixing 10 g of CLM with 90 ml of a 1% sterilized peptone-water solution. The mixture was homogenized at 350 rpm for 2 minutes at room temperature using an Ultra Homogenizer (T50, VWR Scientific Homogenizer, Germany, LLC). Subsequent dilutions were made by adding 1 ml of each dilution to 9 ml of a 0.1% sterilized peptone water solution. The total floral bacteria (TFB) were determined after 72 hours of incubation at 30°C using a plate count agar medium. Clostridium perfringens was determined on a Polymyxin-Sulfadiazine Agar (PSA) medium after 24 hours of incubation at 37°C. Total Coliforms were determined on a Violet Red Bile Agar (VRBA) medium after 24 hours of incubation at 37°C. Streptococcus aureus, yeast, and mold were surface cultured on Baird Parker Agar (BPA), Dichloran Rose-Bengal (DRB), and Chloramphenicol Agar (CA), respectively. Incubation was done at 37°C for 24 hours and 25°C for 5 days. The results were expressed as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$ (colony forming units per gram of meat). All measurements were performed in triplicate.

2.7. Sensory analysis

Sensory characteristics, including color, aroma, flavor, and overall acceptance, were assessed one day after preparation by 10 trained panelists using the method described by by Siripatrawan and Noipha (2012). Samples were evaluated at room temperature using a 5-point hedonic scale (1: poor, 5: excellent).

2.8. Statistical analysis

The statistical significance of the results was evaluated using one-way analysis of variance (ANOVA). Duncan’s multiple range tests were used to identify differences between analyses with a 95% confidence limit (P < 0.05). All tests were conducted in triplicate, and the results were presented as mean ± standard deviation. Data analysis was performed using SPSS 16 software (SPSS Inc, USA).

3.1. Measurement of AST content

The AST levels in CLMs supplemented with free and microencapsulated AST were examined over different AST initial concentrations: FAST (50 ppm and 100 ppm), MFAST (200 ppm and 400 ppm), and MSAST (200 ppm and 400 ppm), monitored over 1 day, 15 days, 30 days, and 45 days (Fig. 1). As observed, AST levels decreased over the storage period. However, microencapsulation significantly preservation more AST in CLM about 2 times compared to CLM, containing free AST (p < 0.05), suggesting that microencapsulation effectively protects AST from the physical effects of oxygen, light, and heat (Mendis Pinto et al., 2001). In contrast, FAST, although initially effective (10 to 15 mg/kg), experienced a rapid decline in AST levels, dropping to 3–4 mg/kg. Higuera-Ciapara et al. (2004) reported that microencapsulated AST in glutaraldehyde-linked chitosan prevented isomerization and pigment degradation during 8 weeks of storage at 25, 35, and 45°C. Similarly, Liu et al. (2019) demonstrated that microencapsulated AST in polylactic acid reduced deterioration by 12.4%, 16.5%, and 67.5% after 6 months of storage at 0, 25, and 40°C, respectively, compared to free AST, which showed deterioration rates of 29.3%, 35.9%, and 100%, respectively. Microencapsulated treatments (MFAST and MSAST) at higher concentrations (400 ppm) displayed the highest and most stable antioxidant activities over the 45 days of cold storage. At 400 ppm, initial values were 53.4 and 48.2, reducing to 38.1 and 33 by day 45 for MFAST and MSAST, respectively. Our findings also indicated approximately 5 units higher AST content in freeze-dried samples than in spray-dried samples. Freeze drying uses significantly lower temperatures and eliminates oxygen exposure, providing better protection for AST than spray drying (Buljeta et al., 2022).

3.2. DPPH radical scavenging assay

Figure 2 illustrates the antioxidant capacity of CLM supplemented with free and microencapsulated astaxanthin (FAST, MFANS, and MSANS), compared to samples containing sodium nitrate and control samples over 45 days of cold storage (4°C). Sodium nitrate (1000 ppm) maintains higher antioxidant values than the control, gradually decreasing from 11.1–5.17%. Although antioxidant activity declined over time, samples containing ASN showed significantly higher antioxidant activity than the control and sodium nitrate samples (p < 0.05). This is attributed to ASN's ability to capture free radicals through its conjugated polyunsaturated bonds and terminal rings, effectively scavenging free radicals and heavy metals (Montero et al., 2016; Yuan et al., 2011). In line with our results, Seo et al. (2021) found that AST's antioxidant activity in pork sausage was similar to the antioxidant activity of BHT. The DPPH radical scavenging of FAST with 50 ppm and 100 ppm AST were 26% and 29.1% and decreased to 12% and 16.1% by day 45 respectively.

In comparison, microencapsulated forms exhibit more robust radical scavenging, with values exceeding those of free forms by over 10 units, especially in CLMs supplemented with freeze-dried ASN ( about 44% higher). These values were significantly improved by 1.11 times and 1.67 times with a two-fold increase in the concentration of ASN in free and microencapsulated forms, respectively. This was due to the minimal degradation of AST during freeze drying, attributed to the prolonged exposure at low temperatures (-55°C). Research by Ahmed et al. (2015) explored the effects of spray drying versus freeze drying (under vacuum or air) at temperatures ranging from − 20°C to 37°C on the astaxanthin-rich alga over 20 weeks. Their findings indicated that freeze drying offers a distinct advantage in terms of AST yield, resulting in 41% higher AST recovery compared to spray drying. Therefore, remaining higher amounts of AST led to higher antioxidant properties and these results agreed with AST levels monitored over 1 day, 15 days, 30 days, and 45 days.

2.3. Microbial Analysis of CLM

Table 1 presents the total floral bacteria (TFB) of CLMs over 45 days at a temperature of 4°C. The table compares the effects of different treatments, including free and microencapsulated forms of AST FAST, MFANS, and MSANS, synthetic sodium nitrate, and a control sample. The TFB values were measured at four storage time points: 1 day, 15 days, 30 days, and 45 days. The results indicate that the different treatments did not significantly impact the TFB of the CLMs during the 45 days of cold storage. However, it is worth noting that the microbial growth rate increased throughout the storage period and remained within acceptable microbial limits, ranging from 2.82 ± 0.03$\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$ on the first day to over 5.35 ± 0.04$\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$ on day 45. The initial microbial count in the meat primarily results from contamination during the slaughter, transportation, and further processing stages. The bird itself can serve as a potential source of contamination (Van Ba et al., 2018). The samples treated with sodium nitrate demonstrated the lowest microbial growth, ranging from 2.42 ± 0.02 to 4.10 ± 0.05$\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$. The FAST MFAST and MSAST treatments at any concentration showed TFB scores similar to the control sample throughout the storage period varied from 2.90 ± 0.00 to 5.54 ± 0.02. Therefore, it can be concluded that AST in both forms free and microcapsulated didn’t affect the TFB of CLM. In contrast with our results, previous studies by Reyhani Poul et al. (2024), revealed that tomato pastes containing 6% pure astaxanthin and nanocapsules carrying astaxanthin had different bacterial counts. The total bacterial count in the tomato paste with pure astaxanthin was 8.9$\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$, while the count in the paste with nanocapsules was 4.8$\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$.

Table 2 shows the effects of different treatments on the yeast and mold cell numbers of CLMs during 45 days of cold storage at 4°C. After 15 days of storage, there were no significant differences (p > 0.05) in the yeast and mold cell counts between the control sample (1.43 ± 0.12 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$) and the samples treated with free and microencapsulated forms of AST (FAST, MFANS, and MSANS), which were 1.43 ± 0.04 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$. By day 30, the yeast and mold counts had increased to approximately 2.74 ± 0.01 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$across all samples, and by day 45 they had further increased to around 3.26 ± 0.03 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$. These results indicated that the free and microencapsulated AST treatments were not able to effectively delay the growth of yeast and mold in the CLMs compared to the untreated control during the 45-day storage period.

Table 1

The TFB of CLMs as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$during 45 days of cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus synthetic sodium nitrate and control samples.
Treatment	Concentration (ppm)	Storage time (day)
Treatment	Concentration (ppm)	1	15	30	45
Control	-	2.90 ± 0.07^a	3.30 ± 0.02^a	4.89 ± 0.02^a	5.54 ± 0.08^a
NaNO₃	1000	2.42 ± 0.02^a	3.08 ± 0.05^a	3.70 ± 0.09^a	4.10 ± 0.05^b
FAST	50	2.90 ± 0.06^a	3.31 ± 0.08^a	4.90 ± 0.02^a	5.54 ± 0.02^a
FAST	100	2.90 ± 0.00^a	3.28 ± 0.04^a	4.89 ± 0.00^a	5.53 ± 0.07^a
MFAST	200	2.89 ± 0.01^a	3.25 ± 0.02^a	4.89 ± 0.04^a	5.54 ± 0.02^a
MFAST	400	2.86 ± 0.05^a	3.25 ± 0.04^a	4.88 ± 0.03^a	5.53 ± 0.06^a
MSAST	200	2.86 ± 0.01^a	3.25 ± 0.02^a	4.87 ± 0.01^a	5.54 ± 0.03^a
MSAST	400	2.86 ± 0.04^a	3.25 ± 0.05^a	4.88 ± 0.02^a	5.54 ± 0.06^a

* Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

Table 2

The yeast and mold cell numbers of CLMs as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$during 45 days of cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus control samples.
Treatment	Concentration (ppm)	Storage time (day)
Treatment	Concentration (ppm)	15	30	45
Control	-	1.43 ± 0.12^a	2.75 ± 0.02^a	3.27 ± 0.02^a
FAST	50	1.42 ± 0.04^a	2.74 ± 0.01^a	3.27 ± 0.01^a
FAST	100	1.43 ± 0.04^a	2.74 ± 0.02^a	3.27 ± 0.02^a
MFAST	200	1.43 ± 0.04^a	2.74 ± 0.00^a	3.27 ± 0.10^a
MFAST	400	1.43 ± 0.05^a	2.75 ± 0.02^a	3.23 ± 0.05^a
MSAST	200	1.43 ± 0.10^a	2.73 ± 0.01^a	3.24 ± 0.03^a
MSAST	400	1.43 ± 0.01^a	2.74 ± 0.02^a	3.27 ± 0.04^a

*Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

Table 3 presents the coliform counts of CLMs during 45 days of storage at 4°C, comparing the effects of free and microencapsulated forms of AST (FAST, MFANS, MSANS) against a control sample. After 15 days of storage, the coliform counts were statistically similar across all samples, ranging from 0.71 ± 0.21 to 0.75 ± 0.05 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$ (p > 0.05). By days 30 and 45, the coliform counts had increased to around 1.42–1.46 and 1.60–1.62 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$, respectively, with no significant differences observed among the control and the AST-treated samples (p > 0.05). These results suggested that the free and microencapsulated forms of AST did not provide any significant antimicrobial effects against coliforms in CLMs compared to the untreated control during the 45-day cold storage period.

According to Tables 4 and 5, the free and microencapsulated forms of AST (FAST, MFAST, and MSAST) did not demonstrate any significant antimicrobial effects against Staphylococcus aureus and Clostridium perfringens in CLMs during the 45-day cold storage period at 4°C (p > 0.05). The Staphylococcus aureus and Clostridium perfringens counts remained statistically similar across the control, AST-treated samples throughout the 45-day storage, ranging from 0.85 ± 0.14 to 1.50 ± 0.03 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$for Staphylococcus aureus and ranging from 0.90 ± 0.16 to 1.31 ± 0.03 $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$ for Clostridium perfringens. This indicates that the different AST treatments were not effective in reducing the levels of these pathogens compared to the untreated control. These results align with the findings of Alonso-Calleja et al. (2004), which reported zero prevalence of Staphylococcus aureus in vacuum-packed retail ostrich meat.

Table 3

The coliform numbers of CLMs as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$during 45 days of cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus control samples
Treatment	Concentration (ppm)	Storage time (day)
Treatment	Concentration (ppm)	15	30	45
Control	-	0.75 ± 0.05^a	1.43 ± 0.04^a	1.60 ± 0.05^a
FAST	50	0.74 ± 0.12^a	1.42 ± 0.04^a	1.60 ± 0.01^a
FAST	100	0.73 ± 0.04^a	1.42 ± 0.08^a	1.61 ± 0.05^a
MFAST	200	0.72 ± 0.10^a	1.45 ± 0.02^a	1.62 ± 0.02^a
MFAST	400	0.72 ± 0.04^a	1.43 ± 0.08^a	1.61 ± 0.01^a
MSAST	200	0.75 ± 0.04^a	1.46 ± 0.05^a	1.60 ± 0.02^a
MSAST	400	0.71 ± 0.21^a	1.42 ± 0.04^a	1.60 ± 0.04^a

*Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

Table 4

The *staphylococcus aureus* numbers of CLMs as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$during 45 days of cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus control samples.
Treatment	Concentration (ppm)	Storage time (day)
Treatment	Concentration (ppm)	15	30	45
Control	-	0.85 ± 0.14^a	1.30 ± 0.02^a	1.50 ± 0.03^a
FAST	50	0.85 ± 0.20^a	1.30 ± 0.08^a	1.47 ± 0.02^a
FAST	100	0.85 ± 0.15^a	1.30 ± 0.03^a	1.48 ± 0.05^a
MFAST	200	0.86 ± 0.09^a	1.29 ± 0.10^a	1.47 ± 0.02^a
MFAST	400	0.85 ± 0.13^a	1.30 ± 0.04^a	1.48 ± 0.01^a
MSAST	200	0.86 ± 0.09^a	1.30 ± 0.11^a	1.47 ± 0.01^a
MSAST	400	0.85 ± 0.20^a	1.30 ± 0.09^a	1.49 ± 0.07^a

*Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

2.4. Sensory analysis

Table 6 displays the sensory evaluations of CLMs conducted one day after production using a 5-point hedonic scale (1: poor, 5: excellent). The evaluations compared different treatments, including free and microencapsulated forms of AST FAST, MFANS, and MSANS, synthetic sodium nitrate, and a control sample. The control sample had the lowest scores, ranging from 2.90 to 3.30, indicating a less desirable product. The CLM treated with sodium nitrate at a concentration of 1000 ppm performed better than the control and achieved moderately acceptable scores (3) in all sensory attributes. This indicates that the addition of sodium nitrate improved the color of the CLM. On the other hand, the FAST treatment at concentrations of 50 ppm and 100 ppm resulted in moderately unacceptable levels (3) and acceptable levels (4) for all sensory attributes, respectively. This suggests that increasing the AST concentration from 50 ppm to 100 ppm could be effective in enhancing the sensory properties of the CLM. Microencapsulated forms of AST, particularly MFAST at 400 ppm, received the highest scores across all sensory attributes, with a flavor and aroma score of 4.71, a color score of 4.30, and an overall acceptance score of 4.65. These results indicate that these treatments significantly improved the sensory attributes of the CLM. Notably, the color attribute showed significant differences between the encapsulated and free AST with sodium nitrate, as well as the control sample, due to the presence of AST red pigments. This is consistent with the findings reported by, Pogorzelska et al. (2018) where the use of Haematococcus pluvialis extract, rich in AST, altered the color of meat to a desirable pinky-red shade.

Table 5

The *clostridium perfringens* numbers of CLMs as $\:{\text{l}\text{o}\text{g}}_{10}^{\text{C}\text{F}\text{U}/\text{g}}$during 45 days of cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus control samples.
Treatment	Concentration (ppm)	Storage time (day)
Treatment	Concentration (ppm)	15	30	45
Control	-	0.92 ± 0.08^a	0.99 ± 0.09^a	1.31 ± 0.03^a
FAST	50	0.92 ± 0.09^a	1.00 ± 0.04^a	1.25 ± 0.13^a
FAST	100	0.91 ± 0.13^a	0.98 ± 0.03^a	1.28 ± 0.04^a
MFAST	200	0.90 ± 0.05^a	0.99 ± 0.11^a	1.28 ± 0.06^a
MFAST	400	0.92 ± 0.03^a	1.01 ± 1.17^a	1.28 ± 0.05^a
MSAST	200	0.90 ± 0.16^a	0.95 ± 0.05^a	1.30 ± 0.07^a
MSAST	400	0.91 ± 0.12^a	0.94 ± 0.03^a	1.29 ± 0.03^a

*Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

Table 6

The sensory attributes of CLMs one day after production at cold storage (4°C), influenced by free and microencapsulated (AST FAST, MFANS and MSANS) versus synthetic sodium nitrate and control samples.
Treatment	Concentration (ppm)	Flavor and aroma	Color	Overall acceptance
Control	-	3.00 ± 0.08^g	2.90 ± 0.05^f	3.30 ± 0.05^g
NaNO₃	1000	3.20 ± 0.03^f	3.48 ± 0.10^e	3.63 ± 0.06^f
FAST	50	3.50 ± 0.07^e	3.70 ± 0.05^d	3.80 ± 0.04^e
FAST	100	4.10 ± 0.03^d	4.00 ± 0.04^c	4.11 ± 0.05^d
MFAST	200	4.20 ± 0.03^c	4.20 ± 0.06^b	4.56 ± 0.02^b
MFAST	400	4.71 ± 0.04^a	4.30 ± 0.08^a	4.65 ± 0.01^a
MSAST	200	4.18 ± 0.04^c	4.13 ± 0.07^b	4.27 ± 0.01^c
MSAST	400	4.25 ± 0.04^b	4.18 ± 0.04^b	4.38 ± 0.11^c

* Different letters indicate significant differences between samples at p < 0.05. NaNO₃, sodium nitrate; FAST, free AST; MFAST, freez-dried AST; MSAST, spray-dried AST.

In conclusion, the findings of this study highlight the effectiveness of microencapsulation in enhancing the stability of AST levels in CLM during 45 days at 4°C. While the AST levels decreased over time, the microencapsulated forms preserved approximately twice as much AST compared to CLMs containing free AST. Freeze-dried samples at higher concentrations (400 ppm) exhibited approximately 5 units higher AST content than spray-dried samples. Sodium nitrate (1000 ppm) maintained higher antioxidant values than the control, gradually decreasing from 11.1–5.17% over time. However, samples containing AST showed significantly higher antioxidant activity than the control and sodium nitrate samples. The antioxidant activity of free FAST with 50 ppm and 100 ppm AST declined from 26% and 29.1–12% and 16.1% respectively by day 45. In contrast, microencapsulated forms exhibited stronger radical scavenging activity, surpassing the free forms by more than 10 units, particularly in CLMs supplemented with freeze-dried ASN (approximately 44% higher). Increasing the concentration of AST by twofold resulted in a significant improvement of antioxidant properties, with a 1.11 times increase in free forms and a 1.67 times increase in microencapsulated forms. The different treatments, including free and microencapsulated forms of AST (FAST, MFAST, and MSAST), as well as synthetic sodium nitrate, did not significantly impact the TFB, yeast and mold, coliforms, Staphylococcus aureus, and Clostridium perfringens counts in CLM during the 45-day cold storage period at 4°C. The microbial growth in all samples, including the control, increased over the storage period but remained within acceptable microbial limits. The control sample, without any additives, showed the lowest scores across all sensory attributes, suggesting a less desirable product. However, the microencapsulated forms of AST, particularly MFAST at 400 ppm, showed the highest scores across all sensory attributes. The application of microencapsulated AST can contribute to the development of high-quality meat products with improved sensory attributes and extended shelf life.

Conflicts of Interest

No potential conflict of interest was reported by the authors.

Ethics statement

The use for human subjects in this research study was approved by the Agricultural Research, Education and Extension Organization (AREEO).

Consent to participate

Written informed consent was obtained from the sensory panellists.

Author Contribution

Parvin Sharayei: [email protected] - Project administration, Investigation, Conceptualization, Supervision, Writing - original draft. Elham Azarpazhooh: [email protected] Investigation, Data analysis.Fatemeh zare: [email protected], Writing - review & editing. Yeganeh Sabeghi: [email protected], Writing and editing

Data Availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Preserving quality and enhancing sensory attributes of chicken lunch meat: free and microencapsulated shrimp shell extract

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials

2.1.1. Raw materials

2.1.2. Chemicals, reagents, and bacterial culture media

2.2. Methods

2.2.1. Ultrasound-assisted extraction of AST

2.2.2. Microencapsulation of AST

2.2.2.1. Preparation of Microcapsule Wall Materials

2.2.2.2. Spray-drying and freeze-drying of AST extract

2.2.3. Preparation of Chicken Lunch Meat (CLM) and Formulation with AST

2.4. Measurement of AST content

2.5. DPPH radical scavenging assay

2.6. Microbial analysis of CLM

2.7. Sensory analysis

2.8. Statistical analysis

3. Result and discussion

3.1. Measurement of AST content

3.2. DPPH radical scavenging assay

2.3. Microbial Analysis of CLM

2.4. Sensory analysis

4. Conclusions

Declarations

Conflicts of Interest

Ethics statement

Consent to participate

Author Contribution

Data Availability

References

Additional Declarations

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