Improving quality of smoked sardine fillets by soaking in cuttlefish ink

Abstract

Cuttlefish ink, a by-product of food processing, is a promising source of natural compounds with antioxidant and antimicrobial activities and it can be used as a natural additive for sea food products. Therefore, this work aimed to extend the shelf life of smoked sardine by soaking in cuttlefish ink solution. No antibacterial activity of cuttlefish ink solution with different concentrations (1%, 2% and 3%) has been determined against Staphylococcus aureus, Listeria monocytogenes and Pseudomonas aeruginosa. DPPH radical scavenging activity for the ink samples ranged from 91% to 95% for the three ink solutions. Fresh fillets were steamed, smoked and soaked in the three ink solutions and then stored at 4 °C for 35 days. During storage, total viable counts, total volatile basic nitrogen values and peroxide values increased significantly only for control samples and those soaked with 1% of ink solution. Soaked samples showed the lowest amount of histamine when compared to the control one which may be related to a better controlled microbial proliferation. Ink solutions did not affect the whiteness of fillets, whereas the hardness increased significantly during the two first weeks of storage. Obtained results support using cuttlefish ink to lengthen smoked sardine shelf life.

Keywords:
Sepia ink; antioxidant activity; antimicrobial activity; Sardinella aurita, stability

1 Introduction

In Tunisia, sardine production is high but has a low commercial value. Despite its high nutritional value, sardines are very perishable fishes (Cyprian et al., 2015). Smoking may extend the shelf life of this species and give it a higher value. Gómez‐Guillén et al. (2009) reported that cold smoking both extended shelf life and improved the sensorial quality of sardines. However, cold smoked sardines are known for their high concentration in salt which decreases their nutritional value (Cyprian et al., 2015). Steaming before cold smoking could be an alternative to preserve quality and reduce the amount of salt used in cold smoked fishes. To extend the shelf life of smoked sardine fillets and to prevent lipid oxidation, natural and safe antioxidants may be used. Using antioxidants from the by-products of seafood processing is beneficial for the industry and allows reducing problems related to the environment pollution. In Tunisia, cuttlefish (Sepia officinalis) is one of the major seafood resources and one of the main marine species exported. During processing, ink sacs are manually removed and thrown without appropriate management. This by-product is considered as a potential threat for the environment. Many authors have revealed that cuttlefish ink has antimicrobial, antioxidative, antitumor, antileukemic and antiviral properties (Derby, 2014; Fahmy et al., 2014; Nithya et al., 2011; Vate & Benjakul, 2013; Vennila et al., 2010; Vivek et al., 2002; Zaharah & Mohd Salleh, 2017). Due to these properties, cuttlefish ink has many therapeutic applications, especially in Asia, where homeopathic medicine is often practiced (Derby, 2014). Karim et al. (2016), Sadok et al. (2004), Vate et al. (2015) reported that melanin-free cuttlefish ink could extend the shelf life of some seafood products. The application of crude cuttlefish ink in the food industry is limited because of its black color, while it remains used in some traditional foods Mediterranean and Asian countries such as rice, pasta and soup (Derby, 2014; Sadok et al., 2004).

In this work, cuttlefish ink was used as a natural preservative to add value and to extend the shelf life of cold smoked sardine fillets. The aim of this work was: (i) to determine the antimicrobial and antioxidant properties of cuttlefish ink, (ii) to evaluate the effect of the cuttlefish ink solution on the microbiological, biochemical and textural properties of smoked sardine fillets during storage at 4 °C.

2 Material and methods

2.1 Preparation of ink solutions

Fresh cuttlefish (Sepia officinalis) were purchased from a local market (Tunis, Tunisia) and quickly transferred to a laboratory in an ice polystyrene box within one hour after purchase. Means of dorsal mantle length (DML) and total body weight were 136 ± 27 mm and 305 ± 79 g, respectively. All samples were assigned to a maturity stage III according to the relative size and color of reproductive organs (International Council for the Exploration of the Sea, 2010). The ink-sacs were cut out aseptically and transferred to a sterilized test tube. They were then washed and ruptured with a sterilized instrument. A quantity of 253 g of ink was obtained from the cuttlefishes (n = 65). Three ink solutions of 1% (S1), 2% (S2) and 3% (S3) were prepared by diluting 1, 2 and 3 g of crude ink in 100 mL of salted water (3.5%), respectively.

2.2 Preparation of smoked sardine fillets

20 kg of sardines (Sardinella aurita) were purchased within one day of capture from a local market in Tabarka (Tunisia), put on ice and rapidly transferred to the Transformation of Sea Products laboratory unit of the National School of Fisheries of Tabarka. The sardines were immediately beheaded, eviscerated, filleted and washed. To minimize the use of salt, the fillets were steamed at 70 °C for 30 min, then cold smoked at 22-25 °C for 90 min using industrial smoking chamber with an external smoke generator (Räucher F V2A, Germany). Oak wood was used for the smoking process (Sciure de fumage Chêne, France). The smoked fillets were put in glass jars filled each with one of the three ink solutions. The ratio of fish to ink solution was 1:1.5 (w:v) so that the fillets were totally emerged in the ink solutions throughout the experiment. For control samples, smoked sardine fillets were soaked only in salt water (3.5%). A total of 7 glass jars (300 mL of capacity with metal cap) were prepared for the control samples and for each ink solution (1%, 2%, and 3%) and stored at 4 °C for 35 days. One jar of each solution is used each day of analysis. The experiment was replicated twice.

2.3 Analysis

Antibacterial effect of ink solutions

The ink solutions (1,2 and 3%) and the salt water (3.5%) were screened against two gram-negative bacteria (Escherichia coli ATCC 8739 and Pseudomonas aeruginosa ATCC 27853) and two gram-positive bacteria (Staphylococcus aureus ATCC 6538 and Listeria monocytogenes ATCC 19115) using the disc diffusion method (McCaffrey & Endean, 1985). The microorganisms tested were cultured in a nutrient broth at 37 °C (Accumix, Belgium). After 24 h of incubation, the bacterial suspensions were centrifuged at 12,000 g for 15 min (Universal, 320) and the pellets were washed with sterile distilled water and resuspended in 10 ml of the nutrient broth. Finally, the number of bacterial cells in each suspension was adjusted to reach the range of 8 log CFU/mL by using a spectrophotometer (Jenway 6305). A 500 µL suspension of each microorganism tested was spread on a solid agar medium in Petri dishes (Mueller-Hinton agar, Accumix, Belgium). Sterile filter paper discs (6 mm dia. Whatman No. 1) were soaked in 20 µl of the ink solutions (1%, 2% and 3%) and then placed on the inoculated plates and allowed to dry for 15 min, then incubated at 37 °C for 24 h. The diameters of the inhibition zones were measured in millimeters and the antimicrobial efficacy was graded based on the zone diameter as high activity (> 15 mm), moderately active (9-14 mm), and no activity (< 8 mm) (Ponce et al., 2003). Antibiotic discs (Bio-Rad, France) ampicillin (10 µg) and Doxycycline (30 µg) were used as standards.

Antioxidant activity of ink solutions

The antioxidant activity of ink solutions was studied by evaluating the free radical scavenging effect on the 1,1-diphenyl-2-picrylhydrazine (DPPH) radical as described by Vate & Benjakul (2013). 1 mL of various dilutions of the ink solutions was mixed with 2 ml of 0.3 mM DPPH in methanol. The mixture was vigorously shaken, then allowed to stand in the dark at room temperature for 30 min. Absorbance was measured at 517 nm by a spectrophotometer (6715 UV/Vis Spectrophotometer, Jenway). The absorbance value of the methanolic solution of DPPH was also measured at 517 nm. The reaction mixture without DPPH was used for background correction. The DPPH radical scavenging effect was calculated by the following Equation 1:

$$DPPHscavengingactivity=\left[1-\left(\begin{array}{l}Abssample517nm/\\ Abssamplecontrol517nm\end{array}\right)\right]\times 100$$ (1)

Bacteriological analysis

Ten grams of each sardine sample were diluted with 90 ml of sterile peptone water (Accumix, Belgium) using a Stomacher 80 Biomaster. Serial 10-fold dilutions were prepared in sterile peptone water. Total viable counts were determined on Plate Count Agar (PCA) (Accumix,

Belgium) after 48 h of incubation at 30 °C. Coliforms were enumerated on Desoxycholate Agar (Accumix, Belgium) after 24 h at 37 °C for total coliforms and 24 h at 44 °C for fecal coliforms. Staphylococcus aureus strains were enumerated on Baird Parker agar (Accumix, Belgium) after 48 h of incubation at 37 °C (Guiraud, 1998). Analyzes were carried on days 0,7, 14, 21 and 35 of storage at 4 °C.

2.4 Chemical analysis

Chemical analyses were carried out on fresh sardine fillets, after steaming-smoking and during soaking in cuttlefish ink solutions at 4 °C on days 0,7, 14, 21 and 35. The moisture percentage was determined according to AOAC method (Association of Official Analytical Chemists, 2000). Total volatile base nitrogen (TVB-N) was determined by Conway’s micro-diffusion method (Cobb et al., 1973). The sardine content in TVB-N was expressed as mg TVB-N per 100 g of sample. Peroxide values were measured according to AOCS method (American Oil Chemists Society, 2009). The peroxide value of fish oil is expressed as milliequivalents of peroxide per kg of oil. The histamine content was determined using the colorimetric method described by Patange et al. (2005). Histamine was extracted from fish muscle as described by Patange et al. (2005). The histamine concentration in the sample was estimated (on days 0,15 and 35 of soaking) using the following Formula 2:

$$Histamine\left(mg/100g\right)=A\times 2\times 25\times 100=Amg/100g5\times 1000$$ (2)

where A is the value of histamine obtained in l g/ml from the standard curve.

2.5 Color and texture

A colorimeter (Minolta Chroma Meter CR-300) with D65 as illuminant and an observer angle of 10°, previously calibrated against a standard white ceramic reference plate was used to measure color directly on cuttlefish ink and samples of fresh, smoked and soaked fillets. The lightness (L*), redness (a*) and yellowness (b*) parameters were recorded. Whiteness as described by the National Fisheries Institute (1991) was calculated using the following Formula 3:

$$Whiteness=100-{\left[\left(100-L*\right)²+a*²+b*²\right]}^{1/2}$$ (3)

Sardine fillets were subjected to textural analysis with a texture analyzer (TA-XT2 stable Micro-systems, Haslemere, UK). Measurements were done on the dorsal part of fish muscles. Each sample was compressed with a cylindrical probe at 1 mm/s speed and the level of compression was 50% of the thickness of the sample. Hardness was determined by peak force during compression and expressed in gram.

Whiteness and hardness were carried out on days 0, 15 and 35 of storage.

2.6 Statistical analysis

Results are reported as means ± standard deviation. Statistical analyses were conducted by using STATGRAPHICS Centurion (XV) 16.1.15 software (Manugistics Inc, Cambridge, MA). Data were subjected to analysis using a two-way ANOVA procedure. Duncan’s multiple range test was used to determine any significant difference between mean values, and evaluations were based on a significance level of P < 0.05. Coefficient of Pearson (r) was used to evaluate relationships between TVB-N and TVC parameters.

3 Results and discussion

3.1 Antibacterial activity of ink solutions

Inhibition zones were detected only for Staphylococcus aureus, Listeria monocytogenes and Pseudomonas aeruginosa (6-8 mm). The inhibition zones were affected only by the ink solution concentration (P = 0.01) and not by type of microorganism (P = 0.09) (Table 1). The inhibition zones for the positive control doxycycline and ampicillin ranged from 21 to 34 mm. No antibacterial activity was detected for three concentration ink solutions (1,2 and 3%) against all the tested strains, since all the diameters of inhibition zones are less than 8 mm (Ponce et al., 2003). Many other studies have shown antibacterial activity of cephalopod ink against a variety of Gram positive and Gram negative pathogenic bacteria (Girija et al., 2012; Girija et al., 2014; Nithya et al., 2011; Sadayan et al., 2013; Vennila et al., 2010; Vivek et al., 2002; Zaharah & Mohd Salleh, 2017). Our results supposed that salted water used as solvent negatively affected the antibacterial activity of ink solutions. In fact, the antimicrobial activity of cephalopod ink is affected by the solvent used for extraction (Derby, 2014; Sadayan et al., 2013; Zaharah & Mohd Salleh, 2017). Zaharah & Mohd Salleh (2017) reported that water and ethanol extracts of squid ink gave the highest value of antibacterial activity, however, there was no inhibition activity with hexane extract. Vennila et al. (2010) reported that molluscan ink samples contain a large number of compounds with various biological and chemical properties which are responsible for the antimicrobial activity. Some authors reported that antimicrobial activity of the cephalopod ink could be due to tyrosinase activity (Vate et al., 2015; Vennila et al., 2010). Chen et al. (2008) isolated a non-sulfated glycosaminoglycan-like polysaccharide from melanin free ink of the squid, having antibacterial activity. A protein extracted from squid ink showed antimicrobial activity against Staphylococcus aureus (Vate & Benjakul, 2013).

Table 1
Antibacterial activity of cuttlefish ink solutions.

Diameter (mm)
Microorganism	DO	AM	S1	S2	S3	NC
Staphylococcus aureus	29 ± 2.54	-	6 ± 0.52a1	6 ± 1.05a1	7.5 ± 0.86a2	-
Escherichia coli	-	21 ± 1.89	-	-	-	-
Listeria monocytogenes	34 ± 2.75	-	6 ± 0.94a1	7 ± 0.98a1	7 ± 1.02a2	-
Pseudomonas aeruginosa	29 ± 2.57	25 ± 2.08	7 ± 0.86a1	7.5 ± 0.12a1	8.5 ± 0.95a2	-

• Mean value ± standard deviation (n = 3). DO: Doxycycline; AM: Ampicillin; S1: cuttlefish ink solution (1%); S2: cuttlefish ink solution (2%); S3: cuttlefish ink solution (3%); NC: negative control. Means with the same parameter and the same column with different superscript letters (a) are different (P < 0.05). Means with the same parameter and the same line with different superscript numbers (1,2) are different (P < 0.05)
• S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%.

3.2 Antioxidant activity of ink solutions

DPPH radical scavenging activity for the ink samples were 91.78 ± 3.5%; 94.56 ± 4.9% and 95.45 ± 2.3% for S1, S2 and S3 respectively. Antioxidant activity was affected by ink solution concentrations (P = 0.02). Results obtained here are comparable to those of Zaharah & Mohd Salleh (2017), who found that DPPH radical scavenging activity of squid ink was 96.24%. Fahmy et al. (2014) reported that sepia ink extract produced dose dependent inhibition of DPPH radical ranging from 86.3% to 94%. The antioxidant activity of cephalopod ink resides in both the melanin and melanin free ink fractions (Derby, 2014). Chen et al. (2007) reported that melanin of squid ink acts as a superoxide dismutase and it is able to prevent free radical production by catalyzing oxygen to hydrogen peroxide. For the melanin free ink, the antioxidant activity could be related to the hydroxyl group of L-dopa and L-dopamine component which are able to donate hydrogen atom to the test radical (Vate & Benjakul, 2013). Liu et al. (2011) found that squid ink improved the antioxidant ability of growing broiler chickens. In the same way, Vate & Benjakul (2013) reported that melanin free ink inhibited lipid oxidation in sardine surimi gel.

3.3 Biochemical and microbiological characterization of fresh and smoked sardine fillets

Moisture decreased significantly (P = 0.008) from 78.06 ± 0.5% to 64.33 ± 0.7% after steaming and smoking. This reduction is explained by the evaporation of water on the fish surface and the diffusion of water from the sardine muscle to the surface during the steaming and smoking process (Cyprian et al., 2015). Peroxide value of fresh fillets (0.36 ± 0.07 meq/kg oil) increased significantly after steaming and smoking, to reach a value of 1.24 ± 0.04 meq/kg fish (P = 0.005). This increase is probably due to the steaming temperature which generally accelerates lipid oxidation (Cyprian et al., 2015). In addition, smoked fillets were more dehydrated and therefore more saturated with lipids than fresh fillets (Idah & Nwanko, 2013). A significant increase (P = 0.01) in TVB-N value from 2.79 ± 0.03 mg/100 g fish (fresh fillets) to 5.36 ± 0.91 mg/100 g fish was noted after steaming and smoking, probably due to an autolytic process which produces volatile amine compounds (Mosarrat et al., 2016). Under the effects of temperature, dehydration and antimicrobial activity of smoke constitution, TVC counts decreased from 4.95± 0.26 to 1.74± 0.1 log cfu/g (P = 0.02) after steaming and smoking.

3.4 Quality of ink soaked sardine fillets during storage

Microbiological analysis

TVC counts of soaked sardine fillets in the three ink solutions during storage are presented in Figure 1. TVC counts increased significantly (P < 0.05) from 1.72 ± 0.01 and 1.73 ± 0.03 log cfu/g to reach values of 4.85 ± 0.31 and 3.98 ± 0.25 log cfu/g for control samples and those soaked with 1% of ink solution after 35 days of storage at 4°C, respectively. However, the increase of TVC counts in samples soaked with 2 and 3% of ink solution was not significant (P > 0.05) and the values reached remained below 10³cfu/g (Table 2). These results showed that TVC counts were significantly (P < 0.05) affected by ink solution concentrations (Table 2). In much the same way, Karim et al. (2016) and Sadok et al. (2004) showed the effect of ink free melanin concentrations on TVC counts of seafood products during storage. Total and fecal coliforms and Staphylococcus aureus strain were not detected in all samples during storage. This result can be explained by the sensitivity of these strains to salted mediums.

Figure 1
Total viable counts of smoked sardine fillets soaked in three different cuttlefish ink solutions (1,2 and 3%) during storage at 4 °C.

Table 2
Effect (P-values) of the time of storage and the type of the soaking solution on microbiological and biochemical parameters of smoked sardine fillets.

	P- time				P- soaking solution
	C	S1	S2	S3
TVC	0.001	0.009	0.827 (NS)	0.775 (NS)	0.001
TVB-N	0.008	0.007	0.431 (NS)	0.652 (NS)	0.021
PV	0.011	0.031	0.258 (NS)	0.658 (NS)	0.038
Histamine	0.025	0.061 (NS)	0.312 (NS)	0.625 (NS)	0.041

• S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%; C: samples soaked in salt water (3.5%); TVC: Total Viable counts; TVB-N: Total Volatile Base Nitrogen; PV: Peroxide Values; NS: not significant.

TVB-N

TVB-N value increased significantly (P < 0.05) from 5.36 ± 0.91 and 5.38 ± 0.2 mg/100g to 17.27 ± 0.6 and 10.4 ± 0.3 mg/100g fish for control samples and those soaked with 1% ink solution after 35 days of storage at 4°C, respectively (Table 2). However, TVB-N values for these two samples remain under the limit which is 35 mg/100 g (Connell, 1995). This increase is due to a microbiological degradation of nucleotides and free amino acids as well as microbial reduction of TMAO to TMA (Sallam et al., 2007). Indeed, a significant positive correlation (r = 0.98; P < 0.05) was obtained between TVB-N values and TVC counts for control and S1 samples during storage. The evolution of TVB-N values during storage of S2 and S3 was not significant (P > 0.05) (Table 2). At the end of storage, S2 and S3 samples showed the lowest values of TVB-N (Figure 2), thus confirming the effect of ink concentrations on microbial growth. Similarly, Karim et al. (2016) found a significant decrease of TVB-N values storing squid coated with squid ink compared to the control. Sadok et al. (2004) showed that free melanin squid ink had a significant effect on TVB-N values of shrimp during storage.

Figure 2
TVB-N (a) and peroxide values (b) of smoked sardine fillets soaked in three different cuttlefish ink solutions (1,2 and 3%) during storage at 4 °C. S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%; C: samples soaked in salt water (3.5%).

Peroxide value

Peroxides produced from lipid oxidation lead to odor formation and also loss of taste and texture (Maltar-Strmečki et al., 2013). PV increased significantly up to day 14 from 1.35 ± 0.05 and 1.37 ± 0.04 meq/kg oil to 7.2 ± 0.98 and 6.54 ± 0.87 meq/kg oil for control samples and those soaked with 1% ink solution, respectively (Figure 2b). Until the end of storage, PV decreased gradually as hydroperoxide decomposed to other secondary compounds (Vate et al., 2015). For the S2 and S3 samples, PV did not show a significant increase (P > 0.05) during storage (Table 2). At the end of storage PV values of S2 and S3 samples were 1.5 ± 0.01 and 1.43 ± 0.03 meq/kg oil, respectively. These results suggest that ink solutions of 2 and 3% acted as antioxidants. Vate & Benjakul (2013) stated that melanin free ink solution had radical scavenging and metal chelating activities and were able to lower the formation of lipid oxidation products. Similarly, Vate et al. (2015) reported that melanin free ink prevented lipid oxidation in surimi gel in sardines (Sardinella albella). In all samples, peroxide values did not rise to 20 meq/kg oil, generally considered necessary for oils to become rancid (Hraš et al., 2000).

Histamine

Histamine values were determined on days 0, 15 and 35 (Figure 3). In all samples, values did not develop beyond the limit (20 mg/100 g) set by the EU (Council of the European Union, 1991). Lower histamine values found in this work are related to the low storage temperature. Indeed, Lehane & Olley (2000) reported that histamine is produced at a very low rate at refrigeration temperature. Initial histamine values were 0.85 ± 0.02; 0.84 ± 0.03 mg; 0.86 ± 0.04 and 0.83 ± 0.03 mg /100 g to 2.1 ± 0.05 mg /100 g for control, S1, S2 and S3 samples, respectively (P > 0.05). After storage, control samples showed the highest value of histamine (P < 0.05) when compared to S1 (1.01 mg/100g), S2 (1.05 mg /100 g) and S3 (1.08 mg /100 g) samples (P > 0.05) (Table 2). This result can be explained by the higher count of TVC found in the control samples leading to histamine formation. The antimicrobial effects of the ink solution reduce histamine formation in smoked sardine fillets.

Figure 3
Histamine content of smoked sardine fillets soaked in three different cuttlefish ink solutions (1%,2% and 3%) during storage at 4 °C. S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%; C: samples soaked in salt water (3.5%). ^a–c Means within histogram with different superscripts significantly differ among time (P < 0.05) for each soaking solution.^1-3 Means within histogram with different superscripts significantly differ among soaking solution (P < 0.05) for each day of storage.

Whiteness

The whiteness value of the fresh fillets was 50.16 ± 2.6 (Table 3). Due to a large amount of myoglobin, fresh sardine has quite dark color (Vate et al., 2015). This value deceased significantly after steaming and smoking to reach a value of 43.05 ± 2.01. Cardinal et al. (2001) explained this decrease of whiteness by a loss of water in fish muscle. Cuttlefish ink plays a role as a natural food coloring agent (Ahamed et al., 2018). The whiteness value of the cuttlefish ink used in this work was 13.98 ± 1.05. These results are similar to those of Ahamed et al. (2018) who found that the whiteness value of cuttlefish ink was 14.16. Just after soaking, due to the melanin pigment, a significant difference was observed between the sardine control sample and those soaked with the ink solution (P < 0.05). Whiteness values were affected only by storage time and not by ink solution concentrations (P > 0.05). In fact, whiteness values deceased significantly (P < 0.05) after storage to reach values of 33.01 ± 0.9, 32.25 ± 1.7 and 31.64 ± 2.1 for S1, S2 and S3 samples, respectively (Table 3). This decrease is related to the denaturation of melanin. The whiteness of the control samples did not change during storage (P > 0.05).

Table 3
Whiteness of fresh, smoked and ink soaked sardine fillets.

Sample	Whiteness
Fresh fillets	50.16 ± 2.6a
Smoked fillets	43.05 ± 2.01b
Soaked fillets	Day 0	Day 35
C	44.12 ± 2.9a1		42.54 ± 2.01a1
S1	40.26 ± 2.1b1		33.01± 0.9b2
S2	41.56 ± 1.8b1		32.25 ± 1.7b2
S3	39.82 ± 2.5b1		31.64 ± 2.1b2

• S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%; C: samples soaked in salt water (3.5%). Mean value ± standard deviation (n = 3). Means with the same parameter and the same column with different superscript letters (a, b) are different (P < 0.05). Mean with the same parameter and the same line with different superscript numbers (1, 2) are different (P < 0.05).

Hardness

The evolution of the value of hardness (g) during storage is presented in Figure 4. During the first fifteen days of storage, hardness values increased significantly from 4935 ± 99.25, 4853 ± 92.28 and 4753 ± 99.37 g to reach values of 5125 ± 102.25 g, 5236 ± 110.36 g, and 5369 ± 120.11 g for S1, S2 and S3 samples, respectively (Figure 4). While, for the control samples, hardness decreased from 5553 ± 95.25 to 4885 ± 100.58 g. The increase in the experimental samples, can be explained by the activity of tyrosinase which catalyzes the oxidation of tyrosine to DOPA and diquinone. The reaction of these latter with amino acid chains leads to the formation of inter-and intra-molecular cross links (Vate et al., 2015). A similar result was obtained by Vate et al. (2015) who showed that the tyrosinase activity of free melanin ink plays a role in cross-linking proteins which leads to increase hardness of sardine surimi gel incorporated with free melanin ink during storage at 4 °C. From the 15^th day until the end of storage, hardness values decreased significantly (P < 0.05) to reach values of 3828, 3985 and 3852 g, for S1, S2, and S3 samples, respectively. This decrease was not affected by the ink solution concentration and was probably due to the action of muscle proteases causing destruction of the collagen structure (Gómez‐Guillén et al., 2009). For the control samples, hardness decreased significantly throughout the storage.

Figure 4
Hardness of smoked sardine fillets soaked in three different cuttlefish ink solutions (1,2 and 3%) during storage at 4 °C. S1: samples soaked in cuttlefish solution 1%; S2: samples soaked in cuttlefish solution 2%; S3: samples soaked in cuttlefish solution 3%; C: samples soaked in salt water (3.5%). ^a–c Means within histogram with different superscripts significantly differ among time (P < 0.05) for each soaking solution.^1-2 Means within histogram with different superscripts significantly differ among soaking solution (P < 0.05) for each day of storage.

4 Conclusion

On the basis of the obtained data it can be concluded that cuttlefish ink, a by-product from cuttlefish processing, could be utilized to improve the stability of smoked sardine fillets during storage at 4 °C. Soaking smoked sardine fillets in cuttlefish ink solution led to reduce TVC counts during storage and then improve microbiological quality. Due to this microbial effect, samples soaked with 2 and 3% solutions of cuttlefish ink showed the lowest values of TVB-N and histamine. Moreover, the cuttlefish ink used in this study led to reduce lipid oxidation of smoked sardine fillets. The use of crud ink can economically and technically be used for a preservation of smoked sardine, however a sensorial and a consumer study needs to be done to ascertain products acceptability.

• Practical Application: Extending shelf life of smoked sardine fillets by valorization of cuttlefish ink, a by-product of the seafood industry.

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Publication Dates

• Publication in this collection
  14 Mar 2022
• Date of issue
  2022

History

• Received
  05 Dec 2020
• Accepted
  25 Nov 2021