Characterization of fish sausage manufactured with combination of sunflower oil and fish oil stabilized with fish roe protein hydrolysates

Abstract

The present study aimed to determine the effect of adding protein hydrolysates obtained after 30, 60, and 90 min enzymatic hydrolysis of fish roe on properties of silver carp sausages enriched with fish oil during storage at 4 °C for 30 days. Properties of the fortified sausages were determined by assessment of primary and secondary oxidation, fatty acid composition, microbial spoilage, texture, and organoleptic properties. The results indicated that the hydrolysates could retard oil oxidation and microbial spoilage and preserve n− 3 fatty acids in fish sausages during the refrigerated storage. Also, they rendered firmer microstructure with smaller oil droplets to the sausages. Fish sausages fortified with the hydrolysates were lighter and exhibited better textural and sensory properties. It can be concluded that enzymatic hydrolysates from discarded fish roe can be added to fish sausages containing fish oil to retard oil oxidation and microbial spoilage and improve sausage properties.

Electronic supplementary material

The online version of this article (10.1007/s13197-019-04179-6) contains supplementary material, which is available to authorized users.

Introduction

Current biotechnological developments have paved the way to consider beneficial possibilities of value-added products from marine wastes. These by-products are regarded as potentially nourishing resources and they usually possess valuable protein content (Ennaas et al. 2015). In this sense, one of the most important trends in food biotechnology is enzymatic hydrolysis of marine by-products to yield bioactive agents (Intarasirisawat et al. 2014). Protein hydrolysates from marine by-products were produced using underutilized species (Nikoo et al. 2015), head (Chi et al. 2015), and roe (Chalamaiah et al. 2015).

Fish roe is a valuable nutritional source with high protein content (11% albumins, 75% ovoglobulin, and 13% collagen) (Chalamaiah et al. 2015) and substantial amounts of essential amino acids (Intarasirisawat et al. 2011). Fish roe hydrolysates with functional and antioxidant properties were developed from different species such as common carp (Cyprinus carpio) (Chalamaiah et al. 2015), striped snakehead (Channa striatus) and Rohu (Labeo rohita) (Galla et al. 2012), and skipjack tuna (Katsuwonous pelamis) (Intarasirisawat et al. 2014). Chalamaiah et al. (2015) stated that roe protein hydrolysate had lower fat content but higher ash content than fish protein hydrolysate; they explained that higher ash content of roe protein hydrolysate might be due to high content of minerals in fish roe. In addition, roe protein hydrolysate showed comparable antioxidant properties to fish protein hydrolysate (Chalamaiah et al. 2015; Intarasirisawat et al. 2014).

Fast-paced lifestyles have urged individuals to consume ready-to-eat meat products. In this regard, sausage is one of the most popular meat products due to its various tastes and flavors (Souissi et al. 2016). Nevertheless, there have been growing concerns over negative influence of unhealthy fats and saturated fatty acids in sausages and the risk of obesity and/or cardiovascular disease (Feng et al. 2013). Moreover, due to the risk issues regarding the frequent use of red meat, seafood and processed fish products have recently gained more publicity. However, susceptibility of marine products to quality loss caused by oil oxidation has limited the consumption of these products (Tayel 2016).

Owing to the potential side effects of using synthetic agents in food products (Nasri et al. 2013), natural products, e.g. protein hydrolysate, have been suggested as functional and antioxidant agents to fortify marine products (Elias et al. 2008). Along with oxidation, food-borne infection caused by microorganisms is another chief concern (Jemil et al. 2014). These microorganisms may cause health problems due to spoilage, toxins, and quality deterioration in foods (Sokmen et al. 2004). In addition, frequent incorporation of antibiotics in food products will lead to side effects and resistance of pathogens (Sila et al. 2014). Protein hydrolysate is one of the healthy and natural antibacterial agents to incorporate in food products. Protein hydrolysates with antibacterial effects were extracted from marine resources (Pu and Tang 2017).

Taken together, the present study was formulated in order to determine the effect of fortification of silver carp sausage with protein hydrolysate from fish roe and its influence on physicochemical, microbiological, and textural properties of the sausages.

Materials and methods

Chemicals

Alcalase 2.4 L FG (2.4 AU-A/g) was purchased from Novozymes (Bagsværd, Denmark). Hexamethyldisilazane (HMDS) was obtained from Sigma-Aldrich (Steinheim, Germany). Glutaraldehyde was purchased from Sigma (St Louis, USA). Chloroform and methanol were provided from Lab-Scan (Dublin, Ireland). All other chemicals and solvents were of analytical grade obtained from Merck (Darmstadt, Germany).

Production of protein hydrolysate

Pulverized common carp roe (5 g) was suspended in distilled water (150 ml) and mixed for 2 min. After thermal pre-treatment at 50 °C for 15 min, Alcalase 2.4L FG was added at 1.5% (v/w) of protein content of roe powder in optimum condition for the enzyme (pH = 8 and 50 °C). The reaction continued for 30, 60, and 90 min to yield different degrees of hydrolysis (DH). The mixture was placed in a water bath at 100 °C for 15 min to terminate the reaction. The slurry was centrifuged at 13,000g at 4 °C for 30 min and supernatant was lyophilized (Alpha 1–2 LD plus, Martin Christ, Osterode, Germany).

Characterization of protein hydrolysate

Lipid content was determined by Bligh and Dyer (1959) method and Moisture, ash and protein were examined through AOAC (2006) method. In brief, moisture and ash contents were gravimetrically measured by heating the samples until constant weight at 105 °C and 550 °C, respectively. Protein content was acquired by the Kjeldahl method by considering a nitrogen-to-protein conversion factor of 6.25 (Kjeldahl 1883). Measurements were done in duplicate. The amino acid composition was assessed by HPLC–MS following hydrolysis and derivatisation using EZ:faast Amino Acid Kit (Phenomenex, Torrance, CA, USA).

After thermal inactivation of Alcalase 2.4L, the reaction mixture was mixed with one volume (v/v) of 20% trichloracetic acid (TCA) followed by centrifuging at 6700g at 4 °C for 30 min to collect the 10% TCA-soluble materials. DH (%) was calculated as follows:

$$\text{DH}\left(\%\right)=\left(10\%\text{TCA}-\text{soluble}\text{nitrogen}\text{in}\text{substrate}\right)/\left(\text{total}\text{nitrogen}\text{in}\text{substrate}\right)\times 100$$

Preparation of silver carp sausage

Silver carp fillets were minced by a meat grinder (MK-G1300P Panasonic, Japan). Fish mince was blended with NaCl (20 g/kg) and sunflower oil (150 g/kg) at 4 °C for 5 min. Afterwards, four types of sausages were prepared:

1. control: added with fish oil (150 g/kg) without hydrolysate;

2. hydrolysate-30: added with fish oil (150 g/kg) and hydrolysate (3 g/100 g) obtained after 30 min reaction time;

3. hydrolysate-60: added with fish oil (150 g/kg) and hydrolysate (3 g/100 g) obtained after 60 min reaction time; and

4. hydrolysate-90: added with fish oil (150 g/kg) and hydrolysate (3 g/100 g) obtained after 90 min reaction time.

Commercial cod liver oil was donated by Maritex A/S, subsidiary of TINE, BA (Sortland, Norway) and stored at − 40 °C until use. The oil’s 16:0, C16:1, C18:1, C20:1, C20:5, and C22:6 contents were 9.5%, 8.7%, 16.3%, 12.6%, 9.2%, and 11.4%, respectively. Moreover, its α-, β, γ-, and δ-tocopherol contents were 200 ± 3, 5 ± 1, 96 ± 3, and 47 ± 1 μg/g oil, respectively.

Other ingredients were tripolyphosphate (2 g/kg), potato starch (24 g/kg), and crushed black pepper (1 g/kg). The mixtures were ground for 3 min and the pastes were stuffed into cellophane casing (22-mm diameter). The samples were incubated at 55 °C for 40 min and then cooked at 80 °C for 15 min. the sausages were cooled in iced-water for 30 min, sealed in polyethylene bags, and stored at 4 °C. Samples were randomly selected at 0, 10, 20, and 30 days of refrigerated storage for physicochemical, microbiological, and textural analyses.

Lipid oxidation

Lipid oxidation in samples was determined by measuring PV and TBARS values. PVs were measured on lipid extracts via the colorimetric ferricthiocyanate method at 500 nm (Shantha and Decker 1994). TBARS values were evaluated according to the method of Buege and Aust (1978).

Fatty acid composition

Fatty acid profile of control and hydrolysate-containing fish sausages was determined through gas chromatography of fatty acid methyl esters (FAMEs). Toluene and heptane with internal standard (C23:0) (400 μl, 1:3v/v) were introduced into Bligh and Dyer (1959) lipid extracts. After a one-step methylation using a microwave oven (Multiwave3000 SOLV, Anton Paar, Graz, Austria) with a 64MG5 rotor (5 min at 500 W and 10 min cooling), the methyl esters were dissolved in n-heptane at 20 mg/ml. Afterwards, 1.5 μg of FAMEs was injected into an HP 5890A a gas chromatograph (Agilent Technologies, Palo Alto, CA, USA) in split mode (1:70) with a volume of 0.2 μl. Separation was carried out on a DB127-7012 column (10 m × ID 0.1 mm × 0.1 μm; Agilent Technologies) and helium was used as carrier gas (21 cm/s) with injection and detection at 250 °C and 240 °C, respectively and initial oven temperature at 160 °C which was raised to 200 °C for 0.3 min, 220 °C for 1 min, and 240 °C for 3.8 min at 10.6 °C/min. Fatty acids were quantified by comparing integrated areas and were expressed as area percentage of total fatty acids.

Microstructure

Microstructure of fish sausages containing fish oil and protein hydrolysate from discarded roe was assessed through a Phenom Pro desktop scanning electron microscope (SEM) (Phenom-World, Netherlands). Four-mm thick samples were fixed with 2.5% (v/v) glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) at room temperature for 2 h. The samples were subsequently rinsed in distilled water and subjected to dehydration using ethanol series (20%, 50%, 70%, 80%, 90% and 100%) for 10 min at each concentration. The dehydrated samples were post-fixed in 100% HMDS for 2 h. Rinsing and dehydration were carried out in the similar way as mentioned above. The samples were allowed to dry overnight under a fume hood and then mounted on an aluminum pin stub using double-sided carbon adhesives. The specimens were observed at an acceleration voltage of 5 kV.

Microbiological analysis

Aseptically sepaarted samples (25 g) were subjected to homogenization for 90 s in 225 ml peptone water (0.1%) with 0.5 g NaCl in a Stomacher 400 (Seward Medical, London, UK). The homogenates were pour-plated in decimal dilutions on Plate Count Agar (PCA) (Merck, Darmstadt, Germany). The plates were incubated at 37 °C for 4 days and at 4 °C for 7 days for determination of the total viable count (TVC) and the psychrophilic bacterial count (PBC), respectively. Microbial counts were presented as logarithms of colony-forming units per gram (Log cfu/g).

Color determination

Color of the sausages was determined using a Chroma Meter (Minolta^® Camera Co. LTD, CR-200, Osaka, Japan) with the CIE L* a* b* color system. The instrument was standardized by use of a black and white Minolta calibration plate. The values were shown as L* (i.e. lightness), a* (i.e. redness), and b* (i.e. yellowness).

Texture profile analysis (TPA)

TPA was performed on a TA.XT.plus Texture Analyzer (Stable Micro Systems Ltd, Godalming, Surrey, UK) using a cylindrical probe (50 mm diameter). The sample sores (diameter 3 cm, height 2.0 cm) were axially compressed to 50% of their original height at ambient temperature. Force–time curves were obtained with a load cell of 25 kg applied at a crosshead speed of 50 mm/min. The tests were run with two compression cycles with a 10-s interval between the cycles. Hardness, cohesiveness, springiness, gumminess and chewiness were measured according to the curve.

Sensory analysis

Sensory evaluation was carried out by twenty-eight untrained panelists (25–35 years old) with regular sausage consumption habit. Informed written consents were obtained from the panelists. The panelists were asked to examine the sensory properties of fish sausages (color, odor, taste, appearance, and overall acceptability). They were served with fish sausage slices (2 mm thick) on white polystyrene plates in a well-lit room. For mouth cleaning purpose, they were also provided with water and unsalted crackers. The scoring was performed through a 9-point hedonic test (1 = extremely unpleasant, 5 = neither pleasant nor unpleasant, and 9 = extremely pleasant) (Intarasirisawat et al. 2014). All the analyses were repeated three times and the panelists were allowed to rest for 60 min between servings to avoid exhaustion.

Statistical analyses

Statistical analyses were performed through Analysis of Variance (ANOVA) and the Least Significant Difference (LSD) test. All the operations were carried out in IBM SPSS package (version 21.0, IBM Corp., Armonk, NY, USA). Differences were considered significant at p < 0.05.

Results and discussion

Characterization of protein hydrolysate

According to Table 1, protein content of fish roe was around 19%, which is consistent with a previous study (Balaswamy et al. 2009). However, the protein content of hydrolysates was much higher (68–78%). removal of insoluble solid matter by centrifugation and solubilization of protein might be responsible for the high level of protein in hydrolysates (Chalamaiah et al. 2012). DH increased sharply within the first 30 min reaching 7.59 and increased significantly after another 30 min reaching 9.70 (p < 0.05); although it continued to increase reaching 10.06 after 90 min reaction, DH difference between 60 and 90 min reaction times was not significant (p > 0.05). The reduced rate of hydrolysis might be due to the reduction of substrate molecules (Chalamaiah et al. 2015) and formation of reaction products restricting enzyme activity (Ovissipour et al. 2009). Due to the lyophilization carried out after enzyme deactivation, moisture of hydrolysates dramatically decreased. Furthermore, lipid and ash were found to be circa 11% and 6–9%, respectively. In addition, the hydrolysates contained higher levels of valine, lysine, arginine, leucine, glutamic acid, aspartic acid, and glycine, which is consistent with previous reports (Intarasirisawat et al. 2011; García-Moreno et al. 2016; Morales-Medina et al. 2016; Chalamaiah et al. 2015).

Table 1.

Characterization of fish roe and hydrolysates

	Discarded roe	Hydrolysate-30	Hydrolysate-60	Hydrolysate-90
DH (%)	–	7.59b	9.70a	10.06a
Protein (wt%)	19.26 ± 1.06b	68.94 ± 0.98a	71.51 ± 0.74a	73.16 ± 1.20a
Lipid (wt%)	3.22 ± 0.06b	11.76 ± 1.03a	11.26 ± 1.41a	11.37 ± 1.18a
Moisture (wt%)	71.84 ± 0.26a	9.90 ± 0.05b	6.29 ± 0.86c	5.30 ± 0.33c
Ash (wt%)	1.32 ± 0.09d	6.34 ± 0.50c	7.25 ± 0.02bc	8.56 ± 0.27ab
Amino acid composition (g/100 g protein)
ARG	3.9 ± 1.27	3.65 ± 0.93	4.6 ± 0.57	6.62 ± 1.91
HIS	0.06 ± 0.04	0.04 ± 0.00	0.03 ± 0.01	0.06 ± 0.01
ILE	1.46 ± 0.45	2.10 ± 0.68	1.13 ± 0.39	1.53 ± 0.06
LEU	4.05 ± 0.76	3.95 ± 0.06	3.18 ± 0.74	3.47 ± 0.55
LYS	4.86 ± 1.11	3.2 ± 1.02	6.5 ± 0.61	6.16 ± 1.70
MET	1.10 ± 0.25	1.15 ± 0.30	1.00 ± 0.24	1.04 ± 0.06
PHE	1.75 ± 0.20	1.71 ± 0.22	1.45 ± 0.51	1.54 ± 0.22
THR	2.81 ± 0.53	2.67 ± 0.52	2.54 ± 0.49	2.75 ± 0.44
TRP	0.28 ± 0.03	0.25 ± 0.01	0.39 ± 0.02	0.37 ± 0.05
VAL	5.55 ± 1.00	6.46 ± 0.77	6.2 ± 1.03	6.93 ± 1.66
ALA	1.83 ± 1.23	0.22 ± 0.19	1.01 ± 0.71	2.63 ± 1.08
ASP	3.65 ± 0.71	3.62 ± 0.58	3.46 ± 0.64	3.68 ± 0.57
CYS	0.22 ± 0.02	0.34 ± 0.06	0.16 ± 0.06	0.19 ± 0.03
HYP	3.87 ± 0.72	3.70 ± 0.07	2.89 ± 0.61	3.40 ± 0.04
GLY	4.49 ± 0.10	5.06 ± 0.46	3.76 ± 0.78	4.74 ± 0.94
GLU	5.66 ± 1.31	5.80 ± 0.65	6.40 ± 0.07	6.87 ± 1.43
PRO	2.45 ± 0.59	2.43 ± 0.25	1.83 ± 0.41	2.45 ± 0.21
SER	2.87 ± 0.59	3.21 ± 0.45	2.64 ± 0.39	3.04 ± 0.56
TYR	1.25 ± 0.15	1.27 ± 0.21	1.15 ± 0.25	1.14 ± 0.14

Results are the average of duplicate determinations ± standard deviation. The superscripts ‘a’, ‘b’, ‘c’, and ‘d’ indicate significant differences among the samples; No significant difference was observed among the samples in terms of amino acids

Lipid oxidation and fatty acid composition

There was significant increase in PVs of all the sausages after 10 days of refrigerated storage (p < 0.05). Control sample underwent the highest amount of oil oxidation at Day 10 with PV reaching from 7.58 meq/kg oil at Day 0 to 46.07 meq/kg oil at Day 10. The increasing trend in PV of control sausage continued until Day 20 thereafter it started to decrease significantly until Day 30 (p < 0.05). The reduction of PV in control sausage at the end of refrigeration period can be justified formation of secondary oxidation products such as aldehydes, ketones, etc. because of hydroperoxides decomposition (Intarasirisawat et al. 2014). Protein hydrolysates from discarded fish roe could substantially decrease PV in fish sausages during the refrigerated storage. Although hydrolysate-30 had a better performance to block decomposition of hydroperoxides, no significant difference was detected between the experimental samples (p > 0.05). Higher antioxidant activity of hydrolysates with lower DH was witnessed in previous studies and was attributed to molecular weight of peptides and amino acids as well as abundance and placement of hydrophobic amino acids (García-Moreno et al. 2016; Morales-Medina et al. 2016). Although no single consensus has been attained yet regarding the exact mechanism of action of protein hydrolysate to render its protective properties against oxidation, it is widely agreed that molecular size of peptides and amino acids composition and sequence in hydrolysates are the determinant of their antioxidant activities (García-Moreno et al. 2016).

TBARS values in the beginning of the storage (Fig. 1b) show that oil oxidation happened when the sausages were produced. This is plausible since the sausages contain high amount of unsaturated fatty acids. TBARS of all the sausages increased significantly until Day 10 (p < 0.05). The most prevalent increase in TBARS was detected in control sample reaching from 4.36 mg MDA/kg sample at Day 0 to 39.65 mg MDA/kg sample at Day 10. However, no significant increase happened in the samples afterwards (p > 0.05). TBARS is formed in samples because of interactions among secondary oxidation products (Intarasirisawat et al. 2014) and formation of Schiff bases due to reaction with free amino acids, proteins, and peptides (Maqsood et al. 2012). The results of this experiment revealed that protein hydrolysates from discarded fish roe could successfully retard formation of oxidation products as evidenced by significantly lower TBARS values of the hydrolysate-containing sausages compared to control sample (p < 0.05). However, as it was the case for PVs, no significant difference was detected among the sausages in terms of TBARS values (p > 0.05). It is true that low fat sausages (10–20%) are prioritized over high-fat sausages (20–30%) because of health concerns, but it is mainly because of high saturated fat content in the latter type of sausages. However, in the present study, two types of healthy oil were incorporated, namely sunflower oil, which is known to have lower level of saturated fats, and fish oil, which is known as healthy oil with high content of polyunsaturated fatty acids with numerous health effects for human.

Fig. 1.

Oil oxidation and microbiological analysis in silver carp sausages containing fish roe protein hydrolysates in a 30-day refrigerated storage; a PV, b TBARS, c TVC, d PBC. Bars indicate standard deviation (n = 2)

At Day 0, PUFAs were the most abundant as evidenced by high content of oleic acid (18:1 n − 9) and docosahexaenoic acid (22:6 n − 3; DHA). Furthermore, a rather high content of eicosapentaenoic acid (20:5 n − 3; EPA) was also detected at Day 0. Abundance of n − 3 fatty acids (EPA + DHA) in fish sausages can be explained by the addition of fish oil in fish sausages. Also, linoleic acid (18:2 n − 6) was found in approximately higher values, which is owing to the addition of sunflower oil in fish sausages. At Day 30, the content of PUFAs decreased in all fish sausages, which is due to the susceptibility of PUFAs to oxidation (García-Moreno et al. 2016). Overall, hydrolysate-containing fish sausages had significantly higher PUFAs than control fish sausage at Day 30 (p < 0.05), which could be attributed to antioxidant activity of protein hydrolysates from discarded fish roe (Morales-Medina et al. 2016). Fish sausage fortified with hydrolysate-30 showed higher level of EPA + DHA, which is in accordance with lower PV and TBARS of the sausages containing this hydrolysate (Fig. 1a, b). It seems that higher level of the hydrophobic amino acids isoleucine and glycine accounted for protective capability of hydrolysate-30; Chalamaiah et al. (2012) mentioned that the hydrophobic amino acids in hydrolysates might be responsible for their antioxidant capacity. However, no significant difference was detected between hydrolysate-containing fish sausages in terms of their EPA + DHA (p > 0.05). Therefore, protein hydrolysate from discarded fish roe can be used to preserve valuable fatty acids, especially EPA and DHA, in fish sausages. See Supplementary Material for thorough results regarding the fatty acid composition of control and hydrolysate-containing fish sausages.

Microstructure of sausages

The microstructure of the sausages is shown in Fig. 2. Control sample contained larger oil droplets while the samples containing protein hydrolysates had smaller oil droplets. Furthermore, among the sausages with hydrolysates, the sample containing hydrolysate-30 had more compact structure and fewer voids. This is logical because increase in hydrolysis time and therefore achieving higher DH will lead to reduction in emulsifying activity of hydrolysate. Shorter-length peptide chains, which are yielded when the hydrolysis protracted, are less efficient in decreasing interfacial tension and therefore have weaker emulsifying activity (Kristinsson and Rasco 2000). The result of this study is in agreement with previous reports. Intarasirisawat et al. (2014) found that skipjack roe protein hydrolysate resulted in smaller fat globules and more compact microstructure in fish sausages containing fish oil. Besides, the compact structure of the experimental sausages may be owing to the antioxidant and antimicrobial effects of hydrolysates. Microorganism, free radicals, and oxidation products might degrade protein matrix and damage the microstructure of final products (Maqsood et al. 2012).

Fig. 2.

Scanning electron microscopy micrographs of control and hydrolysate-containing silver carp sausages; control (top left), hydrolysate-30 (top right), hydrolysate-60 (down left), and hydrolysate-90 (down right)

Microbial analysis

Microbial counts of control and hydrolysate-containing fish sausages during refrigerated storage are depicted in Fig. 1c and d. Although TVC in all the fish sausages rose significantly during the storage (p < 0.05), significant differences were seen between TVC of control and hydrolysate-containing fish sausages (p < 0.05). In other words, protein hydrolysate from discarded fish roe retarded microbial spoilage in fish sausages. At Day 30, there was a significant difference between TVC of the fish sausage containing hydrolysate-90 and the other two experimental sausages (p < 0.05); however, no other significant differences were seen between the hydrolysate-containing fish sausages (p > 0.05). Higher antibacterial effect of hydrolysate at higher DH is in agreement with the results of Sila et al. (2014). Smaller peptides yielded by prolonged hydrolysis are presumably more potent inhibitors of microbial growth. This is presumably because smaller peptides possess a better capability of interacting with microbial cell membranes; such an interaction might occur through the peptides’ ability to create discrete channels in lipid bilayers, disturbing lipid bilayer via carpet-like peptide binding, phase separation through particular peptide–lipid interaction, and detergent-like solubilization of microbial membrane (Sila et al. 2014).

It is noteworthy that TVC of the fish sausage fortified with hydrolysate-90 at the end of the refrigerated storage was still below the suggested threshold for meat products (i.e. 5 log cfu/g) (Stannard 1997). There were no psychrophilic bacteria in the sausages when the storage began (Fig. 1d); however, PBC increased significantly after 10 days of refrigerated storage in all the samples (p < 0.05). Also, PBC of control sausage significantly exceeded that of hydrolysate-containing fish sausages (p < 0.05), which is likely because of antibacterial properties of bioactive peptides in the hydrolysates.

Color

The color parameters of control and hydrolysate-containing fish sausages during the storage period at 4 °C are shown in Fig. 3. Fish sausages fortified with protein hydrolysates from discarded fish roe were significantly lighter than control sausage (p < 0.05) while there were no significant differences between the experimental sausages (p > 0.05). The higher lightness of fish sausages containing protein hydrolysates could be owing to the interaction of fish oil and hydrolysates, which reduces oil droplet size and raise light reflectance from the sliced surfaces (Chen et al. 2015). Fish sausages had low redness (α*); this is plausible since myoglobin in fish muscle is not as much as that in red meat. Redness might also be reduced because of addition of fish oil in sausages as reported by Cáceres et al. (2008). No clear trend was detected as to the effect of protein hydrolysate from discarded fish roe on the b* values of fish sausages. Therefore, protein hydrolysate resulted in lighter sausages but had no obvious influence on redness and yellowness of fish sausages.

Fig. 3.

Color parameters in control and hydrolysate-containing silver carp sausages during a 30-day refrigerated storage. Bars indicate standard deviation (n = 3)

Textures

In most of the cases, hardness of the sausages containing protein hydrolysate from discarded fish roe was found to be higher than that of control samples. The sausages containing hydrolysate-60 and -90 showed significantly higher hardness values than control samples at the end of the refrigerated storage (p < 0.05). The higher hardness of hydrolysate-containing fish sausages could be due to the fact that functional peptides in the hydrolysates cover oil droplets and elevate consistency of sausages (Cáceres et al. 2008). Also, gumminess and chewiness were found to be significantly higher in the sausages with hydrolysates yielded at higher reaction times (p < 0.05). The higher gumminess and chewiness of hydrolysate-containing fish sausages could be attributed to the amphiphilicity of hydrolysates, which render higher stability to the final product (Rahali et al. 2000). There was significant difference in terms of springiness values among fish sausages (p > 0.05). The rather similar springiness of the fish sausages is justified by equal oil content in them because elasticity in meat products is related to their oil content (Youssef and Barbut 2011). Cohesiveness values had no obvious trend in fish sausages, which could be owing to the fact that other factors such as moisture and water activity are responsible for cohesiveness (Lorenzo et al. 2013). Future studies should be directed toward determining more textural characteristics, e.g. gelling quality, in sausages prepared from freshwater fish species and toward finding alternatives to improve these qualities. Overall, addition of hydrolysates from fish discarded roe improved textural properties of fish sausages during refrigerated storage (Table 2).

Table 2.

Texture analysis of control and hydrolysate-containing silver carp sausages during a 30-day refrigerated storage

Storage period (day)	Samples	Hardness (N)	Cohesiveness	Springiness (cm)	Gumminess (N)	Chewiness (N cm)
0	Control	34.96 ± 0.41Ba	0.30 ± 0.00Bc	0.70 ± 0.00	10.58 ± 0.37Ba	7.47 ± 0.18Ba
	Hydrolysate-30	34.39 ± 1.05Bb	0.31 ± 0.00Bb	0.71 ± 0.00	10.53 ± 0.24Bb	7.19 ± 0.26Cb
	Hydrolysate-60	27.34 ± 1.68Cb	0.36 ± 0.00Aa	0.70 ± 0.00	9.29 ± 0.46Cb	6.81 ± 0.48Cb
	Hydrolysate-90	51.48 ± 0.79Aa	0.30 ± 0.00Bbc	0.70 ± 0.00	14.84 ± 1.09Aa	10.88 ± 0.72Aa
10	Control	24.97 ± 0.53Cb	0.34 ± 0.00Bb	0.70 ± 0.00	8.67 ± 0.60Cb	5.49 ± 0.13Cb
	Hydrolysate-30	39.36 ± 0.70Ba	0.26 ± 0.00Cc	0.71 ± 0.00	10.93 ± 1.71Ba	7.90 ± 0.14Ba
	Hydrolysate-60	20.93 ± 0.43Dc	0.37 ± 0.00Aa	0.70 ± 0.00	7.40 ± 0.21Dc	5.31 ± 0.48Dc
	Hydrolysate-90	42.89 ± 1.36Ab	0.30 ± 0.00BCc	0.69 ± 0.00	12.64 ± 0.83Ab	9.14 ± 0.40Ab
20	Control	21.05 ± 0.74Cc	0.26 ± 0.00Bd	0.70 ± 0.00	6.02 ± 0.18Cc	4.07 ± 0.11Cc
	Hydrolysate-30	23.96 ± 0.81Bc	0.31 ± 0.00Aab	0.69 ± 0.00	8.06 ± 0.37Bc	5.38 ± 0.24Bc
	Hydrolysate-60	38.93 ± 1.21Aa	0.31 ± 0.00Ab	0.71 ± 0.00	12.36 ± 1.36Aa	9.10 ± 0.29Aa
	Hydrolysate-90	38.44 ± 1.73Ac	0.31 ± 0.00Ab	0.70 ± 0.00	12.20 ± 1.67Ab	8.78 ± 0.22ABb
30	Control	23.71 ± 0.96Bb	0.32 ± 0.00Ba	0.69 ± 0.00	8.71 ± 1.05Cb	6.01 ± 0.43Cb
	Hydrolysate-30	19.84 ± 0.50Cd	0.34 ± 0.00Ca	0.71 ± 0.00	6.86 ± 0.68Dd	4.85 ± 0.75Dd
	Hydrolysate-60	27.06 ± 1.02Ab	0.33 ± 0.00Ba	0.70 ± 0.00	9.23 ± 0.044Bb	6.74 ± 0.18Bb
	Hydrolysate-90	27.43 ± 0.76Ad	0.43 ± 0.00Aa	0.71 ± 0.00	11.98 ± 1.09Ab	9.03 ± 0.67Ab

Values are the means of three iterations ± standard deviation. Capital and small letter superscripts indicate significant differences among the samples at each sampling point and for a same sample at different sampling points, respectively. No significant difference was detected in springiness

Sensory analysis

Sensory properties of control and hydrolysate-containing fish sausages scored by untrained panelists during a 30-day refrigerated storage are shown in Fig. 4. At Days 0 and 10, no significant variation was observed in sensory properties of fish sausages (p > 0.05). It should be noted that panelists gave slightly higher score to the odor and taste of control sausage at Day 0, but there was not any significant difference between control and hydrolysate-containing fish sausages in terms of odor and taste Day 0 (p > 0.05). The slightly lower taste scores of experimental sausages could be attributed to bitter amino acids of hydrolysates liberated during hydrolysis (Linde et al. 2009). Furthermore, low color scores of fish sausages could be owing to the relatively low redness values of the sausages (Fig. 3). However, at Days 20 and 30, fish sausages containing protein hydrolysate from discarded fish roe showed significantly higher sensory properties than control sausages (p < 0.05). This can be explained by antioxidant activity of hydrolysates blocking the appearance of oxidative products and unfavorable organoleptic characteristics (García-Moreno et al. 2016). No significant difference was detected at these sampling points between the hydrolysate-containing fish sausages in terms of sensory properties (p > 0.05). Therefore, protein hydrolysate from discarded fish roe can be incorporated in fish sausages in order to preserve sensory qualities during refrigerated storage.

Fig. 4.

Sensory properties of contorl and hydrolysate-containing silver carp sausages during a 30-day refrigerated storage

Conclusion

Fish sausages containing fish oil were found to be susceptible to oil oxidation and microbial spoilage during the refrigerated storage. Protein hydrolysates from discarded fish roe could retard oil oxidation and bacterial quality degradation and preserve PUFAs in fish sausages. The hydrolysates also rendered a firmer microstructure with smaller oil droplets to the fish sausages. Incorporation of roe hydrolysates in fish sausages improved textural and sensory properties, especially during the later parts of refrigerated storage. Therefore, protein hydrolysate from discarded fish roe can be used as a natural additive to improve properties of fish sausages enriched with fish oil.

Electronic supplementary material

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