Raw meats and fats

Fresh beef, pork meat, and pork back-fat were purchased from a local Manta City (Ecuador) market. Tuna heads and tails were obtained from a commercial tuna processing plant (Manta-Ecuador).

Tuna sausage preparations

Tuna and control sausages were produced according to a standard procedure (Figure 1).

Figure 1 Sausages procedure scheme 

The basis of the formulations was similar to the control and tuna formulations, except for meat for the control compared with tuna sausages (Table 1).

Beef, pork, tuna, and pork back-fat were minced separately through an 8 mm diameter disc using a meat grinder (PM-12, Mainca-Spain), then refrigerated at 4 °C. The ground beef, pork, and tuna meat used in each formulation was mixed, homogenized, and emulsified using a meat mincer (Mainca CM-21, Spain). The batter was chilled with ice (0℃) to maintain the temperature of the batter (4 °C). Then, additives, spices, and non-meat ingredients (Alitecno, Ecuador) were added to the meat and mixed. Finally, pork back-fat was added to the mixture, and the batter temperature was maintained below 10℃. The emulsified mix was stuffed into 12 mm diameter cellophane casing # 240, NIPPI Inc. (Tokyo, Japan), using a stuffer (FC-12, Mainca-Spain). Cooking was done in a water bath at 80-85 °C for 20 min until an internal temperature of 80 °C. Finally, the samples were cooled at ~7 ºC and stored at ~4 ºC until further analyses.

Cooking loss

The losses in processing were determined during heat treatment. The sausages were cooked (approximately 0.5 kg) and weighed before and after cooking. Cooking loss was expressed as a percentage of the initial weight before cooking minus the final weight multiplied by one hundred after cooking. The tests were carried out in triplicate according to the methodology proposed by Salazar, Arancibia ².

Proximate composition and pH analysis

Proximate composition (moisture, crude protein, crude fat, and crude ash) was performed according to the Association of Official Analytical Chemists (AOAC, 2005): Moisture was determined by drying in an oven at 105 ºC ± 2 ºC until constant weight; crude protein was evaluated by nitrogen content using Kjeldahl method, and content estimated by multiplying the nitrogen content by 6.25; crude fat content was determined by the Soxhlet method using petroleum ether; crude ash was determined by incineration in a muffle at 550 °C; The determination of fiber was carried out using the enzymatic-gravimetric method (AOAC 985.29) (PRT-701.03-019, 2011) (AOAC, 2005). The sample size was reduced to 0.5 g, and reagent volumes were reduced by half. Incubation with heat-stable alfa-amylase in a boiling water bath was extended to 30 min. The pH of the cooled assay solution was adjusted to 7.5 with 0.5 M NaOH. The pH of the suspension after protease incubation was adjusted to 4.5 with 0.5 M HCl. The residue and Celite were removed from the crucible, ground, and mixed well, and only portions (25 mg) were used for micro-Kjeldahl Nitrogen determination. Carbohydrates were estimated by difference. All determinations were performed in triplicate using three samples for each treatment.

The pH of the samples was measured in a solution of sample and water using a digital potentiometer (HANNA HI 9126, Rhode Island, USA). All tests were carried out in triplicate.

Microbiological determinations

Samples of sausages were collected and weighed aseptically, homogenized, and grown in a specific medium. Mesophilic aerobic bacteria were determined and evaluated according to the official ISO 4833-1:2013 method. Escherichia coli by the ISO 7251:2005 method. Staphylococcus aureus is used by the official ISO 6888-3:2003 method and total coliforms by the official ISO 4832:2006.

Color determination

Color CIE Lab parameters, L* (lightness), a* (red/green), b* (yellow/blue), of cross-sections of sausages, were determined with a Hunter Lab Colorimeter (mini Scan 4500L EZ, Hunter Associates Laboratory INC, Reston, Virginia, USA) calibrated with an illuminator D65 (natural light) and standard observer D10. The results were expressed as Hue and Chroma values. The chroma polar coordinate or saturation C* was calculated from the expression C* = √ (a* ² + b* ²) and Hue (hº) = arc-tan (b*/a*) to a* and b* positives. Furthermore, the whiteness index was determined according to the equation W = 100-[(100-L*)² + (a*² + b*²)]^1/2. At least 15 measurements were performed in different sample areas.

Sensory attributes

The sensory properties were performed in the sensory laboratory equipped with individual cabins. The sausages were previously grilled until the internal temperature reached 90°C and kept at 70°C in an electric oven. Three pieces of 2 cm along the length of grilled samples without casing were provided with water at room temperature and salted crackers for palate cleansing. Sensory attributes such as smell, color, flavor, texture, and overall acceptability were evaluated by 20 semi-trained judges. They used a 5-point hedonic scale (5 - I liked very much; 4 - like moderately; 3 - neither liked nor disliked; 2- disliked moderately; 1 - I do not like it). Also, sausages were evaluated in hard texture using a scale of 1 for "hard" and 5 for "very soft." Attributes of smell, color, flavor, texture, and overall acceptability were evaluated. The acceptability index (AI) was calculated using Equation 1, according to the method proposed by Dutcosky ⁴⁰.

Eq. 1

Statistical analysis

Data were presented as means and standard deviations (SD) and analyzed using the GraphPad Prism 5.0 program (GraphPad Software, San Diego, California, USA). One-way analysis of variance (ANOVA) and Tukey test with a significance level of P< 0.05 were done to determine the differences between samples. Shapiro and Wilks' test was applied to the data for sensory analysis to establish if the dates had a normal distribution, and the Friedman test was used for statistical analysis.

Physicochemical properties

The pH of sausages shows a significant difference in storage time (p<0.05) during the 24-day storage period (Table 2). The pH showed different behavior between control and tuna sausages; in control, the values tended to decrease; in two types of tuna, these values increased. Control sausage started with a pH of 6.77, decreasing to 6.67 from 0 to 24 days. The decrease in pH values could be attributable to the lower quality characteristics of meat, such as water holding capacity, cooking loss, and emulsion stability because of decreased ionic strength between myofibrillar in meat protein ^41-43. The pH started at 6.00 (BET6) and 5.69 (SJT2) and increased to 6.47 and 6.34 at the end of 24 days of storage. The increased pH values in tuna sausages could have occurred due to the growth and development of other types of bacteria competing with lactic acid bacteria, thus increasing the pH of sausages. For fish meat to be fit for human consumption, the pH must be below 6.8 ²⁸. In concordance with this analysis, the tuna sausages would be fit for consumption for 24 days, which was the evaluation period. The behavior of tuna sausages was similar to those observed in sausages made with vongole (Anomalocardia brasiliana), with increasing values between 5.20 and 5.27 ⁴⁴.

Cooking loss

The cooking loss was similar in tuna sausages and different from the control (p<0.05), although none differed more than 1% from the control, so these differences can be considered despicable (Table 3). The cooking loss obtained in this study was relatively low (~5%) and could be attributable to cassava starch content and a stable emulsion of components. The high cooking yield (<10% cooking loss) is indicative of the excellent quality of meat products because of the high water-holding capacity during cooking ⁴⁵. A fish sausage from an unwashed minced blend of low-cost marine fish has a higher cooking loss (13.3%) ⁴⁶. Likewise, when the effects of replacing pork with tuna levels were studied on the quality characteristics of Frankfurters, the cooking losses ranged from 12.86 to 16.77% ⁴⁷. Other authors have observed different cooking losses in fish sausages because, generally, the cooking loss depends on the formulation, emulsion stability, and water holding capacity ⁴⁸.

Proximal analysis

The results of the proximate composition analysis of the sausages are shown in Table 3. A significant difference was observed between tuna sausages and control in moisture content (P<0.05). Tuna sausages showed higher moisture content (~72 %) than the control (~ 59 %). The moisture content in tuna sausages was similar to that of Frozen South African hake (Merluccius capensis), which reported 72.9% ³⁴. Tuna meat has high amounts of protein, balanced essential amino acid compounds, and good digestibility ^49,50. Thereby, meat products elaborated with meat fish tend to have good quality protein. In this sense, sausages prepared with the meat of Big Eye and Skip Jack tuna presented, on average, 18 % protein and were close to sausage prepared from marine catfish (Sciades herzbergii) stored under low temperatures (18.98%) ⁵¹, with meat from Tetradon fahara (18.61%) and Clarias lazera + Tetradon fahara (18.93%) ⁵², and fish sausages produced from fillets of crimson snapper (Lutjanus erythropterus) (19.7%) ⁵³.

The variability of the composition of meat raw materials, the formulations, and the addition of fat could influence the amount of fat in the final product. The tuna sausages contained an average of 4.1% of fat. The higher fat content in the control sample could be attributable to the composition of pork meat that was not added to tuna sausages. Also, the fat results in the present work were low in contrast with sausages prepared with fresh bull’s eye fish (Priacanthus hamrur) (5.62%) ⁵⁴, sausages produced with fillets of crimson snapper (Lutjanus erythropterus) (12.2%) ⁵³, and sausages with Pangas fish (Pangasius Pangasius) (10.70%) ⁵⁵. No significant difference (P>0.05) was observed in ash, fiber, and carbohydrates. The Ecuadorian legislation ⁵⁶⁾ does not prescribe the ash concentration for sausage, so there is no way to compare the composition with any set limits.

Concerning the caloric content of sausages (Table 3), there were significant differences between control and tuna sausages (P<0.05). The samples BET6 and SJT2 had less caloric content than the control. However, despite having the same proportion of fat in all formulations, the pork meat included in the control could have produced this difference. The lower caloric content obtained in this study is less than those reported in sausages with fat replaced with olive oil, flax, or konjac gels (up to 165 kcal/100 g product) ⁵⁷, or about 139.30 kcal/100 g product with makgeolli lees fiber ²⁴. The World Health Organization (WHO, 2003) recommends that for a balanced diet, the energy content should be composed of a variable contribution of 55-57% carbohydrates, 15-30% fat, and 10-15% protein.

Although each product consumed can have a dietary balance since a series of products are consumed daily, there is no doubt that some consumers consider meat products a vital source of their diet. So, achieving figures that allow reducing the energy content of fat (from 79.05% to 12.95% in tuna sausages) represents an essential advance to achieving foods whose caloric protein intake is more important than the caloric intake of fat.

Microbiological analysis and shelf life

Sausages showed a count of < 3 CFU/g during 17 days of evaluation for total coliforms. However, from this point of the analysis, the presence of this bacteria (9.2 CFU/g in BET6 and 6.4 CFU/g in SJT2) was detected. On the control sample, there was no coliform presence for 24 days. The results established that although adequate hygienic conditions were considered, sausages began to develop more rapidly at the end of the storage, inferring that this evaluation point defined the end of the lag phase. On the other hand, E. coli counts showed the absence of this microorganism during 24 days of chilling storage. The Ecuadorian Technical Standard NTE 1338:2012 establishes that the count of E. coli for cooked sausages shall be < 10 CFU/g. Therefore, tuna sausages stored for 24 days in refrigerated storage comply with the requirements. The results of E. coli were similar to those reported in sausages from Nile tilapia carcasses (Oreochromis niloticus) ⁴². The absence of Staphylococcus aureus during 24 days of tuna storage and control sausages was observed when refrigerated. The Ecuadorian Technical Standard NTE 1338:2012 allows up to 1,0x10³ CFU/g of Staphylococcus aureus in cooked sausages. The results show that the sausages comply with hygienic procedures; the packaging and the refrigeration storage temperatures conserved and maintained the quality. The results of S. aureus were similar to those reported in sausages made from Nile tilapia carcasses (Oreochromis niloticus), which do not evidence the presence of Staphylococcus aureus⁴². Also, in sausages produced with the meat of Clarias lazera, the Staphylococcus aureus counts during 30 days of storage at 5°C were imperceptible ⁵². Mesophilic aerobic bacteria count showed absence in tuna sausages, and in the control sample, there were 5 CFU/g at 24 days of storage. These results indicated the excellent quality of the tuna meat. The Ecuadorian Technical Standard, NTE 1338:2012 specifications for cooked sausages, recommend that these bacteria in food intended for human consumption do not exceed 5.0 *10⁵ CFU/g. Studies of sausages produced with Indian sardines (Sardinella longiceps) found similar results for mesophilic aerobic bacteria ⁵⁸. The microbiological results showed that the control of the tuna post-capture microbial load could be minimal to generate products of high quality from marine raw materials.

Color

The results of the color parameters of sausages are shown in Table 4. Lightness (L*) does not show significant differences (P>0.05) between the two types of tuna sausages, but they differed from the control. Lightness in control declined, being slightly darker and colorless. The redness (a*) was similar for all samples during the evaluation, which could be attributed to the red color added in all formulations. Regarding the yellowness (b*), there were no differences. However, the control showed a slightly higher value, while the BET6 and SJT2 exhibited the lowest. The Chroma and Hue do not show differences (P>0.05) between the treatments. The scarce difference in coloration among the different batches of sausages may be due to color differences in the mass formed by the emulsified muscle protein and the natural color added to the dough. The color values of tuna sausages reported in this study were similar to those reported in Frankfurter sausages with different tuna levels ⁴⁷. The values reported in sausages with added surimi, fat, and water content were not significantly different (P>0.05) regardless of the addition of fish meat protein or fat.

Sensory analysis

The sensory analysis results were performed to establish the normality in dates, and the results showed, as was expected, no normal distribution. In this sense, dates were analyzed using the Friedman test to establish differences. The sensory results showed differences between tuna sausages and the control sample. The sensory attributes and acceptability results are shown in Figures 2A and 2B, respectively. The color of the sausages prepared with tuna meat was scored 4, "liked moderately"; also, the control sample was scored similarly. The acceptability index (AI) for the color parameter of the tuna sausages was 80%. Values above 70% indicate that the judges accept the product ⁴⁰. The acceptability in the color parameter of this type of product is interesting because it is not a standard product in Ecuador. The judges could not compare it with the color of commercial fish sausage; they could only compare it with the control sample. The color acceptability of the sausages formulated in the present work was similar to Frankfurters with combined pork meat and yellowfin tuna ⁴⁷⁾ and sausages from marine catfish (Sciades herzbergii) ⁵¹.

The smell of sausages received a rating equivalent to "liked moderately." This result could be caused by the fact that spices were added to the dough commonly used in the production of sausages and contributed to developing a good smell in the final product. The result of "liked moderately" on the acceptability of the smell of the sausages was surprising because the premise with new products, especially with fish, is that consumers reject the new product. The rejection by the consumer could be expected because the smell of marine fish is usually powerful; in these species, more nitrogen components are not concentrated, which are low molecular weight volatile compounds ⁴⁴. The tuna sausages smell was almost similarly appreciated to sausages from bull eye fish (Priacanthus hamrur) ³⁸⁾ and sausage from marine catfish (Sciades herzbergii) ⁵¹. The texture of the tuna sausages produced in the present work was scored as 4," liked moderately," and categorized as "soft moderately." The result in the hardness of texture of the tuna sausage was satisfactory because all procedures, raw materials, and conditions were adequate according to the standards for sensory evaluation. Regarding flavor and overall acceptability, the SJT2 sample scored the highest compared to BET6 and the control sample; the panelist qualification was "liked very much." This result is significant because the tuna meat used to produce tuna sausages comes from the head and tails of commercial tuna, which habitually needs to be more utilized.

Figure 2 A) Sensory evaluation; B) Acceptability index. Control (sausage with beef, pork, and pork back-fat), BET6 (sausage with Big Eye tuna meat and pork back-fat), SJT2 (sausage with Skip Jack and pork back-fat). Different letters in AI (a. b) of each parameter indicate significant differences between samples (P < 0.05).