Development of quality index method for eviscerated and non-eviscerated octopus ( Octopus insularis ) 1

This work aimed to develop a Quality Index Method (QIM) for octopus species Octopus insularis and apply it to samples of eviscerated and non-eviscerated specimens in order to establish stability in cold storage. Initially we carried out the recruitment of judges, who were trained to verify the changes undergone by the octopus during storage under refrigeration. The development of QIM Table took place in three experiments. At first, a QIM Table for Octopus vulgaris from literature was presented to judges together with specimens of O. insularis, so they could assess whether the parameters were suited to this specie. In the second experiment the objective was to validate the table, verifying if changes were needed in the predefined parameters or if it was also necessary to add new ones. Finally, the QIM Table was applied to determine the shelf life of non-eviscerated (NEV) and eviscerated (EV) octopuses (O. insularis) during refrigerated storage (2 ± 2 °C). The sum of demerit points for all parameters was named Quality Index (QI). To determine the value of QI at rejection point, N-TVB and N-TMA contents were determined. The data were submitted to analysis of variance and Tukey test at 5% significance, besides correlation and regression analysis by the GLM routine. The developed Quality Index Method for Octopus insularis consists of 8 parameters, totalizing 24 demerit points. The NEV samples were rejected at the tenth day of storage, while the rejection of EV samples occurred only at the end of the experiment.


INTRODUCTION
In Brazil, two octopus species have commercial interest: Octopus vulgaris, in the southern region and O. insularis (LEITE et al., 2008) in the Northeast and North regions (LEITE et al., 2009).O. insularis is the main target of octopus fisheries in northeastern Brazil, where several hundred tons are fished each year, mainly in Ceará fishing with long line pots (BRAGA et al., 2007).
In these states, the octopus catch of the species O. insularis has great prospects for growth due to the vast coastline and constant tourism that demands exotic and healthy dishes.The species O. insularis has a high water content and a low lipid content, being considered a very healthy meat (MACHADO, 2011;SILVA, 2009).Along with this growth comes the need to expand the trading of that product, even more by putting it in distant markets from the fishing sites to meet a growing demand from consumers in a convenient and secure way.
According to Amaral and Freitas (2013) food quality and safety are issues of great relevance, especially in the international scenario.In minimalist terms, it can be said that quality refers to the characteristics that make food acceptable to consumers.Sensory evaluation is the most commonly used method for evaluating the quality of fresh fish and has always been considered as the main way to evaluate fish freshness of the fish (BOGDANOVIC et al., 2012).
Several researchers and institutes of the fishing industry have developed methods to evaluate fish freshness based on post-mortem changes associated with sensory, physicochemical and microbiological changes (LI; LI; HU, 2013; OCAÑO-HIGUERA et al., 2011).Appearance, odor, taste and texture are the main sensory attributes used to evaluate the fish freshness (ÁLVAREZ et al., 2008;HERNÁNDEZ et al., 2009).
In recent years, great progress has been made in assessing the freshness of fish and consequently in the marketing and quality assurance of fresh fish, mainly at the international level.The scheme known as the Quality Index Method (QIM) is considered a promising method in the evaluation of fish freshness in a fast and objective way (AMARAL; FREITAS, 2013).Barbosa and Vaz-Pires (2004) established a demerit scoring scheme of 11 items for fresh common octopus (Octopus vulgaris) ranging from 0 to 1 or from 0 to 2, depending on the attribute, totaling a maximum of 16 points.However, the QIM should be developed for each species under study, since different species have particular characteristics (ESTEVES;ANÍBAL, 2007).
Therefore, the objective of this study was to develop the Quality Index Method (QIM) for octopus of the species Octopus insularis and apply it to samples of eviscerated and non-eviscerated specimens, in order to establish its stability in cold storage.

Material
A total of 72 specimens of the specie Octopus insularis weighing 550 + 100 g, were obtained from extractive fishing held with long line pots at Itarema municipality, Ceará, during the months of November 2012, and January and April 2013.After capturing, the fishermen slaughtered the octopuses immediately, by immersion in a NaCl 15% solution.They were then immersed in fresh water to remove salt excess and then transported in inboxes with ice until the Meat and Fish Processing Laboratory of Food Engineering Department of the Federal University of Ceará.After weighing and washing, the samples were separated into two groups.In the first group, the octopuses were kept whole, non-eviscerated (NEV) and in the second, they were eviscerated (EV).All fisheries were placed in nylon/polyethylene packages.The samples were stored in a cooler (Electrolux, H500) at 2 + 2 °C until rejection by physicochemical or sensory indicators.

QIM development
The development of Quality Index Method (QIM) for O. insularis species was based on the QIM for octopus O. vulgaris, proposed by Barbosa and Vaz-Pires (2004).Judges were recruited by means of questions about age, gender, education level, consumption habits and interest in participating.Three women and two men were selected to composse the sensory team, being 2 individuals over the age of 50 years, 2 in the age group of 30-50 and only 1 with less than 30 years old.All judges had higher education level.Only 2 individuals had the habit of consuming octopus, because this food is not a common dish in Ceará diet.On the first experiment sessions were conducted in groups, when 6 octopus, 3 non-eviscerated (NEV) and 3 eviscerated (EV) were placed in white Teflon boards and displayed in a stainless steel table, so that all judges could see them.The judges were asked to verify the changes undergone by the octopus O. insularis under the refrigeration storage and at the same time establish parameters for the construction of the new Table QIM.It was also asked to observe if the parameters shown in the QIM Table proposed in literature for Octopus vulgaris (BARBOSA; VAZ-PIRES, 2004) was suited for the species under study.Parameters that were inadequate for O. insularis were discarded or new standards and levels were established, and thus, a new table QIM was proposed.That first step was carried out in about half an hour each session, in which were explained some biological terms of the species, not known by the judges.At that stage, the specimens were evaluated every 2 days, during 20 days, in a total of ten sessions.The octopuses were removed from the refrigerator for sensory session and immediately after the session were returned to the refrigerator to be used at the next session.
The second experiment had another 6 animals, 3 NEV and 3 EV, each animal being one replicate.After this stage, the analysis was performed individually, it means, a judge at a time.The specimens were displayed in white Teflon boards and coded with random numbers with three digits.The QIM Table constructed in the first step was given to the judges to evaluate three pairs of samples, following a balanced design (AB and BA).The objective was to test the table, verifying if changes were needed in the predefined parameters or if it was necessary to add new ones.The same samples were evaluated every 3 days for 18 days in a total of 7 sessions.As in the first experiment, the octopuses were removed from the refrigerator for sensory session and soon after were returned for use in the next session.Suggestions made by the judges were evaluated by the team and when considered necessary, were included in the Table .In the third experiment were used 30 octopuses in each of two repetitions (EV 15 and 15 NEV), all stored under the same conditions (2 ± 2 °C).In each session we analyzed 3 NEV and 3 EV, which were discarded after evaluation, due to manipulation of the judges of the samples, a possible source of contamination.In the next session, 6 more octopus were evaluated (3 NEV and 3 EV), and so on.It was performed one session every 5 days during 20 days of storage.

Statistical analysis
The data were submitted to analysis of variance (ANOVA) and Tukey test to compare means at 5% level of significance.Correlation analysis and linear regression by GLM routine were also performed.All analyses were carried out using the statistical software SAS® "Statistical Analytical Systems" version 9.2 (SAS INSTITUTE, 2009) for Windows.

Development of the Quality Index Method (QIM) Table for Octopus insularis
The initial version of Table QIM, built to evaluate the freshness of octopus species Octopus insularis, consisted of seven quality parameters with demerit points ranging from 0 to 3, namely: "external color", "color of mouth region", "skin elasticity ","odor", "mucus", "mantle texture" and "eyes".The sum of points of all parameters was named Quality Index (QI), ranging from 0-21, calculated as an average of the sensory team.At this stage, we perceived that the judges had difficulty in defining the parameter "external region color," because they were in doubt as to which region it referred: the dorse or the abdomen (venter).This parameter was then replaced by two other parameters: "color of the dorsal region" (Figure 1A) and "color of the ventral region" (Figure 1B), making a total of eight parameters to be evaluated in the final QIM (Table 1).So, the maximum Quality Index (QI) rose from 21 to 24 demerit points.
In Table 1, fresh octopus was described with the following characteristics: light gray or predominant light brown in the dorsal region, predominantly white on the ventral region, ivory color with light rose spots in the mouth area, elastic skin, sea and seaweed odor, aqueous mucus in small amount, flexible flesh (mantle) to the touch and eyes with translucent cornea and black pupil or dark red, with little turbidity.During the storage, the appearance, odor and texture characteristics suffered changes in different extensions, and, according to the sensory team, reached the maximum conditions of deterioration after 18 days of storage: dorsal region of gray/brown color with strong rose, ventral region of rose color, mouth region of intense rose color, skin without elasticity, intense sour or rotten odor, no mucus, rigid mantle, and opaque cornea and opaque dark red pupil, normally bloodstained.

Determination of octopus O. insularis stability stored under refrigeration by the Quality Index Method (QIM)
Most of parameters showed high positive correlation with the storage time, for both non-eviscerated  (NEV) and eviscerated octopus (EV) (Tables 2 and  3). Figure 2 illustrates the evolution of demerit points (average of sensory team on both repetitions) of each parameter during the 20 days of cold storage.In Figure 2A is possible to observe that for NEV sample, the score for "color of the dorsal region" increased already on the 5 th day, and continued increasing until the 10th day, showing a dark gray /dark brown colored body, level reached by the eviscerated sample only on the 15 th.At the end of storage (20 th day), both samples showed the same score, around 1,5 demerit points.
For the "ventral color" (Figure 2B), the samples differ from each other in 10 th and 15 th day, with the NEV reaching the highest score.However, by the end of storage time, both samples showed the same score, 2,0 demerit points, corresponding to slightly rose.
In the parameter "color of the mouth region" (Figure 2C) it was observed outlier values for a few judges, which influenced the means of demerit points on the 15 th day to NEV sample, but it was still possible to establish a significant linear correlation also for this sample.Therefore, we can infer that, the mouth region of the both samples (NEV and EV) changed to ivory with slightly rose spots (between 1,0 and 2,0 demerit points) at the end of storage.Analyzing these three parameters together, we observe that the octopus non-eviscerated  For the parameter "skin elasticity" (Figure 2D), "odor" (2E) and "eyes" (2H), the rate of evolution of demerit points showed the same pattern for the two samples, increasing faster in the first storage period (10 days) and then more slowly until the 20 th day of storage, but never exceeding 2,0 demerit points (on average).So, it was observed a correlation among these parameters, it means, changes in skin elasticity are accompanied by changes in odor and appearance of the animal's cornea and pupils.The skin loses elasticity and eyes lose transparency, together with development of off odors to the product, all at the same speed.Similar results were observed by Barbosa and Vaz-Pires (2004) in Octopus vulgaris, which odor, skin color and skin elasticity, besides the material of the octopus suckers, were the main parameters that determine the rejection of the product, the time that the odor was metallic and grassy, the skin rose and saggy.

Parameters
The parameters "mucus" and "mantle firmness" did not differ between samples, however, while the "mucus" presented unchanged, viscous, slightly cloudy and in a small amount (1,5 to 2,0 points) from the beginning to the end of the storage (20 days), the "mantle firmness" went from flexible (0,0 point) at the beginning to low flexible (1,0 point) on the 5 th day, and has not increased until the end of storage.
Figure 3 shows the average results of Quality Index (total demerit points QIM) adjusted by means of a linear regression analysis, which resulted in R 2 of 0,92 and 0,99 for the non-eviscerated octopus (NEV) and eviscerated (EV), respectively.Both samples were evaluated by QIM, obtaining 4 demerit points on the first day of storage due to small, but already noticeable changes in all evaluated parameters.It was observed in this stability experiment that samples reached 12 demerit points at the end of the cold storage time (20 days), unlike the initial experiment of Table QIM development when, after 18 days, under the same conditions, the octopus hit the maximum score (24 demerit points).This happened because in the first experiment, we always performed the evaluations with the same samples that were manipulated by the sensory team and returned to the cold room.Even with the use of gloves, this manipulation was a source of contamination, which caused a more rapid deterioration of the samples.When we decided to dispose of the animals and replace them with others that were in the cold chamber under the same conditions, it was obtained more realistic situation storage, where octopuses are kept without any kind of manipulation.
It is also noted that the non-eviscerated octopuses showed slightly higher values than the eviscerated ones, at all analyzed times, indicating that its deterioration was more rapid.Barbosa and Vaz-Pires (2004) observed that non-eviscerated O. vulgaris specimens reached maximum QIM (16 points) in just 8 days of refrigeration, time considered as the product rejection moment.Already for Eledone moschata octopus kept under refrigeration, rejection was detected when the overall sensory score using the European Union (EU) freshness classification system reached 5,5 on the 10 th day of storage (LOUGOVOIS et al., 2007).

Physico-chemical analysis
The physico-chemical analyzes were performed to verify if by the time of sensorial rejection, the octopus The initial N-TMA content in O. vulgaris octopus samples stored under refrigeration, reported by Atrea et al. (2009) was 1,3 mg N / 100 g, being this value lower than those found in this study.However, on the 9 th day of storage, the samples of these authors reached an N-TMA content of 30 mg N / 100 g.It was possible to establish a significant correlation between the demerit points QIM with N-TVB values (α = 0,05) and N-TMA (α = 0,15) for non-eviscerated octopus, but there wasn't significant correlation between the QIM and the physico-chemical parameters for EV samples.It means that while the sensory quality was already compromised, chemical indicators didn't accuse deterioration to the eviscerated octopus.

CONCLUSIONS
The QIM developed for octopus non-eviscerated and eviscerated Octopus insularis consists of 8 parameters with scores from 0 to 3, making a total of 24 demerit points.The QIM  was out of the physico-chemical standards required by law.In Brazil, there are no maximum limits for volatile basis values (N-TVB) and trimethylamine (N-TMA) for cephalopods, but the Agriculture Defense Secretary of the Ministry of Agriculture, Livestock and Supply (BRASIL, 1997) establishes the value of 30 mg/100 g as a maximum value of N-TVB and 4 mg/100 g of N-TMA as limit for fishery, except elasmobranches.Thus, these values were adopted as limit values to indicate the state of freshness of the octopus in this study.
The initial values of N-TVB were around 13 mg N-TVB/100 g, indicating that the samples were in good quality at the beginning of the experiment, according to Ogawa and Maia (1999).By the twentieth day, any of the samples reached the maximum limit of 30 mg/100 g, even though there was a tendency to increasing values, as shown in Figure 4.
According to Vaz-Pires et al. (2008), the N-TVB analysis has been useful as an indicator of deterioration, Figure 3 -Quality Index evolution of octopus non-eviscerated (NEV) and eviscerated (EV) O. insularis during refrigerated storage, determined by the QIM method with 24 demerit points NEV = non-eviscerated; EV = eviscerated having low use as a freshness index.This justifies the fact that the samples from the present study did not reach the established maximum limit, but were rejected sensorially.
As far as N-TMA, on the first day of analysis it was recorded values close to 2,30 mg N-TMA/100 g for both samples, which showed similar behavior until the 10 th day of the experiment.The two samples diverged from the 15 th day of storage, the N-TMA contents to EV octopus remained unchanged until the end of the experiment, while the NEV octopus reached 7,70 mg N-TMA/100 g on the 15t h day, exceeding the limit set by law, and continued to increase until the end of the experiment (Figure 5).
to assess the stability of non-eviscerated and eviscerated specimens stored under refrigeration.The rejection of non-eviscerated samples occurred around the fifteenth day of storage when they reached around 10 demerit points.For the eviscerated sample rejection occurred on the twentieth day of storage, where the samples reached about 11 demerit points, with physico-chemical parameters still within the laws' limits.

Figure 2 -
Figure 2 -Graphical representation of mean QIM values (demerit points) of each freshness quality parameter for non-eviscerated (NEV) and eviscerated (EV) octopus O. insularis stored under refrigeration by 20 days

Figure 4 -Figure 5 -
Figure 4 -Evolution of N-TVB contents during storage of octopus O. insularis

The protocols of sensory tests were previously approved by the Ethics in Research Committee
of the State University of Ceará, No.147,279.

Table 1 -
QIM Table developed for Octopus insularis M. F. Aragão et al.

Table 2 -
Mean QIM values (demerit points)of each freshness quality parameter for non-eviscerated (NEV) octopus O. insularis stored under refrigeration by 20 days and its correlation with storage time *significant ns non-significant (α=0.05)

Table 3 -
Mean QIM values (demerit points)of each freshness quality parameter for eviscerated (EV) octopus O. insularis stored under refrigeration by 20 days and its correlation with storage time Table was successfully applied