Simultaneous Identification and Evaluation of Amino Acid Profiles of the Male and Female Innards of Neopetrolisthes maculatus

. This article reports the amino acid profiles of the innards of the male and female dry samples of Neopetrolisthes maculatus collected from the Atlantic Ocean at Orimedu beach in Ibeju-Lekki, Lagos, Nigeria. The analytical results showed high values of amino acids being observed in both heterosexual samples (g 100g -1 protein): 8.17-8.32 (Leu), 8.35-10.3 (Asp), 17.6-18.2 (Glu) and 7.76-9.55 (Arg) with total amino acid values being greater in female innards (97.6g100g -1 ) than the male innards (95.5g 100g -1 ). These quality parameters were instructive of the quality of the amino acids in the innards of N. maculatus : P-PER 1 , (2.83-3.01), P-PER 2 (2.89-2.96), EAAI (88.7-89.0), BV (85.0-85.5), Lys/Trp (L/T) (3.00-5.01), Met/Trp (M/T) (1.78-3.50) and Phe/Tyr (1.04 - 1.65). The pI values were close at 5.46-5.57. In the amino acid groups (classes), the following trend was observed: class I > IV >V > VI > II > III > VII. For the amino acid scores: serine (0.487-0.511) was limiting in both samples on the total hen’s egg scoring pattern; in provisional scoring pattern, Lys was limiting in both samples with values of 0.820-0.889 and in the pre-school amino acid requirements, Lys was also limiting at 0.778-0.843. In the statistical analyses total amino acid profiles as well as egg scores were significantly different between the two samples whereas quality scores in pre-school amino acid requirements and provisional amino acid scoring pattern were both not significantly different between the two samples all at r =0.01 . Among the EAAs, six out of nine (66.7%) were more concentrated in the male innards and three of nine (33.3%) were more concentrated in the female. Thus the overall summary showed the male innards amino acids were of better quality than in the female as shown: male innards EEA = 46.1 g100g -1 and 46.0 g100g -1 in the female with corresponding TNEAA of 49.3g 100g -1 and 51.6g 100g -1 respectively. cow (whole, nonfat, evaporated or dry), EAAI (88) and BV (84, predicted; 90, observed), human, EAAI (87) and BV (83); eggs, chicken (whole, raw or dried), EAAI (100), BV (97, predicted; 96, observed), whites (raw or dried), EAAI (95), BV (92, predicted; 93 observed); yolks (raw or dried), EAAI (93), BV (89, predicted); shellfish (shrimp, including prawns, raw or canned), EAAI (67), BV (61, predicted). These literature results show the quality position of N. maculatus innards under discussion. EAAI is useful as a rapid tool in the evaluation of food formulation for protein The isoelectric point, pI , was 5.46-5.57 which showed the samples to be in the acidic medium of range. pI calculation from amino acids would assist in quick production of certain isolate of organic product without evaluating the protein solubility to get to the


Introduction
Crab is often recommended for pregnant women as it is consumed by many individuals. Crab innards (insides) have a hepatopancreas which is a part of the crab's digestive system. The colour of the organ is often yellow and it is similar to the deep yellow colour usually found in high-vitamin butter produced from cows grazing on rapidly growing grass. This yellow fatty acid organ has been given a common term known as crab "butter" or "mustard". Due to its colour, this part of the viscera is expected to be rich in fat-soluble activators [1]. Many indigenous groups that understood the necessity for special foods prior to conception, during pregnancy and during lactation; crab used to be one of these foods.
Crab insides have been used in a variety of ways. In Japanese cuisine, one dish with crab organs involves a blended mixture of the viscera, served in the skull of the crab with a raw egg on its top. The natives of Fiji also were aware that a particular specie of spider crab fed to mothers during and prior to pregnancy would produce children "physically excellent and bright mentally".
Special foods of the sea were eaten "day to day" during the time of pregnancy. There is then a message from many wise traditions around the planet: eat crabs during the period of preconception, pregnancy and lactation -and eat the whole crab [1]. The Chinese mitten crabs Eriocheir sinensis had been described to have a delicious taste and unique pleasant aroma, and has high nutritional value [2]. The meat, hepatopancreas and gonads are all edible parts of the Chinese mitten crab. Whilst consumers in western societies often choose to eat the meat alone, Asiatic consumers greatly prefer consuming the hepatopancreas and gonads. Indeed, this contributes to the popularity of mitten crabs as a delicacy in China with the hepatopancreas of males being especially prized, followed by the gonads of both male and female crabs [3].
Porcellanidae family is a group of crab-shaped anomuran crustaceans that belong to the superfamily Galatheoidea together with three other families Galatheidae, Munididae and Munidopsidae [4]. They are commonly found in rocky and coral reefs of temperate and tropical coasts.
The World Register of Marine Species (WoRMS) has given the taxonomic details of Neopetrolisthes maculatus (H. Milne Edwards, 1837) [5]. Further details of the taxonomy classification of N. maculatus had been given in Adeyeye [6]. The distribution of porcellanids had been enumerated [6,7]. Porcellanids places of abode had also been enumerated as they are typically found in a heterosexual pair [8].
Neopetrolisthes maculatus is a spotted crab. Two different colour forms are known, although the ground colour of the bodies of both form is white. In one form, carapace and chelipeds are white, with an uneven pattern of irregular sizes of red blotches; ambulatory legs also white, with some small red spots on meri of the first pair (second pereopod). In the other form, the carapace and chelipeds have a uniform pattern of numerous small, reddish purple spots; meri of ambulatory legs also with numerous small, reddish spots [9].
There is paucity of information on the nutritional data for the organs of N. maculatus. However, literature information can be found in recent works of Adeyeye [6,10]. Whilst reference [6] was on the amino acid profiles of the flesh of the heterosexual pairs of N. maculatus, reference [10] was on the chemical composition (proximate, minerals, vitamins), mineral ratios and mineral safety index of the innards of male and female Neopetrolisthes maculatus. The work reported in this article is an attempt to evaluate simultaneously the amino acid profiles of the viscera of the male and female samples of N. maculatus and also appraise critically the quality superiority of the samples over each other. The colour pattern of the samples was of large and uneven blotches resembling the Pacific Ocean N. maculatus [11].

Materials and Methods
Collection of samples: Samples were collected from trawler catches from the Atlantic Ocean of Orimedu beach in Ibeju-Lekki area of Lagos State, Nigeria. The experiment took place between November 2014 and June 2015. The crabs were washed with distilled water to remove adhering contaminant and transported in ice crushed containers to the laboratory for identification and preservation prior to analysis. The crabs were identified in the Department of Forestry, Wildlife and Fisheries Management of Ekiti State University, Ado-Ekiti, wrapped in aluminium foil and frozen at -4 o C for 2-3 days before analysis.
Sample treatment: More than ten matured crabs were caught with the net but three samples were used in this study. The three whole crabs were separated fresh, two were males and only one was female. For the purpose of analysis, the separated parts were the carapace and cheliped exoskeleton (to constitute the exoskeleton) the muscle from the thoracic sterna and cheliped (to constitute flesh) and the viscera of each crab. The viscera from each sex was later dried and separately blended. Drying was at 105 o C in the oven.
Extraction and analysis: Extraction and the instrumental analysis were carried out by following AOAC method [12] and Danka et al. [13].
The dried pulverized sample was made to be free of water by ensuring constant weight for a period of time in the laboratory. The sample of 10.0g was weighed into 250 ml conical flask capacity. The sample was defatted by extracting the fat content of the sample with 30ml of petroleum spirit three times with Soxhlet extractor that was equipped with thimble. The sample was hydrolyzed three times for complete hydrolysis to be achieved for the totality of amino acids recovery.
The pulverized and defatted sample was soaked with 30ml of 1M KOH solution and was incubated for 48 hours at 110 o C in hermetically closed borosilicate glass container. After alkaline hydrolysis, the hydrolysate was neutralized to get pH in the range of 2.5-5.0. The solution was purified by cation-exchange solid-phase extraction. The amino acids in purified solutions were derivatised with ethylchloroformate by the established mechanism: The derivatising reagent was removed by scavenging with nitrogen. The derivatized amino acid was made up to 1ml in a vial for gas chromatography analysis. The gas chromatographic conditions for the amino acids analysis were as follows: GC: HP6890 powered with HP ChemStation rev. A09.01[1206] software; injection temperature: split injection; split ratio: 20:1; carrier gas: hydrogen; flow rate: 1.0ml/min; inlet temperature: 250 o C; column type: EZ; column dimensions: 10m x 0.2mm x 0.25 µm; oven programme: initial @110 o C, first ramp @ 27 o C/min to 320 o C, second, constant for 5 mins at 320 o C; detector: PFPD; detector temperature: 320 o C; hydrogen pressure: 20 psi; compressed air: 35 psi.
Some calculations were made from the analytical data results: Estimation of isoelectric point (pI): The estimation of the isoelectric point (pI) for a mixture of amino acids can be carried out by the equation of the form [14]: where IPm is the isoelectric point of the mixture of amino acids, IPi is the isoelectric point of the ith amino acid in the mixture and Xi is the mass or mole fraction of the ith amino acid in the mixture.
(iii) Leucine/isoleucine ratio: The leucine/isoleucine ratios, their differences and their percentage differences were calculated. (iv) Estimation of essential amino acid index (EAAI): The method of EAAI calculation due to Oser [16] using the egg protein amino acids as the standard. The amino acid scores were computed using three different procedures: -Scores based on amino acid values compared with whole hen's egg amino acid profile [17]. -Scores based on essential amino acid scoring pattern [18].
-Scores based on essential amino acid suggested pattern of requirements for pre-school children [19]. (viii) The amino acid value differences and their percentage values were calculated between the male and female crab samples.

Statistical evaluation
Data results in Tables 1, 5, 6 and 7 were subjected to statistical analyses of correlation coefficient (rxy), regression coefficient (Rxy), coefficient of determination or variance (rxy 2 ), the coefficient of alienation (CA) and index of forecasting efficiency (IFE). Other calculations were grand mean, standard deviation (SD) and coefficient of variation (CV%). The rxy value was converted to critical Table value (rT) to see if significant differences existed among the two heterosexual sample results at r=0.01 [20].

Results and Discussion
The amino acid profiles of the heterosexual innards of N. maculatus have been depicted in Table  1. They were reported on dry weight basis at g 100g -1 crude protein (cp). Highest concentrated amino acids in both samples were acidic amino acids which were Glu (17.6-18.2g 100g -1 ) followed immediately by Asp (8.35-10.3 g 100g -1 ), the female sample was greater in both amino acids (AA). The two mostly concentrated EAAs in both samples were Leu (8.17 -8.32g 100g -1 ) and Arg (7.76-9.55g 100g -1 cp). Only Trp in the male sample had value of less than 1g 100g -1 cp with a value of 9.75e-1g 100g -1 cp whereas all other AAs had appreciable values. The overall AAs for the male sample added up to 95.4g 100g -1 cp but added to 97.6g 100g -1 cp in the female. However, the total AAs in the male sample might not be a true reflection of the crude protein as the crude protein in the innards male sample was 56.8g 100g -1 whereas it was 55.5g 100g -1 in the female [10]. This observation of lower crude protein (with higher total AAs) and higher crude protein (with lower total AAs) could be due to the fact that the crude protein in the male N. maculatus innards contained less true protein compared to the female innards crude protein. The coefficient of variation (CV%) values were generally low with the lowest value being observed in Pro which had values of 3.31-3.32g 100 -1 cp, mean value of 3.31g 100g -1 (approximately), ±SD value of 0.010 and CV% of 0.295 whereas the highest CV% was observed in Trp with following information: concentration values 9.75e-1 to 1.50g 100g -1 cp, mean value of 1.24g 100g -1 cp, ±SD value of 0.373 and CV% value of 30.1. The low CV% values showed that the concentration values were close in the samples on parameter comparisons. The values discussed above were mostly in tandem with the observations made in N. maculatus flesh amino acid profiles in both the male and female samples. Thus we have: Glu was mostly concentrated (17.7-17.8g 100g -1 cp) closely followed by Asp (9.90-10.0g 100g -1 cp) both also acidic AAs, also in the EAAs, we have Arg (8.70-9.07g 100g -1 cp) and Leu (7.23-7.94g 100g -1 cp) whereas the total AA values ranged from 96.6 -97.1g 100g -1 cp [6]. Unlike in the innards, the flesh of N. maculatus has total AAs as a reflection of the crude protein values: in female flesh, crude protein was 40.2g 100g -1 (and total AAs of 96.6g 100g -1 ) whereas in the male we have 43.7g 100g -1 total AAs [6]. Also in the flesh of N. maculatus, the CV% values of the AAs were close and low with values of 0.124 (observed for Glu) -32.0 (observed for His) [6]. The observations made in the present report for Glu and Asp also corroborated with the observations in the flesh of female West African fresh water crab (Sudananatutes africanus africanus) with Glu (130.2mg g -1 crude protein) > Asp (72.5mg g -1 cp) but almost similar for the positions of Arg and Leu: Leu (66.0mg g -1 cp)> Arg (58.9mg g -1 cp) [21].
The concentration differences and their percentages in the two heterosexual samples could be seen in columns seven and eight respectively in Table 1. Out of the 19 parameters recorded, nine parameters or 9/19 (47.4%) were positive towards male innards, in order words 47.4% of the parameters were more concentrated in the male than the female. It also showed that 10 parameters or 10/19 (52.6%) were more positive towards the female innards. The relative concentration values in the innards among the sexes had opposite values in the heterosexual flesh where the female had
In Table 2, we have the statistical analysis result from the data in Table 1. Table 2 showed female values to be greater than the male in the following parameters: TAAs, mean, SD and CV% although those values in the two samples were relatively close to each other. These values were also high: rxy (0.9790 and significantly different since (rc = 0.9790> rT = 0.590 at r= 0.01), rxy 2 (0.9585), Rxy (1.06), IFE (0.7962) but low CA (0.2038). The Rxy of 1.06 meant that for every unit increase in the male AA parameter, there was a corresponding increase of 1.06g 100g -1 in the female AA parameter. The values of index of forecasting efficiency (IFE) and coefficient of alienation (CA) are always in reverse to each other. The CA is the error of prediction of relationship between two entities whereas the IFE is the value of reduction of error in predicting the relationship between two entities; the higher the value of CA, the higher the difficulty in predicting the relationship between two entities whereas the higher the IFE, the easier the prediction. In these results, since the IFE > CA, it meant that the biochemical functions of the male innards could as well be performed easily by the female innards and vice versa. Table 3 depicted the summary of the concentrations of essential, aromatic, nonessential, neutral, etc. of the amino acid levels in the samples in g 100g -1 . The total AAs values ranged from 95.4-97.6 g100g -1 with the female taking the higher concentration value. These values were close to the values of AAs in heterosexual flesh of N. maculatus having range values of 96.6-97.1 g100g -1 [6], but higher than the total AAs in the flesh of S. africanus africanus (777.0 mg g -1 cp) [21]. The EAA range was 46.0-46.1 g100g -1 and CV% of 47.1-48.4 respectively; note however that the EAAs in the male were higher than those of the female indicating that AAs in the male N. maculatus innards would be of higher nutritional quality than in the female. This observation had been made in China with the hepatopancreas of males being especially prized, followed by the gonads of both male and female crabs [3]. The total sulphur amino acids (TSAA) of the samples were 4.00-4.66g100g -1 which were highly comparable to the values in flesh (4.04-4.85g100g -1 cp) and also close International Letters of Natural Sciences Vol. 75 to the standard value of 58mg g -1 cp recommended for infants [19]. The total aromatic amino acid values range suggested for ideal protein (68-118mg g -1 cp) [19] were found to be lower than in the present results of 11.7-11.8 g100g -1 cp making the samples to be very good sources of ArAA and the samples might also be qualified as supplements to foods of lower ArAA values. The values of 11.7-11.8g 100g -1 ArAA were higher than in the flesh of N. maculatus where values of 7.72-9.67g100g -1 cp were reported [6]. The percentage ratio of EAAs to the TNEAA of 46.0-46.1 were above the 39% considered adequate for an ideal food for infants, 26% for children and 11% for adults [19]. The EAA/TAA in eggs is 50.0% [22].
In Table 3, we have two predicted protein efficiency ratio types (P-PER1 and P-PER2). Values range in P-PER1 were 2.83-3.01 and 2.89-2.96 in P-PER2. These values were lower than in the N. maculatus flesh where P-PER1 range was 3.39-3.69 and P-PER2 range was 3.82-4.14. The in vivo P-PER is in the order of 2.2 [23]; all the present P-PER values were greater than 2.2 attesting to the nutritional quality of the innards. Literature information had the following P-PER values: in the flesh of female S. africanus africanus, P-PER was 3.1 [21]; in Callinectes latimanus (a lagoon crab), P-PER1 was 1.21 and P-PER2 was 1.39 [24]. These literature values showed that N. maculatus might be more physiologically utilized protein source than some of quoted references. In general, it has been found that the better the protein, the lower the level in the diet required to produce the highest protein efficiency ratio. This is a clear reflection of the importance of the proper nutritive balance of all the amino acids to produce optimum metabolic efficiency. The Leu/Ile values ranged from 1.54-1.91 with the difference levels of 2.87-3.97 g100g -1 and % (Leu-Ile)/Leu values of 35.1-47.7. In the flesh of N. maculatus Leu/Ile ratio had values of 1.60-1.63 [6] and in the flesh of S. africanus africanus, the ratio was 1.60 [21]. In literature, the most ideal Leu/Ile is 2.36 [25]. The values of 1.54-1.91 showed that we might not experience concentration antagonism in the samples when consumed as protein source in food. It has been suggested that an amino acid imbalance from excess Leu might be a factor in the development of pellagra [26]. A high Leu imbalance in the diet impairs the metabolism of Trp and niacin, and is responsible for the niacin deficiency in sorghum eaters [27]. Experiments in dogs showed that animals fed sorghum proteins with less than 11g100g -1 protein Leu did not suffer from nicotinic acid deficiency [28]. The present Leu values of 8.17-8.32 g100g -1 were less than 11 g100g -1 protein and therefore considered safe and could be beneficially exploited to prevent pellagra in endemic areas [29]. The percentage Cys/TSAA values were 26.7-33.0 close to the values of 31.9-33.1% in the meat of N. maculatus [6].  [32]. The percentage Cys in TSAA in the diet of the rat, chick and pig is 50% [25] but the value is unknown in man [19]. It is however interesting to note that vegetable protein (e.g. coconut endosperm) has a percentage Cys/TSAA of 62.8% [33]. High percentage Cys/TSAA had also been reported in Anacardium occidentale with a value of 50.51% [34]. The % Cys/TSAA in N. maculatus showed that the crab behaved like a typical animal protein [34]. The presence of cystine and cysteine in the diet would reduce the needs for Met and since all the sulphur in the diet is derived from these three amino acids the sulphur content is sometimes used as an approximate assessment of the adequacy of protein [35]. In the present results, the values range for Met and Cys were respectively 2.68-3.42g 100g -1 and 1.24-1.32g 100g -1 . The  [16]: milk, cow (whole, nonfat, evaporated or dry), EAAI (88) and BV (84, predicted; 90, observed), human, EAAI (87) and BV (83); eggs, chicken (whole, raw or dried), EAAI (100), BV (97, predicted; 96, observed), whites (raw or dried), EAAI (95), BV (92, predicted; 93 observed); yolks (raw or dried), EAAI (93), BV (89, predicted); shellfish (shrimp, including prawns, raw or canned), EAAI (67), BV (61, predicted). These literature results show the quality position of N. maculatus innards under discussion. EAAI is useful as a rapid tool in the evaluation of food formulation for protein quality. The isoelectric point, pI, was 5.46-5.57 which showed the samples to be in the acidic medium of the pH range. The pI calculation from amino acids would assist in the quick production of certain isolate of organic product without evaluating the protein solubility to get to the pI.

ILNS Volume 75
In infants protein requirements, a growth pattern of amino acid requirements was obtained by assigning value of unity to the Trp need [36]. Similar calculation of the amino acid content of mammalian tissues showed that there exists good agreement of growth needs and tissue amino acid patterns. This agreement is good for the Lys The amino acid groupings into classes I to VII have been depicted in Table 4 [38]. The concentration trend of the classes could be seen as shown in g100g -1 protein: class I (28.3-29.3)> class IV (26.0-28.5)> class V (15.8-16.2) > class VI (11.7-11.8)> class II (7.65-7.77)> class III (4.00-4.66)> class VII (3.31-3.32). This trend was as observed in the meat of heterosexual N. maculatus [6].    Table 5 presented the total amino acid scores based on whole hen's egg amino acids profile. These amino acids were more concentrated in the male innards than the whole hen's egg values as shown by their scores: Gly (2.  [6]. Therefore, in order to fulfill the day's needs for all the amino acids in N. maculatus innards samples, 100/51.1 or 1.96 times as much male innards protein, or 100/48.7 or 2.05 times as much female innards protein would have to be consumed (eaten) when they serve as the sole protein source in the diet. Table 6 contained the essential amino acid scores of N. maculatus innards based on FAO/WHO [18] standards. The following scores were greater than 1.00 in both samples: Ile (1.09-1.33), Leu (1. 17-1.19), Met + Cys (1.14-1.33), Phe + Tyr (1.27-1.35) and total AA (1.09 -1.10) but in addition, the female innards had Trp score greater than 1.00 (1.50). In the meat of N. maculatus, all the above AAs including Trp had their scores greater 1.00 each. In both samples Lys was LAA with male value being 0.889 and female value being 0.820. The correction value in the male innard protein was 100/88.9 or 1.12 whilst the correction value in the female was 100/82.0 or 1.22. In the N. maculatus, meat, Lys (0.804) was limiting in the female whereas Val (0.823) was limiting in the male. In Table 7 we have depicted the essential amino acid scores of the innards of N. maculatus, samples based on requirements of preschool child (2-5y). Whilst two scores (Lys = 0.843; Trp = 0.887) were less than 1.00 each in the male innards protein, only Lys had score less than 1.00 in the female innards having score value of 0.778. Hence, Lys was the LAA in both samples and their respective correction values were 100/84.3 or 1.19 and 100/77.8 or 1.29. Results on scores from Tables 5, 6 and 7 were subjected to statistical analyses. In the egg's score comparison, both rxy and rxy 2 were high whereas IFE value was just above 50.0%. The Rxy, mean (for both samples), their SD values were each being less than 1.00. The CV% values were slightly low and close to each other, values being 36.4 -38.9. The rc = 0.8772 > rT = 0.798 at r=0.01 making the rxy being significantly different in the two samples on eggs score basis. Also, the CA (0.480) < IFE (0.520) making the prediction of biochemical relationships easy. For the pre-school AA requirements scores and provisional EAA scoring pattern scores, their rc = 0.6055 < rT = 0.798 (preschool scores) and rc = 0.4547 < rT = 0.798 (EAA scoring pattern) at r= 0.01 showed that no significant differences occurred in the two samples for the two scores. Also, prediction of relationship would also be difficult among the two samples since CA>IFE in both types of scores: CA (0.796) >IFE (0.204) (pre-school child scores) and CA (0.891) > IFE (0.109) (provisional scoring pattern).  The summary of the amino acid profiles into Factors A and B could be seen in Table 9. Factor A means constituted amino acids of the two samples along the vertical axis whilst Factor B means constituted the amino acids values along the horizontal axis as shown in the Table: both containing the essential and non-essential amino acids. It would be observed that the mean of Factor A means and Factor B means gave a value of 48.3g 100g -1 protein. It is interesting to note that the Factors A and B means gave a value of 48.4 in the meat of N. maculatus heterosexual samples [6]. Egg score is significantly different at n-2 and r = 0.01 (critical value = 0.575); pre-school child score is not significantly different at n-2 and r = 0.01(critical value = 0.765); provisional score is not significantly different at n-2 and r = 0.01 (critical value = 0.798).

Conclusions
Neopetrolisthes maculatus innards samples in the male and female were good sources of high quality amino acids with the female total amino acids being more in value than the total male amino acids. In quality parameters, P-PER1, and P-PER2 were high; both EAAI and BV values were high; International Letters of Natural Sciences Vol. 75