1. Objective
In Chapter 2, I suggested that the improvement in gonad taste might be due to phenological changes in the free amino acid composition of cultivated S. japonica fed to sea urchins. The nitrogen content of Macrocystis pyrifera (Stephen and Hepburn 2016) and S. japonica (Fukushi 1988) varied among the sporophyte portions of the plants. The carbon and nitrogen contents of various portions of fronds of Laminaria solidungula, S.
latissima, S. longissima, S. japonica, S. japonica var. ochotensis and S. japonica var.
diabolica differ (Oishi et al. 1967; Dunton and Schell 1986; Sjøtun 1993; Henley and Dunton 1995; Li et al. 2007; 2009; Sato and Agatsuma 2016). Li et al. (2007) reported that the nitrogen and carbon content of S. japonica fronds increased as the fronds matured in summer; they were particularly high in the meristem of the basal portion compared to the apical portion, which is used for storage, in plants that persisted for a second year.
Oishi and Kunisaki (1970) reported that the free amino acid content of the basal and central portions of S. japonica fronds is high compared to that of the apical portion. These findings suggest that feeding of different portions of S. japonica fronds to sea urchins could affect gonad taste. It is possible that gonad taste would be further improved by feeding of the basal or central portions of the kelp.
In the present study, M. nudus were collected from a barren. They were reared in aquaria and fed the basal, middle or apical portions of fresh S. japonica fronds from May–
July, when the gonad quality was most efficiently improved (Chapter 2). The protein and
88
total and free amino acid content of each frond portion was also analyzed. This section aimed to (1) clarify whether the qualities of sea urchin gonads vary when the animals are fed different S. japonica frond portions and (2) verify the relationship between gonad qualities and the constituents of each frond portion.
2. Materials and methods
(1) Experimental design, rearing conditions
A total of 75 adult M. nudus (46–54mm diameter) were collected from a barren by scuba diving at depths of 2.5–3m off Nojima Island, Shizugawa Bay, Miyagi Prefecture (38°40′N, 141°30′E) on 16 May 2016. After collection, the sea urchins were placed in a cool box containing moist urethane mats immersed in seawater and transported to Onagawa Field Center, Tohoku University, in Onagawa, Miyagi Prefecture (38°26′N, 141°27′E) (the transportation time was approximately 1 h). Of 75 sea urchins collected, 45 individuals were used in the feeding experiment. In this experiment, five sea urchins were held in each of nine 10 L aquaria. The sea urchins were reared in running seawater that had been filtered twice using Myclean Filter (AF515, Tanaka Sanjiro Co., Ltd., Fukuoka, Japan) and aerated, and the water was exchanged two or three times per hour.
The seawater was pumped from offshore waters. The sea urchins were reared without food for 4 days until the start of the experiment. The feeding experiment was conducted from 20 May to 11 July 2016. I used fresh S. japonica kelp cultivated off Fudai, Iwate Prefecture (40°01′N, 141°54′E) as feed for the sea urchins. The fronds of S.
japonica were cut into five equal lengths from the basal to the apical portions (Figure 17).
The basal, middle and apical portions of the fronds were fed to sea urchins (three groups for each treatment in nine aquaria) ad libitum every 3–7 days. The aquaria used for each
89
Figure 17. Morphology of individual portions of Saccharina japonica fronds fed to Mesocentrotus nudus.
90
treatment were randomly selected. Thus, the feeding experiment was designed as three treatments of sea urchins fed the apical (AP), middle (MD) or basal portions (BS) of S.
japonica fronds. The remaining 30 sea urchins that were collected were randomly divided into six groups of five individuals (ST) at the start of the experiment; at the end of the experiment, all of the sea urchins were used for measurements and analyses of TD, BW, gonad size, development, hardness and color and free amino acid content of the gonads same as Section 1 of Chapter 2. The temperature of the seawater in the aquaria was measured every 10 min using a wireless data logger (RTR52A, T&D). The survival rate of the sea urchins during the rearing experiment was 100%.
(2) Protein and total and free amino acid content of kelp
On 21 June 2016, the midpoint of the experiment, five samples of each frond portion (approximately 200 g) of S. japonica were frozen at −30 °C. The NaOHsoluble protein content of the frond portions was analyzed by the same method in Section 1 of Chapter 1. The FAA content of the kelp samples was analyzed using the same method that was used to measure the FAA content of the gonads. For total amino acid (TAA) content analyses (total of free amino acids and amino acids in proteins and peptides), freezedried samples (5 mg) were placed in a vacuum hydrolysis tube, and 3 dL of 2mercaptoethanol and 5 mL of 6 N HCl were added. The mixture was hydrolysed at 110 °C for 24 h in vacuo. The hydrolysate was evaporated to dryness and dissolved in 5 ml of lithium citrate buffer (pH 2.2). The solution was filtered through a 0.2μm membrane filter (MillexLG, Merck Millipore, Burlington, MA, USA). Twenty microliters of the filtrate were injected into an amino acid analyzer (L8900, Hitachi HighTechnologies Corporation, Tokyo, Japan).
91
(3) Statistical analyses
The data were tested for normality (ShapiroWilk Wtest) and for homogeneity of variance (Levene's test). Data that were not normally distributed or did not show homogeneity of variance were logtransformed. Significant differences in the TD and BW of the sea urchins in each group at the start and ST were analyzed using the ttest.
Significant differences in TD and BW among the groups at the start of the experiment and differences in TD, BW, gonad index, hardness, L*, a* and b* values and in the FAA content of gonads at the end of the experiment were analyzed using nested ANOVA with R ver 3.4.0 (R Core Team, 2017) through RStudio ver 1.0.143 (Rstudio Inc). Significant differences in TD and BW of ST (n = 6) and in the protein, TAA and FAA contents of the frond portions of S. japonica were analyzed by oneway ANOVA. Tukey's multiple comparison test was performed as a post hoc test. Significant differences in TD, BW, gonad index, hardness, L*, a* and b* values and FAA contents between sea urchins at the start and end of each treatment were tested using the ttest. To evaluate the correlations among the contents of FAA in the AP, MD and BS groups, the FAA content data were analyzed by PCA using Canoco 5 (ter Braak and Šmilauer, 2012). Except for nested ANOVA and PCA, all analyses were conducted using JMP 10 (SAS Institute Inc.).
3. Results
(1) Water temperature
The water temperature varied erratically during the experiment; it decreased to its lowest level of 14.0 °C in early June and then increased sharply to its highest level (20.3 °C) at the beginning of July (Figure 18).
92
Figure 18. Daily water temperatures in aquaria.
12 13 14 15 16 17 18 19 20 21
May June July
93
(2) Protein, TAA and FAA content of kelp
The NaOHsoluble protein contents of the apical, middle and basal portions of S.
japonica fronds fed to M. nudus were 3.23 ± 0.35%, 3.23 ± 0.24% and 3.67 ± 0.12%
(mean ± SE), respectively, indicating that there was no significant difference in this parameter among the portions (df = 2, MS = 0.32, F = 0.97, p = 0.406). The TAA and FAA content of each portion of S. japonica fronds fed to M. nudus are shown in Figure 19.
There were significant differences in the content of eight TAAs and six FAAs among the portions (Table 18). The total TAA and total Glu contents of the basal portion were ca.
475 mg/100 g and 100 mg/100 g higher, respectively, than those of the apical portion. The total Ala, Gly and Val contents of the basal and middle portions were significantly higher than those of the apical portion (p < 0.05). The total Pro content of the basal portion was significantly higher than that of the other portions (p < 0.001). The total Leu and Tyr contents of the basal portions did not differ significantly from those of the other portions, while the Leu and Tyr contents of the middle portion were significantly higher than those of the apical portion (p < 0.05). The total His content of the basal portion was significantly higher than that of the apical portion (p < 0.05).
The total FAA content of the basal portion was 2.0 and 2.3 times higher than those of the apical and middle portions, respectively. Except for Asp, Glu, Ala and Gly, the content of FAAs was markedly lower than the content of TAAs. The free Glu content of the basal portion was 2.4 and 3.2 times higher than those of the apical and middle portions, respectively. The free Gly and Met contents of the apical portion were significantly higher than those of the other portions (p < 0.01).
94
Figure 19. Total and free amino acid content (mg/100 g) of the apical, middle and basal portions of Saccharina japonica fronds (mean ± S.E.). Lowercase letters indicate significant differences among the portions according to Tukey’s test (p < 0.05). Asp, aspartic acid; Glu, glutamic acid; Ala, alanine; Gly, glycine; Pro, proline; Ser, serine; Thr, threonine; Arg, arginine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, lysine; Met, methionine; Phe, phenylalanine; Tyr, tyrosine; Val, valine; Tau, taurine; Cys, cysteine;
Orn, ornithine; PSer, phosphoserine.
Asp Glu Ala 300
0
Gly Pro 250
0
Ser Thr 200
Arg His 150
Ile Leu 100
Lys 50
Phe 0
Tyr 100
Val 50
0 Tau
Cys Orn a
P-Ser a
b a a
b b
a
c
b a a b
a b a b
b a b a a a b
a b b a b b
a
a b b a
a b b a a b a b
150
b
Am ino ac id con ten t (m g/ 10 0 g)
Free amino acids Total amino acids
Apical portion Middle portion Basal portion
a
100 200 300 400 600 800 1000 1200
Tot al of a m ino ac id c ont ent (m g/100 g)
a
b b 0
Total 0 200
Met
95
Table 18. Results of ANOVA of the total and free amino acid contents (mg/100 g) of the apical, central and basal portions of Saccharina japonica fronds (N = 5).
Total amino acids Free amino acids
df MS F P df MS F P
Total Portion 2 185043 3.02 0.087 2 0.09 0.59 0.574
Error 12 61290 12 0.15
Aspartic acid Portion 2 1512.82 0.91 0.427 2 0.30 3.17 0.078
Error 12 1656.43 12 0.10
Glutamic acid Portion 2 0.07 0.90 0.431 2 0.34 0.86 0.446
Error 12 0.08 12 0.39
Alanine Portion 2 0.04 5.63 0.019 2 47.97 0.94 0.418
Error 12 0.01 12 51.07
Glycine Portion 2 0.04 5.62 0.019 2 1.38 19.23 < 0.001
Error 12 0.01 12 0.07
Proline Portion 2 0.30 48.56 < 0.001 2 0.13 0.87 0.443
Error 12 0.01 12 0.14
Serine Portion 2 0.04 3.03 0.086 2 0.01 0.13 0.882
Error 12 0.01 12 0.05
Threonine Portion 2 347.83 3.14 0.080 2 0.06 0.29 0.751
Error 12 110.77 12 0.21
Arginine Portion 2 271.54 3.72 0.055 2 0.61 5.07 0.025
Error 12 72.99 12 0.12
Histidine Treatment 2 44.93 6.07 0.015
Error 12 7.40
Isoleucine Portion 2 282.99 3.95 0.048 2 1.532 0.99 0.399
Error 12 71.68 12 1.543
Leucine Portion 2 0.04 3.92 0.049 2 0.65 5.34 0.022
Error 12 0.01 12 0.12
Lysine Portion 2 0.03 3.84 0.051 2 1.69 1.03 0.387
Error 12 0.01 12 1.64
Methionine Portion 2 18.21 0.77 0.484 2 0.09 20.43 <0.001
Error 12 23.60 12 0.004
Phenylalanine Portion 2 0.03 2.88 0.095 2 0.19 2.59 0.116
Error 12 0.01 12 0.07
Tyrosine Portion 2 158.32 4.55 0.034 2 0.05 0.40 0.680
Error 12 34.77 12 0.12
Valine Portion 2 0.04 6.47 0.012 2 0.53 1.49 0.265
Error 12 0.01 12 0.35
96
Taurine Portion 2 0.32 3.71 0.056 2 0.27 10.19 0.003
Error 12 0.09 12 0.03
Cysteine Portion 2 7.04 4.80 0.029
Error 12 1.47
Ornithine Portion 2 0.90 0.55 0.590
Error 12 1.63
Phosphoserine Portion 2 45.14 85626.74 < 0.001 2 0.05 1.21 0.332
Error 12 12 0.04
Significance levels (p < 0.05) are shown in bold type.
(Continued)
97
(3) Body and gonad size and gonad development
At the start of the experiment, there were no significant differences in TD or BW between ST and the sea urchins in the treatment groups in the aquaria (TD, df = 1, MS = 4.52, F = 1.07, p = 0.304; BW, df = 1, MS = 19.09, F = 0.32, p = 0.574) or among the sea urchins in the various treatment groups (Table 19). There were no significant differences in TD or BW among the five specimens randomly allotted to each of the six ST groups (TD, df = 5, MS = 2.75, F = 0.62, p = 0.683; BW, df = 5, MS = 81.08, F = 1.36, p = 0.273).
No significant differences in TD, BW or gonad indices were detected in the animals in the various treatment groups at the end of the experiment (Table 20). The gonad indices of sea urchins that were fed portions of S. japonica fronds were significantly higher than those of the animals in the ST group (p < 0.001) (Table 21).
The gonadal developmental stages (according to sex) of the M. nudus used in the experiment are shown in Table 22. The gonads of most sea urchins were in the growing stage, with increasing numbers of spermatocytes or early vitellogenic oocytes along the acinal wall and with NPs filling the lumen. Three individuals in the ST group and two individuals in the MD group had recovering gonads. A male individual in the BS group had premature gonads.
(4) Gonad qualities
No significant differences in gonad hardness were detected among the animals in the treatment groups (Table 22). Gonad hardness in each treatment group at the end of the experiment was significantly lower than in ST (p < 0.01) (Table 21).
A significant difference in b* value was found among treatments by nested ANOVA (Table S3), while Tukey's test showed no significant difference (Table 21). No significant
98
Table 19. Results of nested ANOVA of test diameters and body weights of Mesocentrotus nudus that received different treatments at the start of the experiment.
df MS F P
Test diameter Treatment 2 2.27 0.488 0.618 Treatment: aquarium 3 0.94 0.202 0.895 Body weight Treatment 2 9.23 0.149 0.862 Treatment: aquarium 3 33.84 0.547 0.653
99
Table 20. Results of nested ANOVA of the data for test diameter, body weight, gonad index, gonadal moisture, gonad hardness, gonad color and free amino acid content of the gonads of Mesocentrotus nudus in different treatment groups.
df MS F P
Test diameter Treatment 2 0.005 0.001 0.999 Treatment: aquarium 3 0.585 0.130 0.941 Body weight Treatment 2 5.920 0.095 0.909 Treatment: aquarium 3 15.59 0.251 0.860 Gonad index Treatment 2 0.393 0.053 0.949 Treatment: aquarium 3 11.734 1.569 0.212
Hardness Treatment 2 0.002 2.970 0.063
Treatment: aquarium 3 0.002 2.865 0.049
L* Treatment 2 6.203 0.734 0.487
Treatment: aquarium 3 17.626 2.085 0.118
a* Treatment 2 2.795 1.621 0.211
Treatment: aquarium 3 3.570 2.071 0.120
b* Treatment 2 54.03 3.649 0.035
Treatment: aquarium 3 9.95 0.672 0.574 Total FAA Treatment 2 1,193,950 12.631 < 0.001
Treatment: aquarium 3 77,668 0.822 0.490 Aspartic acid Treatment 2 0.007 0.239 0.789 Treatment: aquarium 3 0.042 1.480 0.235 Glutamic acid Treatment 2 3,277 0.958 0.392 Treatment: aquarium 3 2,856 0.835 0.483 Alanine Treatment 2 0.096 15.537 < 0.001
Treatment: aquarium 3 0.002 0.272 0.845 Glycine Treatment 2 166,313 20.150 < 0.001
Treatment: aquarium 3 9,642 1.168 0.334
Proline Treatment 2 823.0 3.262 0.049
Treatment: aquarium 3 52.4 0.207 0.891
Serine Treatment 2 3,419 4.219 0.022
100
Treatment: aquarium 3 684 0.844 0.478 Threonine Treatment 2 1,977.5 8.702 < 0.001
Treatment: aquarium 3 506 2.227 0.100
Arginine Treatment 2 8,102 1.719 0.193
Treatment: aquarium 3 1,023 0.217 0.884 Histidine Treatment 2 398.3 9.896 < 0.001
Treatment: aquarium 3 39.5 0.982 0.411
Isoleucine Treatment 2 2,176.1 8.102 0.001
Treatment: aquarium 3 680.7 2.534 0.071
Leucine Treatment 2 0.165 6.263 0.004
Treatment: aquarium 3 0.04 1.527 0.223
Lysine Treatment 2 3,311 2.453 0.099
Treatment: aquarium 3 755 0.559 0.645 Methionine Treatment 2 687.4 10.042 < 0.001
Treatment: aquarium 3 72.3 1.056 0.379 Phenylalanine Treatment 2 1,191.2 7.454 0.002 Treatment: aquarium 3 283.1 1.772 0.168 Tyrosine Treatment 2 4,295 10.028 < 0.001
Treatment: aquarium 3 753 1.758 0.171
Valine Treatment 2 4,111 5.442 0.008
Treatment: aquarium 3 1,864 2.468 0.076
Taurine Treatment 2 112.42 1.932 0.159
Treatment: aquarium 3 175.24 3.012 0.042 Ornithine Treatment 2 0.185 11.729 < 0.001
Treatment: aquarium 3 0.017 1.048 0.382 Significance levels (p < 0.05) are shown in bold type.
(Continued)
101
Table 21. Test diameters (mm), body weights (g), gonad indices, gonad hardness (N), and L*, a* and b* values of Mesocentrotus nudus gonads at the start and end of the experiment in each treatment group (mean ± SE). Asterisks indicate significant differences between ST and AP, MD, and BS according to the ttest. Asterisks indicate significant differences between ST and AP, MD, and BS according to the ttest. *, p < 0.05; **; p < 0.01; ***, p < 0.001.
Test diameter Body weight Gonad index Hardness L* a* b*
ST 50.0 ± 0.5 58.7 ± 2.0 5.6 ± 0.4 0.43 ± 0.04 45.7 ± 1.2 10.8 ± 0.3 37.5 ± 1.2 AP 50.2 ± 0.5 61.0 ± 1.6 18.9 ± 0.9*** 0.13 ± 0.01** 56.8 ± 0.7*** 9.8 ± 0.4 33.3 ± 0.9 MD 50.2 ± 0.1 62.0 ± 1.2 18.8 ± 1.4*** 0.14 ± 0.01*** 57.3 ± 1.3*** 9.5 ± 0.3* 36.3 ± 1.1 BS 50.2 ± 0.5 60.9 ± 1.1 19.1 ± 0.2*** 0.12 ± 0.01** 56.0 ± 0.5*** 10.3 ± 0.6 36.8 ± 0.9 ST indicates the values obtained at the start (ST) of the experiment, and AP, MD, and BS indicate the values obtained at the end of the experiment for M. nudus fed the apical, middle and basal portions, respectively, of Saccharina japonica fronds.
Test diameter and body weight of ST were replaced by the data obtained from 45 sea urchins in the three treatment groups in aquaria at the start of the experiment.
102
Table 22. Gonadal development stages of Mesocentrotus nudus in each treatment group at the beginning and end of the experiment by sex.
ST, AP, MD and BS are defined in Table 21. I, II and III indicate the recovering, growing and premature stages.
Male Female
I II III I II III ST 2 16 1 11
AP 8 7
MD 8 2 5
BS 8 1 6
103
differences in L* or a* values were detected among treatments (Table 20). The L* values of the gonads of sea urchins fed any of the frond portions were significantly higher than the L* values in the ST group (p < 0.001) (Table 21).
The FAA content of the gonads is shown in Figure 20. There were significant differences among the treatment groups in total FAA content and in the content of 13 individual FAAs (Table 20). The total FAA content of the gonads was significantly higher in the MD and BS groups than in the AP group (p < 0.05). The Glu content of the gonads in the BS group was significantly higher than that in the ST group (p < 0.05). The Ala and Ser contents of the gonads in all treatment groups were significantly higher than those in the ST group (p < 0.001). The Ala content of the gonads in the BS group was significantly higher than that in the AP group (p < 0.01). The Gly content of the gonads in the MD and BS groups was significantly higher than that in the AP group (p < 0.01). The His, Met and Tyr contents of the gonads in the MD group were higher than those in the BS and/or AP groups (p < 0.05). The Arg, Lys and Tau contents of the gonads of the animals in each treatment group were significantly lower than those in the ST group (p < 0.01). The Ile, Phe and Tyr contents of the gonads in the BS group were significantly lower than those in the ST group (p < 0.05).
The results of PCA of the FAA content of the gonads of the animals in each treatment group are shown in Figure 21. The FAA contents of AP were separated from MD and BS along PC 1, which explained 49.7% of the variance in the data. The plots of BS were shifted slightly in the negative direction of PC 2 compared to those of MD. With the exception of Asp, Ser and Ile, all FAAs showed significant negative correlations with PC 1 (p < 0.05). Ala, Glu and Pro had significant negative correlations with PC 2 (p < 0.05).
Only Gly showed a significant positive correlation with PC 2 (p < 0.05).
104
Figure 20. Free amino acid (FAA) content (mg/100 g) of Mesocentrotus nudus gonads at the start of the experiment and in each treatment group at the end of the experiment (mean ± SE). Lower case letters indicate significant differences among treatment groups by Tukey’s test (p < 0.05). Asterisks indicate significant differences between the values obtained at the start and end of each treatment according to the ttest (p < 0.05). The abbreviations ST, AP, MD and BS are defined in Table 21. The abbreviations for the amino acids are defined in the legend to Figure 19.
200
0 400 600 800 1000
Glu Ala Gly Pro Ser Thr Arg His Ile Leu Lys Met Phe Tyr Orn
Asp Val Tau
FAA c on ten t (m g/ 100 g)
b
a *
Tot al FA A c ont en t ( m g/ 100 g)
AP
ST MD BS
b a a
a a
b
b a b a
b b a b b a a b a
a b
a b
Total 0
500 1000 1500 2000 2500
a
a b *
*
* *
* * *
* * *
*
*
*
* *
*
*
* *
*
* * * * *
105
Figure 21. Principal component analysis biplot of compounds detected by free amino acid content analysis of Mesocentrotus nudus gonads. Each arrow indicates the position of a free amino acid. The abbreviations AP, MD and BS are defined in Table 22. The abbreviations for the amino acids are defined in the legend to Figure 19.
106
4. Discussion
In this study, the feeding of different portions of S. japonica fronds to M. nudus did not affect sea urchin somatic (TD and BW) growth, gonadal growth (gonad indices), or gonad hardness or color (L*a*b*). Feeding of whole fronds of S. japonica enhance gonad production, increase L* value and improve gonad color (Agatsuma 1997; Chapter 2). In the animals in the AP, MD and BS groups, the values of these traits were similar to those of M. nudus fed whole fronds of S. japonica from May–July (Section 3, Chapter 2). The lack of significant differences in protein content, which enhances gonad production (de JongWestman et al. 1995; Pearce et al. 2002b; Eddy et al. 2012), among the frond portions of S. japonica is consistent with the similarities in these gonad indices. The color of sea urchin gonads can be improved by βcarotene contained in feed (Robinson et al.
2002; Pearce et al. 2003; Shpigel et al. 2005). Henley and Dunton (1995) reported that the carotenoid content of yearling fronds of S. latissima was lower in April than in July.
In May, the βcarotene content of the apices of fronds of A. esculenta, L. digitata, L.
hyperborea and Saccorhiza polyschides is higher than that of the basal portions (Schmid and Stengel, 2015). In contrast, in S. latissima, the βcarotene content of the apex, middle and base of the frond does not differ (Schmid and Stengel 2015). In the present study, the finding of no significant difference in gonad color in M. nudus in the different treatment groups might be due to a lack of difference in βcarotene content among the frond portions of S. japonica. Gonad hardness, which is affected by gonad development and size (McBride et al. 2004; Section 1 and 3, Chapter 2), also showed no significant differences among the treatment groups, likely due to the similarities in gonadal development and size in the three groups.
The FAA content of gonads is closely associated with their taste (Komata et al. 1962;
107
Lee and Haard 1982; LiyanaPathirana et al. 2002). In M. nudus gonads, high alanine content increases the sweetness and results in preferable taste (Section 1of Chapter 2). In the present study, the Ala content of the gonads of M. nudus in all treatment groups increased significantly compared to the Ala content at the start of the experiment; Ala content was particularly high in the gonads of the BS group. In addition, the higher Gly content in the gonads of BS and MD compared with that of AP and the low Ser content of the gonads of the animals in all of the treatment groups (below the threshold value of 150 mg/dl) (Kirimura et al. 1969) suggest that the sweetness of the gonads in the treatment groups ranked as BS > MD > AP. The higher content of the bittertasting His, Met and Tyr in the gonads of the MD group compared to the BS group would tend to further decrease the relative bitterness of the gonads of the BS animals. The PCA results indicated that the FAA compositions of the gonads varied among the treatment groups. The FAA contents of AP were separated from those of MD and BS along PC 1. In addition, low total FAA content in the gonads of AP would lessen taste enhancement. The PCA biplots also suggest high Ala, Pro and Glu content and low Gly content in the gonads of the BS group compared with the MD group, although no significant differences in Gly, Pro or Glu content were found among the treatment groups. These results suggest that feeding of the basal portion of S. japonica fronds largely enhanced umami and sweet taste, while feeding of the middle portion relatively enhanced bitter taste, and feeding of the apical portion lessened taste compared with other portions.
High levels of bittertasting Arg in gonads make the taste undesirable (Komata, 1964).
The increased Ala and decreased Arg content found in the gonads of the animals in all treatment groups in the present study would be expected to lead to high evaluation of the taste (Section 3 of Chapter 2). The Ala content of gonads of the animals in the BS group
108
(536.1 mg/100 g) was higher than that reported in previous studies of M. nudus (38.0–
433.8 mg/100 g) (Hirano et al. 1978; Hoshikawa et al. 1998; Nabata et al. 1999; Inomata et al. 2016; Chapter 2). In Section 3, Chapter 2, the results showed that gonads of M.
nudus fed whole fronds of S. japonica from May–July were evaluated as having more desirable taste than those from a fishing ground. In that study, the Ala content of testes was 452.6 mg/100 g, and that of ovaries was 419.4 mg/100 g. In contrast, the Arg content of testes was 245.7 mg/100 g and that of ovaries was 192.0 mg/100 g, values that are approximately similar to the level of 245.3 mg/100 g found in the gonads of the animals in the BS group. Therefore, the taste of gonads obtained from animals in the BS group would be evaluated at a higher level in this study than in Chapter 2.
The FAA content of the basal portions of S. japonica fronds is high compared to that of the apex (Oishi et al. 1967). Oishi and Kunisaki (1970) reported that the free Asp, Glu, Ala and Pro contents of the central and basal portions of S. japonica fronds are high compared to those in the apex from April–July. In the present study, the free Asp, Glu and Pro contents of the basal portion were high compared to those of the apical portion. The TAA content of the basal portion was also high. Gonads of E. chloroticus fed feeds containing Glu (glutamate) and Gly are sweet compared to those fed feeds containing Val and Met (Phillips et al. 2009). I suggested that high Ala content in gonads of M. nudus fed S. japonica fronds from May–July would be affected by changes in the FAA content of the mature fronds at the late sporophyte stage in Chapter 2. Glu can be converted to Ala by alanine aminotransferase (Brosnan and Brosnan 2009). The genome sequence of this enzyme in the sea urchin Strongylocentrotus purpuratus has been recorded (NCBI:
LOC580780). Black (1964) identified the enzyme in the sea urchin Lytechinus variegatus from the egg to the pluteus larva stage. These studies suggest that Ala can be produced
109
from Glu in the gonad. Furthermore, the correlated ranking of total Glu content in the basal > middle > apical portions of S. japonica fronds could contribute to the Ala content of gonads. Likewise, correlations of the His, Met and Tyr contents of the gonads of the animals in each treatment group and in the frond portions were not found. The associated factors remain unclear.
In this section, feeding of the basal, middle and apical portions of S. japonica fronds to M. nudus from a barren during May–July improved gonad size, hardness, color and taste. There were no significant differences in gonad size, hardness or color among sea urchins fed different portions of the fronds. The basal portion, which contained high levels of glutamic acid, alanine and proline, markedly increased the alanine and glutamic acid content and decreased the bittertasting amino acid content of the gonads of sea urchins;
this is expected to greatly enhance the sweet and umami taste of the product. In particular, the gonadal alanine content was higher than that reported in previous studies of M. nudus, inferring that the gonad taste would be evaluated at a higher level. The results of FAA analysis suggest that feeding the middle portion of the fronds relatively enhanced bitter taste and that feeding of the apical portion decreased the desirability of the taste compared to feeding other portions. Alanine may be synthesized from total glutamic acid present in S. japonica fronds fed to M. nudus. The basal portion of the frond has the high potential to improve gonad taste.