In this paper, we demonstrate the biochemical parameters of S. droebachiensis, hydrolysate, sediment and shells; the amino acid composition and molecular weight distribution of the hydrolysate, and fatty acid- and lipid compositions of S. droebachiensis and sediment after hydrolysis. Additionally, two enzyme doses are compared in their recovery rates of protein, oil and ash. This was performed to contribute to the discussion in what commercial use the sea urchins may have as an invasive species necessitating removal.
General observation of the hydrolysate includes the following. After spray drying it had a red colour and the relative amounts of the constituents were water > > protein > ash > oil. Due to spray-drying which removes much of the water, the water content is not reflected well in the tables. A coarse taste test reflected a product that was very different from the hydrolysate of any other product previously tested with a citrus-like taste.
Biochemical composition
Water, oil, protein and ash were measured in all relevant fractions; whole animal, dried hydrolysate, sediment and shell fraction, in order to be able to measure the distribution of each in the different fractions obtained (Table 1). Since two different enzyme-concentrations were applied, an increase in protein recovery could also be detected when increasing the enzyme-concentration from 0.1 % to 1 % (Table 2). The moisture content, i.e. water, is substantial in all fractions (the hydrolysate contained 98–99 % water before drying, Table 1) and typically accounts for up to 70 % of the raw material. The hydrolysate and shells contain more ash than the sediment fraction. The shell fraction is expected to contain high levels of ash as shells typically contain much minerals whereas the hydrolysate is somewhat high.
The biochemical composition of two sea urchin species (Echinometra lucunter and Lytechinus variegatus) was reported by Diniz, et al. 5 and it appears the lipid-content (oil) is much larger in these species at 8% than in S. droebachiensis where the lipid content has been approximately 1%.
Haider, et al. 6 studied the biochemical composition of two species of sea cucumber and the results shown are similar to what is presented here.
A study on the composition and amino acid profile of co-products from sea urchin processing plants 3 investigated the urchin digestive tract and non-commercial gonads for proteins, amino acids and fatty acids. The protein contents observed in the digestive tract (processing by-products) were higher than what is presented here for the whole animal (5.3 vs. 3.4%). Gonads displayed a higher protein content. The ash-content varies enormously between the two experiments; where Mamelona, et al. 3 reports ash-contents of 1.6%, the results of this experiment indicates ash-levels of 29.8%. This may be due to the calcareous shell-fraction which is also included in the whole-urchin analyses and presumably excluded from the digestive tract. This would need to be considered in the use of co-product from the sea urchin fishing industry and/or use of whole small sea urchins to produce hydrolysate.
The moisture and ash content in the gonads of sea urchins has previously been shown to vary with changing diets 7–9.
Table 1
Biochemical composition of all the different fractions: whole animal, dried hydrolysates (0.1 and 1% enzyme), hydrolysis sediment and shell fraction.
|
Ash
|
Oil
|
Protein
|
Moisture
|
S. droebachiensis
|
29.8 %
|
±
|
6.1 %
|
0.9 %
|
±
|
0.2 %
|
3.4 %
|
±
|
0.3 %
|
64.1 %
|
±
|
4.9 %
|
0.1% enzyme
|
33.9 %
|
±
|
4.8 %
|
8.2 %
|
±
|
2.0 %
|
38.8 %
|
±
|
0.9 %
|
5.8 %
|
±
|
1.8 %
|
1% enzyme
|
31.9 %
|
±
|
1.4 %
|
5.0 %
|
±
|
0.4 %
|
47.4 %
|
±
|
1.7 %
|
3.3 %
|
±
|
0.2 %
|
Sediment
|
12.7 %
|
±
|
3.1 %
|
6.1 %
|
±
|
1.0 %
|
7.7 %
|
±
|
0.6 %
|
68.0 %
|
±
|
3.8 %
|
Shells
|
56.0 %
|
±
|
2.7 %
|
0.5 %
|
±
|
0.1 %
|
1.3 %
|
±
|
0.1 %
|
38.0 %
|
±
|
2.8 %
|
Table 2
Difference in recovery of the different fractions in the hydrolysate when increasing enzyme concentration 10-fold.
|
Recovery 0.1% enzyme
|
Recovery 1% enzyme
|
Recovery increase
|
Moisture
|
92.0 %
|
86.9 %
|
-5.5 %
|
Oil
|
45.7 %
|
89.9 %
|
96.8 %
|
Ash
|
61.3 %
|
72.5 %
|
18.2 %
|
Protein
|
37.0 %
|
91.3 %
|
146.5 %
|
Recovery
Enzyme efficacy can manifest itself in the recovery of protein in the aqueous hydrolysate compared to the raw material. Recovery in general is calculated based on a recording of all biochemical parameters (i.e. water, ash, oil and protein) in each step of the hydrolysis. Knowing each fraction’s relative contribution to the whole and its content will allow for a tracking of parameters throughout the process. Protein and oil are commonly the two factors that are followed most closely in a commercial perspective due to their role in human consumption and are also the two parameters most commonly affected by change in enzyme concentration or enzyme type. Recovery parameters were affected by the amount of enzyme used. Oil and protein recovery increased substantially, ash somewhat and water recovery remained at a similar level displaying a slight decrease (Table 2) when enzyme dosage was increased.
Amino Acid Analysis
The amino acid analyses of all four hydrolysate samples were similar. An average of all samples is displayed in Table 3.
Similar amino acids as found in the sea urchin dominate the sea cucumbers: Glycine, Aspartic acid, and Glutamic acid 10. Sea cucumber is also high in Alanine and Arginine which appear to be lower in S. droebachiensis. Glutamic acid, Glycine and Alanine are all known to promote sweet and umami flavor in sea urchin roe 11 and two of the three (Glutamic acid and Glycine) are clearly above average distribution both in the total and free form samples from the current study. In free form, Leucine and Glycine dominate whereas Glycine, Glutamic acid and Aspartic acid dominate in total.
The authors suggest a sensory-panel mapping analysis should be undertaken on future sea urchin hydrolysates. Possible uses for the hydrolysate are unclear without further testing but the unusual combination of amino acids and the relatively high levels of Glycine, Aspartic acid, and Glutamic acid should be further investigated in terms of product placement.
Table 3
Distribution of amino acids both total (bound + free) and free (in solution) in the hydrolysate.
|
Total
|
Free
|
Amino acid
|
g/100 g hydrolysate
|
g/100 g hydrolysate
|
Aspartic acid
|
3.25 (± 0.27)
|
0.12 (± 0.08)
|
Glutamic acid
|
4.95 (± 0.44)
|
0.41 (± 0.07)
|
Hydroksyproline
|
0.36 (± 0.08)
|
0.02 (< 0.01)
|
Serine
|
1.85 (± 0.23)
|
0.29 (± 0.10)
|
Glycine
|
7.98 (± 0.97)
|
5.05 (± 0.90)
|
Histidine
|
0.71 (± 0.05)
|
0.13 (± 0.04)
|
Arginine
|
2.33 (± 0.30)
|
0.64 (± 0.16)
|
Threonine
|
1.73 (± 0.19)
|
0.30 (± 0.11)
|
Alanine
|
1.98 (± 0.24)
|
0.64 (± 0.14)
|
Proline
|
1.40 (± 0.27)
|
0.23 (± 0.16)
|
Tyrosine
|
1.14 (± 0.12)
|
0.56 (± 0.09)
|
Valine
|
1.70 (± 0.14)
|
0.46 (± 0.09)
|
Methionine
|
0.92 (± 0.06)
|
0.47 (± 0.08)
|
Isoleucine
|
1.43 (± 0.11)
|
0.41 (± 0.10)
|
Leucine
|
2.33 (± 0.18)
|
1.25 (± 0.17)
|
Phenylalanine
|
1.30 (± 0.07)
|
0.75 (± 0.08)
|
Lysine
|
2.18 (± 0.29)
|
0.61 (± 0.07)
|
Lipid class and fatty acid analysis
The sediment and raw material were subjected to lipid class and fatty acid analyses (Tables 4 and 5). In both samples the lipid class triacylglycerol is most abundant followed by free fatty acids and cholesterol. In total neutral lipids account for 50.15 and polar lipids 1.15 g/100 g extracted fat. Of the typically marine fatty acids (EPA, DPA and DHA), the EPA is most abundant. Other fatty acids of notable amounts compared to the mean are 14:0, 16:0, 18:1 and 20:4 n-6. These analyses were only performed on two samples.
Haider, et al. 6 presented similar distribution of polyunsaturated and monounsaturated fatty acids, in addition to the ratios between the n-3 and n-6.
Table 4
Lipid classes in raw material and sediment. Average of two samples, all amounts are g/100 g extracted fat (Following lipid classes had readings of zero: Diacylglycerol, Monoacylglycerol, Cholesterol esters, Phosphatidylinositol, Phosphatidylserin, Phosphatidylcholin and Lyso-Phosphatidylcholin).
Lipid class/fatty acid
|
Raw material
|
Sediment
|
Triacylglycerol
|
32.5
|
± 5.5
|
25
|
± 3
|
Free fatty acids
|
10.45
|
± 1.55
|
7.7
|
± 1.2
|
Cholesterol
|
6.55
|
± 0.35
|
6.9
|
± 0.5
|
Phosphatidyletanolamin
|
1.15
|
± 0.05
|
2.9
|
± 1.6
|
Total polar lipids
|
1.15
|
± 0.05
|
14.35
|
± 2.55
|
Total neutral lipids
|
50.15
|
± 7.55
|
40.4
|
± 3.3
|
Total sum lipids
|
51.25
|
± 7.45
|
54.65
|
± 5.85
|
Table 5
Fatty acids in raw material and sediment. Average of two samples, all amounts are g/100 g extracted fat.
Fatty acid
|
Raw material
|
Sediment
|
14:0
|
5.25
|
± 0.25
|
4.3
|
± 0.1
|
16:0
|
7.35
|
± 0.15
|
6
|
± 0.1
|
16:1 n-7
|
1.8
|
0
|
1.45
|
± 0.05
|
16:2 n-4
|
0.1
|
0
|
0.1
|
0
|
16:3 n-4
|
0.2
|
0
|
0.2
|
0
|
18:0
|
1.45
|
± 0.05
|
1.25
|
± 0.05
|
18:1 (n-9)+(n-7)+(n-5)
|
5.6
|
± 1.1
|
3.5
|
± 0.1
|
18:2 n-6
|
1.6
|
± 0.1
|
1.05
|
± 0.15
|
18:3 n-3
|
1.15
|
± 0.15
|
0.8
|
± 0.2
|
18:3 n-6
|
0.2
|
0
|
0.15
|
± 0.05
|
18:4 n-3
|
4.45
|
± 0.05
|
5.05
|
± 0.65
|
20:0
|
0.3
|
± 0.1
|
0.25
|
± 0.05
|
20:1 (n-9)+(n-7)
|
4
|
± 0.4
|
3.7
|
± 0.6
|
20:2 n-6
|
1.15
|
± 0.25
|
1
|
± 0.2
|
20:3 n-3
|
1.05
|
± 0.35
|
0.95
|
± 0.35
|
20:3 n-6
|
0.35
|
± 0.05
|
0.25
|
± 0.05
|
20:4 n-3
|
0.65
|
± 0.35
|
0.5
|
± 0.2
|
20:4 n-6
|
5.4
|
± 0.1
|
5.6
|
0
|
20:5 n-3 EPA
|
8.05
|
± 2.05
|
8.25
|
± 2.15
|
21:5 n-3
|
0.1
|
0
|
0.1
|
0
|
22:0
|
0.1
|
0
|
0
|
0
|
22:1 (n-11)+(n-9)+(n-7)
|
1.8
|
± 0.3
|
1.45
|
± 0.35
|
22:4 n-6
|
0.1
|
0
|
0.1
|
0
|
22:5 n-3 DPA
|
0.2
|
0
|
0.15
|
± 0.05
|
22:6 n-3 DHA
|
0.75
|
± 0.25
|
0.6
|
± 0.2
|
24:1 n-9
|
0.15
|
± 0.05
|
0.1
|
0
|
Sum saturated fatty acids
|
14.45
|
± 0.55
|
11.8
|
± 0.1
|
Sum monoenoic fatty acids
|
13.35
|
± 1.85
|
10.2
|
± 0.9
|
Sum total-PUFA fatty acids
|
25.5
|
± 3
|
24.8
|
± 3.9
|
Sum PUFA (n-3) fatty acids
|
16.4
|
± 2.7
|
16.35
|
± 3.45
|
Sum PUFA (n-6) fatty acids
|
8.8
|
± 0.3
|
8.15
|
± 0.45
|
Sum identified fatty acids
|
53.3
|
± 1.7
|
46.8
|
± 3.1
|
Sum unidentified fatty acids
|
18.95
|
± 1.75
|
17
|
± 0.9
|
Size exclusion chromatography
To give a general view of the size distribution of the peptides in the hydrolysate a size exclusion chromatography with a gel filtration column was performed on all hydrolysates. The results were quite similar on all and are presented as mean and standard deviation (Fig. 1). The distribution is based on a standard curve made from known compounds. It appears that most of the sample consists of the smallest range of proteinaceous compounds– from single amino acids to tripeptides.