3.1. Protein solubility of shrimp waste
The Protein solubility of Vannami shrimp waste at different pH was determined (Fig.1). This was similar to the isoelectric pH of other species of shrimp. At pH 4 to 7, the solubility of the protein was low and in the range of 1-3 and 8-10, protein had a higher solubility. At pH 6, the solubility was the lowest (1.8 mg/ml). This pH was determined 5.78 for the brown shrimp [13]. This solubility is extremely related to pH; which at the isoelectric point, the lowest protein solubility is happening. In this point, protein has a net charge to zero, and the highest isoelectric point is at high acid or basic pH [22].
According to Latorres, Rios [23], The low solubility of protein is related to the firm structure of macromolecule to subunits; which bounded by multiple inter and intramolecular disulfide bonds and hydrophobic interactions. Therefore, hydrolysis of shrimp waste may increase the solubility of the protein.
3.2. Effect of different types and levels of enzymes and hydrolysis times on the recovered carotenoprotein from shrimp waste
The effect of different levels of enzymes (alcalase and pepsin) and times of hydrolysis incubation on the recovery of protein and carotenoid from shrimp waste has shown in Fig. 2 & 3.
As expected, with increasing time of hydrolysis and concentration of enzymes, recovery of carotenoid and protein was increased. These recoveries significantly increased at higher units of the enzyme (3U/g). The control batch proved that enzymes in all levels had positive results on the proteolysis of shrimp waste and carotenoid extraction. Nevertheless, in protein recovery a higher level of the enzymes (> 2 U/g) was more effective (p<0.05); which it was previously reported by Sila, Nasri [2]. No significant differences in protein and carotenoid recoveries were considerable at levels of 3 and 4 U/g enzymes. In both enzymes, the hydrolysis of the waste was increased after 2 h; then, a slower rate of hydrolysis was found 180 min up to 240 min and reached to the equilibrium phase (p>0.05). No significant differences in recoveries were observed after 180 min of hydrolysis. The comparison of enzymes showed that the alcalase was more effective than pepsin in the recovery of compounds from white shrimp waste (p<0.05). The highest recovery of protein by 3U/g of pepsin and alcalase was 55.85 and 50.1%, respectively; which had no significant difference by 4 units of the enzymes. In the case of carotenoid recovery, the higher content was 532.14 and 531.21 mg/g for alcalase and pepsin at a concentration of 3.0 U/g of SW. Barse, Chakrabarti [24] reported that trypsin was more effective than papain and pepsin for extracting carotenoprotein from shrimp shell. Related results were also observed by Klomklao, Benjakul [25], Sila, Nasri [2] and Senphan, Benjakul [11] when different concertation and type of proteases were applied for hydrolysis of shrimp wastes to the recovery of carotenoid and protein. The enzyme levels are various in different studies related to the activities of the enzyme. The current study 3 unit of enzymes showed acceptable results of recovery. It was inconsistent with the results of pink shrimp shells hydrolysis by a barbell and bovine trypsin (2 unit/g shell) [2]; while 1.2 units of trypsin enhanced the carotenoprotein extraction of tiger shrimp exoskeleton [25].
3.3. Proximate composition of shrimp waste and carotenoproteins
The proximate compositions and chitin contents of shrimp waste were 35.71% protein, 1.75% lipid, 39.42% ash, and 22.48% (Table 1).
Table 1.
Proximate composition of the raw waste and carotenoprotein extracted with the aid of Alcalase (CP-A) and Pepsin (CP-P) from shrimp (Litopenaeus vannamei) shell
|
Proximate composition
|
Shrimp waste
|
Carotenoprotein
|
CP-A
|
CP-P
|
Protein (%)
|
35.71±1.43b
|
75.32±1.09a
|
72.11±0.42a
|
Lipid (%)
|
1.75±0.25c
|
20.49±0.96a
|
15.72±0.44b
|
Ash (%)
|
39.42±3.92a
|
3.95±0.24b
|
8.30±1.21b
|
Chitin (%)
|
22.48±2.95a
|
1.61±0.37b
|
2.47±0.33b
|
Carotenoids (μg g-1)
|
24.06±2.05c
|
530.37±6.86a
|
330.00±4.07b
|
Data expressed as means of three replicates ± standard errors
Different letters show the significant difference between treatments (P<0.05).
|
Protein in various shrimp shells has been reported about 31 to 49% [2, 11, 26]. Due to the great protein content of the waste, this source could be used directly to the formulations for feed [26]. The max protein content in the CP-A and CP-P was 75.32 and 72.11%, respectively (p>0.05); which is within the range of 71.09 to 80.05 reported by previous studies [2, 27]. Higher protein content in carotenoproteins than the raw waste could be attributed to the removal of insoluble solid matter besides protein solubilization by enzymatic hydrolysis [14, 28]. The carotenoid content in the SW was close to 24.06 μg g-1, which was within the range of 35.8 μg g-1 tiger a prawn (P monodon) from 23 to 331 μg g−1 in the exoskeleton reported by previous studies. It depends on species, nutritional and rearing conditions. Due to effect of proteases, the total carotenoid in CP-A (530.37 μg g−1) and CP-P was significantly higher than raw material; which is in agreement with the other findings [18, 25]. Sila, Nasri [2] reported 140 μg g−1 carotenoid in carotenoprotein extracted of pink shrimp waste. The extracted carotenoprotein was found to have antioxidant activity as the result of the higher content of carotenoid which is bounded to the proteins [11]. Therefore, higher content of carotenoid will cause of the antioxidant activity of the CPs. It was also observed about lipid content in CPs compared with that found in SW; which CP-A had a higher content of carotenoid and lipid in comparison of CP-P. The chitin and ash content was relatively lower in CP-P & CP-A (2.47 and 1.61%) compared with that found in SW (22.48%); which it was inconsistent with Chakrabarti [13] also found that carotenoprotein from tropical brown shrimp shell waste extracted by trypsin had lower ash and chitin contents than shrimp waste. Our findings are in line with previous works reported by Sila, Nasri [2] and Senphan, Benjakul [11] who found high chitin at shrimp residue and the lower content in recovered CP of pink and white shrimps. It has been described that extraction by protease promotes the outgoing of protein, and lipid from shrimp residues, whiles chitin maintained in the shell [11, 25]. According to these results, enzyme-derived CP can be used as a source of pigments, protein, and fats for human dietary supplements and salmon breeding [25].
3.4. Color of shrimp waste and carotenoproteins
The color parameters of SW and recovered CPs are presented in Table 2. L* value (Lightness) was found to be significantly higher in the SW; which was significantly lower in CP-A and CP-P. SW had significantly lower a* and b*values (p<0.05).
Table 2.
Color indexes of the raw waste and carotenoprotein extracted with the aid of Alcalase (CP-A) and Pepsin (CP-P) from shrimp (Litopenaeus vannamei) shell
|
|
Shrimp waste
|
Carotenoprotein
|
CP-A
|
CP-P
|
L*
|
70.40±1.05a
|
36.1±0.00b
|
41.60±0.00b
|
a*
|
9.06±2.05b
|
38.60±0.80a
|
32.05±0.75a
|
b*
|
16.10±2.05c
|
42.90±0.75c
|
40.25±0.50c
|
Data expressed as means of three replicates ± standard errors
Different letters show the significant difference between treatments (P<0.05).
|
The recovered CP-A and CP-P samples were orange-red in color and CP-A showed higher redness of 38.60 (a*) and yellowness of 42.90 (b*). Higher values of a* and b* was related to the extracted astaxanthin along with protein. These results agreed with that reported by Senphan, Benjakul [11] and Sila, Sayari [27] in a studies on obtained carotenoproteins by proteases; which reported lower lightness and higher a* and b* in CPs than shrimp waste. Crustacean color depends on the astaxanthin content that causes a range of color from green, yellow, blue to brown. The deposited astaxanthin is responsibility of the color and hue saturation of crustacean [29]; which could be used as a coloring agent in feed or maybe food.
3.5. Amino acid composition
The essential and non- essential of amino acids were found at Pacific white shrimp waste (Table 3).
The shrimp waste has a significant content of the essential amino acids such as phenylalanine, lysine, valine and leucine (155.34, 73.04, 59.00 and 58.05 mg/g sample, respectively). The non-essential amino acids such as glutamic acid, arginine, aspartic acid, and glycine also have been identified in amino acid profiles of shrimp waste (109.35, 82.05, 73.04, and 62.07 mg/g sample, respectively). Carotenoproteins extracted from shrimp shells had higher total non-essential amino acid (660.06 & 606.23 mg/g) level than shrimp waste (529.67 mg/g). The CPs was a significant source of the non-essential amino acids such as arginine (101.05 & 93.20 mg/g sample, respectively); which was higher in alcalase treated waste.
Table 3.
Amino acid composition of the raw waste and carotenoprotein extracted with the aid of Alcalase (CP-A) and Pepsin (CP-P) from shrimp (Litopenaeus vannamei) shell
|
Amino acid
|
Raw waste
|
CP-A
|
CP-P
|
Histidine
|
38.00±0.09a
|
29.05±0.05c
|
33.00±0.09b
|
Leucine
|
58.05±0.05a
|
57.16±0.05b
|
32.00±2.29a
|
Isoleucine
|
32.05±0.05a
|
22.15±0.25b
|
11.01±0.01c
|
Lysine
|
73.00±0.20a
|
74.01±0.09a
|
54.24±0.33b
|
Methionine
|
18.00±0.10b
|
14.99±0.02c
|
24.70±0.10a
|
phenylalanine
|
155.34±1.35a
|
75.04±0.06c
|
124.74±4.75a
|
Threonine
|
35.01±0.01c
|
55.20±0.10a
|
44.25±0.45b
|
Valine
|
59.00±0.09b
|
61.01±0.00a
|
42.05±0.25c
|
Cystein
|
37.05±0.05a
|
31.00±1.00b
|
31.06±0.04b
|
Aspartic acid
|
73.04±0.05c
|
111.01±1.01a
|
107.13±3.02b
|
Glutamic acid
|
109.35±0.36c
|
142.89±0.08a
|
131.01±0.11b
|
Serine
|
46.05±0.05a
|
38.05±0.05c
|
41.10±0.10b
|
Glycine
|
62.07±2.18c
|
90.89±0.09a
|
72.05±0.05b
|
Arginine
|
82.05±0.05c
|
101.05±0.05a
|
93.20±0.20b
|
Alanine
|
48.05±0.05c
|
83.04±0.05a
|
72.50±0.19b
|
Proline
|
38.95±0.04c
|
42.05±0.05a
|
40.06±0.02b
|
Tyrosin
|
33.05±0.05a
|
20.05±0.05b
|
18.11±0.01c
|
Essential amino acids
|
468.46±1.73a
|
388.62±0.41b
|
366.00±7.57c
|
Non-essential amino acids
|
529.67±2.39c
|
660.06±0.13a
|
606.23±2.97b
|
Total Amino acids
|
998.14±4.13b
|
1048.68±0.28a
|
972.24±10.54c
|
Data expressed as means of two replicates ± standard errors
Different letters show the significant difference between treatments (P<0.05).
|
Due to degradation during hydrolysis, tryptophan was not in any sample. Carotenporoteins recovered by alcalase and pepsin were a significant source of the non-essential amino acids glutamic acid, aspartic acid, glycine, alanine (p< 0.05). The phenylalanine, lysine and valine as essential amino acids were high at alcalase treated waste; while phenylalanine and lysine were at a higher concentration (124.74 & 54.24 mg/g) in CP-P. The non-essential amino acids aspartic acid, glutamic acid, glycine and alanine were found in higher concentrations in both carotenoproteins; which alcalase treated waste displayed higher content amino acids. It was inconsistent by Pattanaik, Sawant [12] reporting between all amino acids, glutamic acid was in the main and high amount in all the CPs extracted from different shrimp species. It was also in agreement with the findings of Senphan, Benjakul [11], Klomklao, Benjakul [25] who reported that the CPs was abundant in these amino acids.
In similar study, the essential amino acids in carotenoprotein of L. vannami shrimp waste reported higher than the current study [11]. Armenta and Guerrero-Legarreta (2009) showed that carotenoprotein extracted from fermented vannami shrimp waste was rich in aspartic acid and glutamic acids. Cremades, Parrado [30] , expressed that carotenoproteins could develop diets for patients who are suffering from specific diseases such as cancer–anorexia-cachexia syndrome and renal damage.
3.6. Fatty acid composition
Fatty acid compositions of shrimp waste and extracted carotenoprotein by different enzymes are presented in Table 4. The fatty acid profile of shrimp shell could be related to species differences and also farming and/or feeding conditions.
Table 4
Fatty acid profile (% of total fatty acids) of the raw waste and carotenoprotein extracted with the aid of Alcalase (CP-A) and Pepsin (CP-P) from shrimp (Litopenaeus vannamei) shell
|
Fatty acids
|
WS
|
Carotenoprotein
|
CP-A
|
CP-P
|
C12:0
|
1.70±0.01
|
1.10±0.00
|
0.81±0.00
|
C14:0
|
3.09±0.00
|
1.11±0.00
|
2.39±0.05
|
C15:0
|
1.70±0.00
|
0.50±0.01
|
1.30±0.01
|
C16:0
|
15.29±0.00
|
6.41±0.01
|
8.60±0.02
|
C18:0
|
6.20±0.00
|
2.25±0.16
|
4.20±0.00
|
C20:0
|
0.39±0.01
|
0.94±0.04
|
0.51±0.00
|
unknown
|
1.45±0.04
|
0.50±0.00
|
0.79±0.01
|
SFA
|
29.85±0.01a
|
12.82±0.11c
|
18.63±0.04b
|
C16:1n-7
|
4.81±0.00
|
5.86±0.08
|
5.79±0.03
|
C16:1n-5
|
1.38±0.01
|
2.50±0.00
|
2.21±0.01
|
C18:1n-9
|
10.31±0.08
|
8.29±0.02
|
7.50±0.00
|
C18:1n-7
|
5.08±0.02
|
7.20±0.00
|
6.58±0.06
|
C20:1n-9
|
2.15±0.05
|
4.20±0.00
|
3.30±0.00
|
C20:1n-7
|
1.51±0.00
|
1.80±0.00
|
2.10±0.09
|
C22:1n-9
|
1.09±0.00
|
2.43±0.03
|
1.93±0.03
|
C22:1n-7
|
0.20±0.00
|
0.52±0.02
|
0.81±0.01
|
unknown
|
0.80±0.00
|
0.49±0.00
|
0.51±0.02
|
MUFA
|
27.37±0.17c
|
33.31±0.04a
|
30.76±0.18b
|
C16:2n-9
|
1.80±0.00
|
2.20±0.00
|
1.59±0.04
|
C16:2n-4
|
2.09±0.02
|
3.23±0.03
|
2.80±0.00
|
C16:3n-4
|
1.22±0.02
|
1.70±0.01
|
0.89±0.02
|
C16:4n-3
|
1.59±0.00
|
0.28±0.01
|
0.83±0.03
|
C18:2n-6
|
1.76±0.08
|
1.51±0.01
|
0.68±0.01
|
C18:2n-4
|
0.60±0.01
|
0.26±0.04
|
0.50±0.00
|
C18:3n-4
|
0.50±0.08
|
0.41±0.01
|
0.80±0.00
|
C18:3n-3
|
0.79±0.06
|
1.80±0.00
|
1.41±0.05
|
C20:2n-9
|
3.10±0.00
|
3.60±0.00
|
3.11±0.01
|
C20:2n-6
|
2.00±0.00
|
2.50±0.00
|
2.14±0.04
|
C20:4n-6
|
4.19±0.00
|
5.21±0.01
|
4.51±0.01
|
C20:5n-3
|
4.79±0.00c
|
6.49±0.00a
|
5.49±0.00b
|
C22:4n-6
|
0.88±0.01
|
1.52±0.00
|
1.39±0.02
|
C22:5n-6
|
1.10±0.00
|
2.20±0.00
|
2.82±0.02
|
C22:5n-3
|
1.69±0.00
|
2.70±0.00
|
2.10±0.09
|
C22:6n-3
|
5.50±0.00b
|
8.52±0.02a
|
5.55±0.15b
|
unknown
|
0.19±0.03
|
0.85±0.05
|
1.80±0.02
|
PUFA
|
33.85±0.22c
|
45.06±0.04a
|
38.58±0.03b
|
Data expressed as means of two replicates ± standard errors
Different letters show the significant difference between treatments (P<0.05).
|
Shrimp waste exhibited a desirable content of PUFAs (33.85%); which were found to be more abundant than MUFAs and SFAs (27.37 and 29.85, respectively). The most abundant SFAs in SW were Palmitic (C16:0) (15.29%) and Stearic acids (C18:0) (6.20%) and the main MUFA was oleic acid (C18:1 n-9); among the PUFAs, DHA (C22:6n-3) and EPA (C20:5n-3) were the main PUFAs (5.50 and 4.79%, respectively) in shrimp waste. It was inconsistent of similar studies on pink shrimp shell fatty acids; which palmitic, stearic and oleic acid was prevail in the shells. Total PUFA in this study was more than the reported of pink shrimp shells (22.48%) [2]. The SFAs were reduced after enzymatic hydrolysis and MUFA and PUFA contents were higher in carotenoproteins (p<0.05). The high content of PUFA and the ratio of PUFA/SFA descending orders were: CP-A> CP-P> WS. The lowest ratio of total ω6 to ω3 was in CP-A samples. Palmitic acid, oleic acid, DHA and EPA were found to be higher than other fatty acids of CP-A and CP-P; which CP-A showed higher DHA and EPA (8.52 and 6.49%) than CP-P (5.55 and 5.49%). According to , Cremades, Parrado [30] recovered CP from crayfish contained significant content of ω3 and ω6 fatty acids which bound to protein and make this compound as a nutraceutical product.
3.7 Mineral and Heavy metal composition
Table 5 shows the mineral and heavy metal compositions of the shrimp waste and recovered carotenoproteins. Minerals are essential to several important biochemical and physiological functions in the body [31]. In the present study, SW contained substantial amounts of calcium, phosphorus, and sodium.
Table5.
Mineral and heavy metals (mg/100g) content of the raw waste and carotenoprotein extracted with the aid of Alcalase (CP-A) and Pepsin (CP-P) from Pacific white shrimp (Litopenaeus vannamei) shell
|
|
Shrimp waste
|
Carotenoprotein
|
CP-A
|
CP-P
|
Ca (calcium)
|
2547.50±27.50
|
Trace
|
Trace
|
Ph (phosphorus)
|
313.00±12.00a
|
101.0±1.00b
|
101.00±1.00b
|
Mg (Magnesium)
|
120.50±3.49a
|
47.5±0.50b
|
47.5±0.50b
|
Na (Sodium)
|
278.50±6.50a
|
100.50±10.50b
|
315.50±15.25a
|
K (potassium)
|
53.00±5.00a
|
39.50±0.50b
|
39.51±0.50b
|
Mn (Manganese)
|
4.95±0.15a
|
1.60±0.50b
|
1.20±0.50b
|
Fe (Iron)
|
4.65±0.55a
|
3.95±0.15b
|
2.87±0.27c
|
Hg (Mercury)
|
Trace
|
Trace
|
Trace
|
Pb (Lead)
|
0.15±0.05
|
Trace
|
Trace
|
Zn (Zinc)
|
4.95±0.25a
|
2.40±0.30b
|
2.26±0.50b
|
Cd (Cadmium)
|
Trace
|
Trace
|
Trace
|
Cu (Copper)
|
10.00±0.20a
|
4.75±0.45b
|
4.32±0.13 b
|
Values are mean ± SD (n = 3)
Different letters in each row denote significant difference (P<0.05).
|
It was likely due to the existence of wide concentrations of the minerals in the raw material. Sodium, phosphorus, magnesium and potassium contents were found to be higher in CPs. The mineral content in CPs was affected by substrate and enzyme. Chalamaiah, Hemalatha [31] also considered the effect of enzyme and substrate type on the mineral type and content in hydrolyzed roe samples. The minerals in the SW were higher than CPs; which calcium mainly found in higher quantity in waste. Furthermore, except of sodium, the mineral contents no significant difference observed among CP-P and CP-A. Due to the neutralization of acidic pH by addition NaOH; sodium was mainly in higher quantity in CP-P [32]. In CP-A and CP-P, phosphorus and magnesium contents were significantly reduced than raw material. Regarding the heavy metals, the lead was in SW; which it was eliminated in the CPs (Table 3). The elements decreased significantly after enzymatic hydrolysis. The presence of heavy metals in shrimp waste depends on the content of metals in the water and the breeding area. Copper, zinc and iron are among essential heavy metals. According to Wu and Yang [33], the amount of Zn and Fe elements are a reflect of aquatic environments. These essential heavy metals decreased in CPs; which were higher in CP-A samples.