Single Factor Experiments
SCPs powder (1 g) was dissolved in 2 mL of deionized water in a beaker, mixed with 3 mL of medical-grade hydrogen peroxide of different concentrations, stirred well, and then placed in constant temperature water baths at different temperatures for a while. Samples were freeze-dried and stored for subsequent experiments. The concentration of commercial medical-grade hydrogen peroxide was calibrated by potassium permanganate titration.
According to the corresponding standards of China, the samples were subjected to a descriptive sensory analysis at room temperature by 10 panelists. Before the evaluation, the panelists were trained three times a week (Selli et al. 2009). The panelists were trained to describe the odor by assigning scores between 1 and 5 (Table 1). Before the test, 20 mL samples were placed in a sealed plastic cup. The panelists were asked to open the cap and sniff the headspace above the sample to determine the intensity of the fishy odor all the samples were presented in random order. The results of the single-factor experiment are shown in Figure 1- 3.
Table1. Sensory Assessment of SCPs
Odor
|
Score
|
The same as untreated hydrolysate (strong fishy odor) Score
|
5
|
Moderately strong
|
4
|
Moderate
|
3
|
slight
|
2
|
None (similar to deionized water)
|
1
|
For single-factor experiments of deodorizing SCPs, the concentration of hydrogen peroxide are 4.1, 16.3, 81.6, 407.0, and 816.2 mmol·L-1 respectively, and the deodorization time is 5, 10, 20, 30, and 60 min respectively, the deodorization temperature are 25, 30, 35, 40 and 45°C respectively. The final products were asked to be sensory scored by the assessor and the results are shown in Figure 1. It was observed that an increase in the concentration of medical-grade hydrogen peroxide concentration had a favorable effect on removing the odor of SCPs. A concentration of 816 mmol∙L-1 hydrogen peroxide achieved a score of 2.1, which indicates a very slight fishy odor. This effect can be achieved because higher concentrations of hydrogen peroxide are more conducive to oxidation reactions with volatile substances in aquatic products, breaking down fishy substances. It can be seen that the concentration of hydrogen peroxide on the SCPs deodorization effect is obvious.
To study the deodorization time, the related tests were performed at different times ranging from 25 to 45 min. At the concentration of hydrogen peroxide of 81.6 mmol·L-1 and temperature of 25 °C, the deodorization time was increased to 5, 10, 20, 30, and 60 min respectively for the deodorization of SCPs. The final product was asked by the assessor for sensory scoring and the results are shown in Figure 2. With the increase in deodorization time, the fishy odor fluctuated between 1.7 - 2.7 and the overall fishy odor tended to be stable. It is worth noting that the lowest value of fishy odor was found at 30 min. It can thus be seen that hydrogen peroxide oxidation has high reaction efficiency for the deodorization of SCPs.
The deodorization temperature was increased to 25, 30, 35, 40, and 45 °C respectively for the deodorization of SCPs at the concentration of 81.6 mmol·L-1 of hydrogen peroxide and reaction time of 20 min, respectively. The final product was requested to be sensory evaluated by the assessor and the results were shown in Figure 3. With the increase in deodorization temperature, the fishy odor score fluctuated between 2.5 - 2.9 and stabilized overall. In the sensory evaluation, the lowest fishy odor score appeared at the temperature of 30 °C, which shows lower deodorization temperature had a more positive effect on the deodorization of SCPs, and vice versa.
Orthogonal Experiments
To ensure the accuracy of the results, the experiment was repeated three times for each factor level and the average of three parallel samples results were adopted for orthogonal analysis. According to the principle of an orthogonal experiment, the degree of influence of different factors on the test results can be characterized by the extreme difference value (R). The size the of R-value is positively correlated with the high influence of factors on the experimental results and should be focused on in experimental and practical applications.
In the actual deodorization treatment process, not all factors play a role alone, but a variety of factors play a role together. Therefore, to optimize the deodorizing effects of the hydrogen peroxide concentration (A), deodorization time (B), and deodorization temperature (C) on the SCPs, three experiments with orthogonal designs L25(35) were conducted using three factors (A, B, and C) and the corresponding levels (five levels for A, B, and C). As shown in Table 2, a sensory value (odor score) was used as an index. The results and ANOVA data of the orthogonal experiments are presented in Tables 3 and 4.
Table 2. Orthogonal table of orthogonal experimental factors
Factors
Levels
|
A[a] concentration (mmol∙L-1)
|
B deodorization time (min)
|
C deodorization temperature (°C)
|
1
|
4.1
|
5
|
25
|
2
|
16.3
|
10
|
30
|
3
|
81.6
|
20
|
35
|
4
|
407.0
|
30
|
40
|
5
|
816.2
|
60
|
45
|
[a] the Configured concentration of medical-grade hydrogen peroxide
Table 3. Results of the L25(35) orthogonal test
Group number
|
Level
|
Result
|
A
|
B
|
C
|
D(Blank)
|
Odor score
|
1
|
1
|
1
|
1
|
1
|
3.0
|
2
|
1
|
2
|
2
|
2
|
2.6
|
3
|
1
|
3
|
3
|
3
|
2.4
|
4
|
1
|
4
|
4
|
4
|
2.3
|
5
|
1
|
5
|
5
|
5
|
2.5
|
6
|
2
|
1
|
2
|
4
|
2.6
|
7
|
2
|
2
|
3
|
5
|
2.4
|
8
|
2
|
3
|
4
|
1
|
2.4
|
9
|
2
|
4
|
5
|
2
|
2.3
|
10
|
2
|
5
|
1
|
3
|
2.5
|
11
|
3
|
1
|
3
|
2
|
2.6
|
12
|
3
|
2
|
4
|
3
|
2.6
|
13
|
3
|
3
|
5
|
4
|
2.5
|
14
|
3
|
4
|
1
|
5
|
2.2
|
15
|
3
|
5
|
2
|
1
|
2.1
|
16
|
4
|
1
|
4
|
5
|
2.3
|
17
|
4
|
2
|
5
|
1
|
2.3
|
18
|
4
|
3
|
1
|
2
|
2.2
|
19
|
4
|
4
|
2
|
3
|
2.4
|
20
|
4
|
5
|
3
|
4
|
2.3
|
21
|
5
|
1
|
5
|
3
|
1.7
|
22
|
5
|
2
|
1
|
4
|
1.5
|
23
|
5
|
3
|
2
|
5
|
1.6
|
24
|
5
|
4
|
3
|
1
|
1.4
|
25
|
5
|
5
|
4
|
2
|
1.5
|
k1
|
2.56
|
2.44
|
2.28
|
2.24
|
|
k2
|
2.44
|
2.28
|
2.26
|
2.24
|
|
k3
|
2.40
|
2.22
|
2.22
|
2.32
|
|
k4
|
2.30
|
2.12
|
2.23
|
2.24
|
|
k5
|
1.54
|
2.18
|
2.26
|
2.20
|
|
R
|
1.02
|
0.32
|
0. 06
|
0.12
|
|
Optimal combination
|
A5B4C3
|
|
|
Order of priority
|
RA > RB > RC
|
|
|
Note: K1, K2, and K3 represent the mean value of experimental results, and R represents the range.
|
Table 4. Analysis of variance
Source of difference
|
S
|
Freedom
|
MS
|
F
|
P
|
α
|
A
|
3.306
|
4
|
0.827
|
132.24
|
0.00
|
*
|
B
|
0.298
|
4
|
0.075
|
11.92
|
0.00
|
|
Error
|
0.050
|
8
|
0.00625
|
|
|
|
Total variation
|
3.66
|
20
|
|
|
|
|
F0.10(4,20) = 2.25 F0.05(4,20) = 2.87 F0.025(4,20) = 3.51
|
Note: MS represents Mean Square; P represents the Critical value of F;
* indicates that this factor has a significant influence.
|
In the orthogonal experiment with the odor score of SCPs as the inspection index, it can be seen that the best combination of experimental conditions is A5B4C3. The odor score is the lowest when the hydrogen peroxide concentration is 816 mmol∙L-1, the reaction time is 30 min and the temperature is 35 °C. The influence order of three factors on the SCP solution is the hydrogen peroxide concentration, deodorization time, and temperature. As the analysis of variance shows F0.05 (4,20) = 2.87 < FA, PA < 0.05, it can be concluded that the effect of the hydrogen peroxide concentration on the odor score of the SCPs solution is significant at the level of α = 0.05, and other factors are not significantly affected.
The best level combination A5B4C3 in 25 orthogonal test protocols had an organoleptic score of 1.4, which shows that the appropriate deodorization time and temperature are conducive to the oxidative deodorization of hydrogen peroxide. Longer deodorization time or higher deodorization temperature may lead to a more concentrated distribution of volatile odors in the space of the main components, and too low to significantly play a role in removing fishy odors.
The total and amino acid nitrogen of SCPs
Amino acid nitrogen content has been considered a primary parameter of the quality level of SCPs as it can reflect the nutritious protein level in it. The total nitrogen (Jamal et al. 2020) and amino acid nitrogen content (Liu et al. 2021) were determined for the original sample of SCPs solution obtained and the sample with optimal deodorization treatment, which was determined and the results are shown in Table 5.
Table 5. Nitrogen content of SCPs before and after deodorization
SCPs Samples
|
Total Nitrogen
(g/100g)
|
Amino acid nitrogen (g/100g)
|
Original sample
|
15.0
|
1.33
|
Deodorized sample (35 °C)
|
15.4
|
1.31
|
Deodorized sample (40 °C)
|
15.3
|
1.32
|
As can be seen from Table 5, the total nitrogen content of SCPs after deodorization by medical-grade hydrogen peroxide was increased from 15.0 to 15.4 (or 15.3), and the amino acid nitrogen content was decreased from 1.33 to 1.31 (or 1.32) slightly. The main reason for this is that the oxidation of SCPs by hydrogen peroxide causes some peptide bonds to break, resulting in a decrease in amino acid nitrogen. Moreover, the oxidation also leads to the formation of a small amount of nitrogen oxides (Yang and CHUNG, 2012; Bergt et al. 2001). In conclusion, the deodorization technique of hydrogen peroxide oxidation of SCPs had little effect on its nitrogen content, and the various nitrogen contents remained relatively stable in general. Medical-grade hydrogen peroxide is likely to have little effect on the main active substances and efficacy of SCPs. Follow-up bioactivity testing of deodorized and deallocated SCPs samples is in progress.
Sensory evaluation of SCPs moisturizer
To observe the acceptability of the fishy odor of deodorized SCPs in a moisturizer, moisturizer samples containing 1% untreated SCPs and 1% treated SCPs with 816.2 mmol∙L-1 hydrogen peroxide at 35 °C for 30 min were sensory evaluated three times: immediately, and 2 and 4 days after the addition of SCPs samples.
Figures 4 show that the fishy odor evaluation of the paste moisturizer samples containing treated SCPs slightly increased from 1.1 to 1.3, while the sample with untreated SCP increased from 1.3 to 2.2. In the meantime, sensory evaluation of moisturizer with SCPs after applying on the skin was carried out (Figure 5), as shown in Figure 5, the sensory scores increased from 1.1 to 1.2, while the others with untreated SCP increased from 1.3 to 1.6. From Figure 4-5, it can be seen that the fishy odor score of moisturizers containing treated SCPs with medical-grade hydrogen peroxide was significantly lower than that of moisturizers containing untreated SCPs. Moreover, the odor score of moisturizer with the deodorized SCPs is close to 1, with almost no fishy odor, which shows that both the paste sample and the application on the skin have obvious deodorizing effects after treatment. It is worth noting that the odor score of the moisturizer without oxidized SCPs increased slightly with time, while the others with oxidized SCPs were more stable, which shows that hydrogen peroxide has a better deodorization effect on SCPs.
Heat resistance test of SCP moisturizer
To test the stability of the moisturizers, 40 g samples of the moisturizer containing 1% and 5% untreated SCPs, and 1% and 5% well-deodorized SCPs were stored at 48 °C for a while, and sensory evaluation was performed after returning to room temperature.
As shown in Table 6, the moisturizer samples with well-deodorized 1.0% SCPs basically had no fishy odor in the first 21 days and a slight fishy after 28 days, while the samples containing untreated 1.0% SCPs were more fish after 28 days. Similarly, the odor score of samples with well-deodorized 5.0% SCPs was 1.2 after 28 days, basically no fishy odor, while the odor score of samples containing untreated 5.0% SCPs was 2.4 after 28 days.
The stability of the moisturizer samples with 1.0% and 5.0% well-treated SCPs was good after 28 days (Table 7). However, the samples containing 1.0% and 5.0% untreated SCPs were found to have slight delamination after 21 and 28 days. Overall, the results of the heat-resistant fishy odor evaluation and stability test showed that the fishy odor of moisturizers containing deodorized SCPs was mild, the spreadability, wetness, stickiness, and softness of the moisturizer after applying on the skin were consistent, and the stability was generally good.
Table 6. Heat-resistant fishy odor sensory evaluation data sheet
Projects
|
Sensory evaluation of fishy odor stability
|
Reference
(RT) [a]
|
3 days
later[b]
|
7 days later[c]
|
21 days later[d]
|
28 days later [e]
|
1
|
1.0
|
1.1
|
1.1
|
1.1
|
1.1
|
2
|
1.0
|
1.1
|
1.1
|
1.1
|
1.2
|
3
|
1.2
|
1.3
|
1.3
|
1.3
|
1.9
|
4
|
1.2
|
1.3
|
1.3
|
2.0
|
2.4
|
[a] Samples at room temperature. [b], [c], [d], [e] The samples were placed in a thermostat at 48 °C and removed after 3, 7, 21, and 28 days.
|
Table 7. Heat Stability Measurement Data Sheet
Projects
|
Evaluation of fishy odour stability
|
Referece (RT) [a]
|
3 days later[b]
|
7 days later[c]
|
21 days later[d]
|
28 days later[e]
|
1
|
uniform
|
uniform
|
uniform
|
uniform
|
uniform
|
2
|
uniform
|
uniform
|
uniform
|
uniform
|
uniform
|
3
|
uniform
|
uniform
|
uniform
|
Slightly layered
|
Slightly layered
|
4
|
uniform
|
uniform
|
uniform
|
Slightly layered
|
Slightly layered
|
[a] Samples at room temperature. [b], [c], [d], [e] The samples were placed in a thermostat at 48 °C and removed after 3, 7, 21, 28 days.
|