Green Astaxanthin Extraction: Microwave and Ultrasound Pretreatment

doi:10.21203/rs.3.rs-5241632/v1

In this study, ultrasonic and microwave extraction were performed to recover astaxanthin from F. merguiensis shrimp and P. maeoticus Gammarus samples, using three solvents, including organic solvent, vegetable oil, and ionic microemulsion. Microemulsions were evaluated in terms of density, conductivity, and particle size. The microemulsion exhibited a density of 0.9715 g/cm3, a conductivity of 312 µSiemens, and an average particle size of 15.8 nm. The results demonstrated that in both samples, the ionic microemulsion solvent showed superior performance in astaxanthin extraction and F. merguiensis shrimp produced more total carotenoid compared to the other sample, In both methods, the recovery efficiency of astaxanthin increased with an increasing amount of solvent and with an increase in power and extraction time, it increased at first, but then a slight decrease was observed. Ultrasonic extraction preserved the structure and antioxidant properties of astaxanthin better, but the microwave method showed a higher extraction efficiency.

Physical sciences/Chemistry

Physical sciences/Engineering

Ionic microemulsion

Astaxanthin

Ultrasonic

Microwave

Antioxidant

Extraction

Astaxanthin, with the molecular formula C₄₀H₅₂O₄ and an orange-red color, is classified as a xanthophyll and possesses the highest concentration among oxygenated carotenoid derivatives [1, 2].

Astaxanthin, with its molecular structure containing carbon-carbon chains with conjugated bonds [3], possesses remarkable properties that include the ability to remove reactive oxygens, inhibit free radicals, and perform specific biological and physiological functions such as anti-aging, antioxidant, anti-diabetic, anti-inflammatory, and cognitive enhancement [4, 5]. However, since humans and animals lack the biochemical pathways to produce carotenoids, including astaxanthin, it must be obtained from external sources [6]. Due to its beneficial attributes, astaxanthin finds extensive application across several industries. It is commonly utilized in the food, pharmaceutical, cosmetic and health sectors, where it appears in various forms such as capsules, gels, tablets, powders, creams, energy drinks, oils and extracts [7, 8]. Moreover,astaxanthin is a widely-used food additive in aquaculture, enhancing the attractive body color of aquatic animals like salmon, shrimp, and crustaceans while also supporting their growth and survival [9]. Astaxanthin can be obtained from two primary sources: natural sources, including algae, fungi, salmon, krill, crustaceans, shrimp, and lobster, and commercial sources such as fafia yeast, Haematococcus, and chemical synthesis [1, 10]. There is a growing trend of using natural ingredients in the food and nutritional market as consumers are becoming more concerned about the safety and potential adverse effects of chemical products [11]. Shrimp Significant amounts of raw material are discarded as waste during shrimp processing, contributing to economic losses [12]. However, these waste materials still contain valuable compounds like astaxanthin and other nutrients [10]. Fenneropenaeus merguiensis (F. merguiensis), a species of banana shrimp, is widely distributed in tropical and subtropical waters along the Indo-West Pacific coast [13] and represents an ideal source for astaxanthin extraction, as it contains a high proportion of this carotenoid [14]. Similarly, P. Maeoticus, an abundant species of Gammarus in the Caspian Sea, serves as a valuable carotenoid source in the region [15, 16]. Various methods have been employed for astaxanthin extraction in recent years, including microwave, ultrasound, supercritical fluids, ionic liquids, Soxhlet, microbial fermentation, and high pressure processes [1–3, 17–19]. Each of these methods offers unique advantages and plays a crucial role in obtaining astaxanthin from different sources. New technologies, such as microwave and ultrasound, are considered green techniques due to their ability to reduce extraction time, minimize solvent usage, and increase extraction efficiency [1, 19, 20]. Astaxanthin can be effectively extracted using green solvents to address issues related to solvent separation, complete recycling s, and environmental threats [21]. Ionic liquids are a class of green solvents known for their unique physical and chemical properties such as low vapor pressure, non-flammability, high thermal stability, and renewability [22]. Recently, a novel study focused on astaxanthin extraction using microemulsions based on ionic liquids [23]. Microemulsions, characterized by their stability, low viscosity, and high capacity to dissolve hydrophilic and lipophilic compounds, offer selective extraction possibilities for biomolecules in the food and chemical industries [21].

This research aims to extract astaxanthin from F.merguiensis and P.maeoticus using microwave and ultrasound as pretreatment methods, in combination with organic solvents, green solvents, and vegetable oil. To optimize the extraction process, the response surface method (RSM) was employed under various microwave and ultrasound conditions. Additionally, the antioxidant properties and other characteristics of the extracted astaxanthin were analyzed. Notably, this study is the first to investigate the use of shrimp and Gammarus crustose sources for astaxanthin extraction. The combination of new extraction methods with suitable solvents has shown promising potential to enhance extraction efficiency. Moreover, two solvents (sunflower oil and ionic liquid) which are non-toxic, non-volatile and environmentally friendly are discussed, and the innovative application of ionic liquids as a green solvent in the form of microemulsions during the extraction process represents a significant advancement in the field.

2.1. Materials

F.merguiensis and P.maeoticus were prepared fresh, with species approval, procured from a reputable Iranian Research Institute in winter. Commercial astaxanthin (> 98% purity), α-diphenyl-β-picrylhydrazyl (DPPH), and butylated hydroxytoluene (BHT) were obtained from Sigma-Aldrich (USA). HPLC grade ethanol, propanol, ethyl acetate, tributyloctylphosphonium bromide, N-butanol and triton X-100 with high purity were sourced from Merck Chemicals Co. (Germany). Furthermore, we acquired high-quality, antioxidant-free refined sunflower oil from a reputable Iranian company.

2.2. Preparation of samples

The Shells of F. merguiensis and P. maeoticus samples were thoroughly washed with distilled water, and subsequently freeze-dried (Christ-Alpha 1–4, LD freeze dryer, Germany) for 48 h at -50°C. After freeze-drying, the powders were sieved through a laboratory sieve with a mesh size smaller than 15 µm. The resulting powders were then stored at -18°C for further use.

2.3. Preparation of solvents

In the experiment, we employed a blend of ethanol and ethyl acetate in a 1:2 ratio as the organic solvent, while refined sunflower oil functioned as the vegetable oil solvent. As for the environmentally friendly solvent, we prepared an ionic liquid microemulsion in water using the following method: a mixture of tributyloctylphosphonium bromide, Triton X 100, n-butanol (in a 3:1:0.62:0.25:0.13 mass ratio) was combined and thoroughly stirred. These compounds represented the non-polar phase, surfactant-co-surfactant, and polar phase of microemulsion, respectively [23].

2.4. Characterization of microemulsion

The density of the microemulsion was measured with a pycnometer and its conductivity was measured with a conductivity meter (Inolab, WTW, Germany). The particle size of microemulsions was determined by a zeta sizer (Nano SZ-100, Horiba, Japan) based on the dynamic light scattering (DLS) technique.

2.5. Experimental design and Optimization

The response surface method (RSM) was used to determine the extraction conditions and optimize the method for obtaining the highest amount of astaxanthin. This method allows for the examination of the main and mutual effects of variables to identify the optimal extraction conditions. In the ultrasound extraction process, three independent variables were considered: the ratio of solvent to sample (X₁) is in the range of 5 to 20 times, the power of the ultrasound device (X₂) is in the range of 60 to 200 W, and the extraction time (X₃) is in the range of 5 to 30 min. Similarly, for the microwave extraction process, the ratio of solvent to sample (X₁) in the range of 5 to 20 times, the power of the microwave device (X₂) in the range of 100 to 300 W, and the extraction time (X₃) in the range of 5 to 110 s were considered as independent variables. The selection of these variable ranges was based on the analysis of various studies on astaxanthin extraction from different sources. The amount of extracted astaxanthin was measured using a spectrophotometer and was considered as the dependent variable in the experimental design. A central composite design was utilized for the response surface method. Each independent variable was set at five levels and was coded as values of 0, ± 1, and ± α, which increased the accuracy of the experiment. Minitab 20 software was used to calculate the extraction conditions for each solvents in the ultrasound and microwave methods. Additionally, six replicates were performed at the central point to estimate the experimental error. The application of the response surface method and central composite design allowed for efficient optimization and determination of the most effective extraction conditions for each solvent in both ultrasound and microwave methods.

2.6. Ultrasound-assisted extraction

The extraction process was conducted by mixing the solvents with the powder samples at the specified ratios mentioned in the previous section. The extraction was carried out at a temperature of 25°C using an ultrasound bath (Sono Swiss, SW3H, 280W) operating at a frequency of 40 kHz and at 41°C, with varying ratios of solvent to sample, power, and time. After the extraction, the resulting extract was subjected to centrifugation at 8500 rpm for 10 minutes, maintaining a temperature of 25°C. This process separated the supernatant of the sample from the settled part. The settled part was then subjected to re-extraction using the same method. Finally, the obtained supernatant was filtered using a 0.45 µm filter to prepare it for analysis [24].

2.7. Microwave-assisted extraction

Firstly, the powder samples were mixed with solvents at the specified ratios at 25°C and the extraction process was done by microwaves (MW domestic multiwave oven, LG, Korea) with a frequency of 2.45 GHz and different values of radiation power and time. After extraction, the obtained extract was centrifuged at 10,000 rpm for 15 min at 25°C, and then the supernatant was separated and filtered with a 0.45 µm filter. For extraction with green solvent or vegetable oil the resulting extract was diluted with ethanol and then filtered to prepare for further analysis [25, 26].

2.8. Analysis

2.8.1. Determination of total carotenoid content (total astaxanthin)

The absorbance of the extract samples was measured by and UV–vis spectrophotometer (UV-1601, Shimadzu, Japan) at a wavelength of 450 nm. It is noteworthy that, at this specific wavelength, the majority of the total carotenoid content detected comprises astaxanthin .

The calculation of the total carotenoid content (TCC) was performed using the following equation, as referenced from the study conducted by [27]:

Total carotenoid content (TCC) = (Absorbance at 450 nm × Dilution factor × 100) / (Sample weight × E1%)

By utilizing this equation, the total carotenoid content in the extract samples was determined, which encompasses the predominant astaxanthin content, among other carotenoids, measured at 450 nm. To calculate the total astaxanthin content, a specific extinction coefficient (E1%) of 2100 was applied. It should be noted that the total astaxanthin content represents the majority of the total carotenoid content detected at this wavelength. Finally, the total carotenoid content (TCC) was calculated by the following equation [27]:

TCC (µg/g DW) = (V ×A ×100 )/(21 ×W) × 100 (1)

Where, V is volume (mL) of solvent used, A is absorbance and W is dry weight (g) of biomass in the solvent.

2.8.2. Spectrophotometric determination of astaxanthin

To create the standard curve of astaxanthin, various dilutions of pure astaxanthin were prepared with concentrations ranging from 0 to 1 µg/ml. For this, 2-propanol was used as the solvent for the pure astaxanthin pigment.The astaxanthin absorbance of the extracts was measured at 478 nm by UV-vis spectrophotometer. The astaxanthin concentration (AC) of the extracts was calculated using the following equation [28]:

AC (mg/mL) = (Absorbance )/1.97 (2)

2.8.3. Determination of astaxanthin recovery yield

After the analysis of extract samples, the astaxanthin recovery efficiency was calculated by the following equation [29]:

Astaxanthin recovery yield (%) = (Extracted Astaxanthin)/(Total Astaxanthin) × 100 (3)

2.8.4. Determination of antioxidant activity

To initiate the DPPH radical scavenging activity test, a DPPH ethanolic solution was prepared with a concentration of 0.2 mM. Then, different concentrations of the extracts (ranging from 0.05 to 1 mg/mL) were mixed with 2 mL of the DPPH ethanolic solution. The mixing was allowed to occur for 30 minutes. After the 30-minute incubation period, the absorbance of the sample mixture was measured using a UV–vis spectrophotometer at a wavelength of 517 nm. The DPPH radical scavenging activity was then calculated based on the following formula. This test was compared with the antioxidant capacity of synthetic antioxidant BHT in similar concentrations [30].

Free radical scavenging activity %= (〖Abs 〗_control- 〖Abs 〗_sample)/Abs_( control) ×100 (4)

Where Abs _control and Abs _sample represent the absorbance of the blank and the sample at 517 nm, respectively.

2.9. Statistical analysis

The results of the astaxanthin analysis obtained using the spectrophotometer were subjected in Minitab 20.2.2 software for the purpose of optimizing the extraction with the response surface method. The significance level for the statistical analysis was set at 5%, meaning that any p-value less than 0.05 is considered statistically significant. Statistical analysis of the data from other tests was performed using IBM SPSS Statistics 22 (IBM Corporation, Armonk, NY, USA) based on one-way analysis of variance (ANOVA). For all tests conducted, including the astaxanthin analysis and other experiments, each test was performed three times to ensure reliability and consistency of the results. The reported results were presented as the mean ± standard deviation.

3.1. Physical properties of microemulsion

The density value of the microemulsion was found to be approximately 0.9715 g/cm3, which was quite similar to that of water. This similarity in density suggests that the microemulsion is stable and well-dispersed, making it a promising candidate for various applications. The microemulsion conductivity was 312 µSiemens and the particle size was about 15.8 nm (Fig. 1)

3.2. Optimizing the extraction of astaxanthin carotenoid pigment by ultrason-

ic-assisted extraction

The results of optimizing the extraction of astaxanthin carotenoid pigment using the ultrasound technique with the response surface method are summerized in Tables 1 and 3. The extracted total carotenoid amounts from the samples of F. merguiensis shrimp and P. maeoticus Gammarus using different solvents are as follows. Total carotenoid amounts for organic solvent were obtained between 28.476–64.142 and 9.745–33.501, for vegetable oil between 8.682–45.364 and 1. 459 − 7.11 and by ionic microemulsion between 44.904–90.454 and 76.269–76.269 µg/g, respectively. Astaxanthin concentration for F. merguiensis shrimp and P. maeoticus Gammarus samples by Ionic microemulsion solvent was between 41.237–85.475 and 26.555–68.142 mg/mL, respectively and Astaxanthin recovery yield was between 91.33–98.33 and 84.66–96.66%, respectively (Tables 1 and 3).

3.3. Optimizing the extraction of astaxanthin carotenoid pigment by microwave-assisted extraction

The extracted total carotenoid amounts from the samples of F. merguiensis shrimp and P. maeoticus Gammarus using different solvents are as follows. Total carotenoid amounts for Organic Solvent between25.634-69.253 and 26.094–12.174, for Vegetable oil between 17.602–50.698 and 2.968–10.158 and by Ionic microemulsion was between 46.872–87.967 and 28.475–89.396 µg/g, respectively. Astaxanthin concentration for F. merguiensis shrimp and P. maeoticus Gammarus waste extracted by Ionic microemulsion solvent was between 44.951–84.348 and 27.856–71.38 mg/mL and Astaxanthin recovery yield was was between 76.66–96.66 and 66.66-98%, respectively (Tables 2 and 4).

3.4. DPPH antioxidant properties assay test

Figure (5A) illustrates that the ultrasound method yields superior results. Although BHT exhibits the highest inhibition percentage at the same concentration (94%), the ultrasound method outperforms, with the oil solvent achieving 83% inhibition, surpassing the ionic solvent (71%) and the organic solvent (67%) in effectiveness. In the microwave method depicted in Figure (5B), results are comparatively weaker than ultrasound. Nevertheless, the oil solvent in this method exhibits a notable 79% inhibition, outperforming the ionic solvent (65%) and the organic solvent (58%) in inhibitory efficacy.

Previous research on microemulsions based on ionic liquids has been limited, prompting the current study to investigate their physical properties. Specifically, the study focused on examining the density, conductivity, and particle size in these microemulsions. Gao et al. (2020) studied the recovery of natural astaxanthin from shrimp waste by ultrasound method and using microemulsion. They evaluated the efficiency of microemulsions containing tributyloctylphosphonium bromide ([P4448] Br), tributyloctylphosphonium trifluoroacetate ([P4448] CF3COO), or tetrabutylphosphatylphosphonium ([P4448] CF3COO)) in astaxanthin extraction compared to organic solvents (ethanol, acetone, and Dimethyl sulfoxide)[23]. The obtained results were similar to our results. Density values of pure ionic liquids and their microemulsions were almost similar to water in the temperature range of 303–323 K. The viscosity of the studied ionic liquids was 20–400 times that of organic solvents (ethanol, acetone and DMSO) and the conductivity for the studied microemulsions was similar to the density and viscosity but in the opposite direction. Higher conductivity corresponds to more dissolve ionic species that are freely present and moving in solution.The small particle size indicates that the microemulsion possesses excellent stability and uniformity.

Among the factors affecting by the ultrasonic method in both F. merguiensis shrimp and P. maeoticus Gammarus samples, only the ratio of sample to solvent factor was found to be significant at a significant level of p < 0.05 (Tables 5 and 7). The best conditions for the extraction of astaxanthin by ultrasonic method and with ionic microemulsion solvent from F. merguiensis shrimp and P. maeoticus Gammarus samples were, the sample to solvent ratio was 1:5, the power was 60 W and the time was 30 minutes. The astaxanthin concentration obtained for the F. merguiensis shrimp sample was 76.30 mg/ml, the total carotenoid content was 79.77µg/g and the extraction efficiency was 95.33%, and for the P. maeoticus Gammarus sample, these parameters were 68.12 mg/ml, 76.2 µg/g and 89.33%, respectively. The results indicate that among the three solvents used for extraction in both F. merguiensis shrimp and P. maeoticus Gammarus, the ionic microemulsion solvent exhibited the best performance in extracting astaxanthin. Moreover, in this experiment, the F. merguiensis shrimp yielded a higher amount of total carotenoid compared to the other sample. These findings suggest that the use of ionic microemulsion as a solvent in the ultrasound extraction technique could be a favorable choice for obtaining higher amounts of astaxanthin from the samples of F. merguiensis shrimp and P. maeoticus Gammarus. The solvent ability to extract the targeted ingredient is primarily influenced by its electrostatic and hydrophilic/hydrophobic interactions with target molecules[23]. Therefore, due to the hydrophobic and electrostatic interactions between the cationic head groups of microemulsion and astaxanthin, and on the other hand, the interaction of microemulsion anions to both hydrophilic ends of astaxanthin by hydrogen bonding can be considered as the reason for the better performance of microemulsions in the extraction of astaxanthin. Gao et al. (2020) recovered natural astaxanthin from shrimp waste with ultrasonic-assisted extraction and using microemulsion. The effect of ultrasonic power, ultrasonic time, and microemulsion composition on extraction efficiency (EEAst) and extraction yield (EAst) of astaxanthin was studied. The results showed that compared to organic solvents (ethanol, acetone, and dimethyl sulfoxide), the microemulsion containing tributyloctylphosphonium bromide, tributyloctylphosphoniumtrifluoroacetate, or tetrabutylphosphatiylphosphonium led to a significant increase in astaxanthin extraction due to stronger electrostatic interactions and more hydrogen bonding interactions [23]. In ultrasound method, the astaxanthin recovery yield increased with the increase in the amount of solvent (Figs. 3 and 4). The high ratio of solvent to sample can activate the diffusion movement of the intracellular matrix to the outside, which ultimately improves the efficiency of pigment extraction. These results are due to the fact that in a higher solvent volume, the sample can be dissolved in a better and more effective way, and the effect of the cavitation phenomenon is better manifested. However, a decreasing trend was observed in high ratios of solvent due to the limited capacity of solvent dissolution. It was determined that the ideal ratio of sample to solvent should be 1:5. This decision was made to prevent any unnecessary wastage of the solvent. The astaxanthin recovery yield increased with the increase of ultrasound power and extraction time at first, then it decreased to some extent with the increase of two parameters (Figs. 3 and 4). On the other hand, we have seen similar destructive results from the long-term ultrasonic method due to the increase in the cavitation phenomenon and the creation of intense local heat, so it is desirable to use the ultrasonic extraction method in a short time. By increasing the power of the device, we see the easier breaking of tissues and cell matrix, which increases the rate of mass transfer and the amount of intracellular compounds to the outside, and better extraction is achieved [24]. The power of the device will cause the rapid release of pigment and intra-tissue materials as target compounds to the outside. However, at high powers, due to the excessive increase of bubbles, the effect of cavitation phenomenon decreases, and ultrasound waves are scattered and reduce the extraction efficiency. Also, at high powers, the local heat created will be the cause of the destruction of the extracted pigment. Zou et al. (2013) studied the ultrasound-assisted extraction of astaxanthin from Haematococcuspluvialis. They showed that as the liquid-to-solid ratio increased, the yield of astaxanthin increased. However, when the liquid-to-solid ratio was increased from 20:1 to 30:1, the yield of astaxanthin remained almost unchanged. They considered the reason for this to be the more effective solubility of the constituents, which can lead to an increase in extraction efficiency. However, increasing the solvent-to-sample ratio caused solvent wastage. Therefore, due to the lack of change in astaxanthin efficiency after 20:1, the liquid-to-solid ratio of 20:1 was used in the next experiments. They also investigated the effect of extraction time on astaxanthin yield, which was consistent with our results. So that the efficiency of astaxanthin increased from 5 minutes to 15 minutes and then the efficiency decreased from 15 to 30 minutes. In fact, the extraction efficiency increases with time before establishing a balance for the target components inside and outside the plant cells. But after establishing the balance, increasing the time cannot increase the extraction yield [24]. Hu et al. (2019) investigated the extraction of astaxanthin from shrimp shells and the effect of different treatments on its concentration. They reported that for astaxanthin extraction, the sample-to-solvent ratio should be 1:7 and a 30-minute ultrasound gives the most favorable results. Additionally, they found that reducing the ratio of solid to liquid will not significantly increase the output [31]. Zhang et al. (2014) conducted a study on the extraction of astaxanthin from shrimp by-products using deep eutectic solvents and ultrasound. The results of extraction using eutectic solvent indicated that the astaxanthin obtained from the shell was 146 µg/g of waste, while from the head, it was 218 µg/g of waste. Meanwhile, the extraction with ethanol organic solvent showed that the amount of extracted astaxanthin from the shell was 102 µg/g of waste and 158 µg/g from the head. The amount of extracted astaxanthin increased with increasing ultrasonication time [20]. Bi et al. (2010) successfully extracted astaxanthin from shrimp waste using ionic liquids and ultrasound. This study examined seven types of uniimidazolium liquids, with different cationsand anions. The results determined that an ionic liquid in ethanol solution with a concentration of 0.50 mol/L was the most appropriate solvent. By optimizing the ultrasound extraction conditions, the amount of extracted astaxanthin increased by 98% (92.7 µg/g) compared to the conventional method (46.7 µg/g) [22].

For the sample of F. merguiensis shrimp, only the factor of sample-to-solvent ratio was significant at the significance level of p < 0.05, and for the sample of P. maeoticus Gammarus, none of the factors were significant (Tables 6 and 8).

The best conditions for the extraction of astaxanthin by microwave method and with ionic microemulsion solvent from F. merguiensis shrimp sample was, the sample to solvent ratio was 1:5, the power was 100 W and the time was 91.81 s. The astaxanthin concentration obtained for the F. merguiensis shrimp sample was 80.22 mg/ml, the total carotenoid content was 83.60 µg/g and the extraction efficiency was 93.733%, and for extracted astaxanthin from F. merguiensis shrimp sample using vegetable oil, these parameters were 41.96 mg/ml, 48.01 µg/g and 88.24%, respectively. Based on the findings from microwave extraction, it was found that the ionic microemulsion solvent was more effective compared to the other two solvents used in both samples. Additionally, when comparing the two samples, the F. merguiensis shrimp sample had a higher quantity of extracted total carotenoids. Considering the fact that astaxanthin pigment is part of heat-sensitive carotenoid pigments, it can be said that due to the destructive effect of long-term microwave radiation on astaxanthin pigment, the shorter the extraction time is the better the pigments are preserved. Also, preliminary experiments with extraction using microwaves show the dependence between temperature and microwave power. As a result, these two parameters cannot be controlled simultaneously. Therefore, increasing the power of the machine has a negative effect on the durability of astaxanthin pigment and reduces the overall extraction efficiency. However, according to the results, the microwave method was more effective in extracting astaxanthin than ultrasound. Zhao et al. (2009) in the optimization of microwave-assisted extraction of astaxanthin from Haematococcuspluvialis showed that microwave power and extraction time showed a similar quadratic effect on extraction content of astaxanthin and the content of astaxanthin extraction increased with the increase of these two factors at first and then It decreased to some extent. Therefore, higher power and longer extraction time were not suitable for the extraction of astaxanthin, which was attributed to the increase in temperature at high powers, which affected the stability of astaxanthin and may disrupt the structure of astaxanthin [25]. Zou et al. (2013) extracted astaxanthin from Haematococcusplevialis with Ultrasound-Assisted. Extraction solvent, liquid to solid ratio, extraction temperature and extraction time were optimized in this study by response surface method. The optimal extraction conditions included ethanol 48.0% in ethyl acetate, liquid-to-solid ratio 20:1 (mL/g) and extraction for 16 min at a temperature of 41.1°C under 200 W ultrasonic radiation. In this condition, the yield of astaxanthin was 27.58 ± 0.40 mg/g [24]. Zhao et al. (2019), the extraction of astaxanthin from Haematococcuspluvialis was optimized using microwaves, and the antioxidant activity of the extracts was investigated. Four independent variables of microwave power (W), extraction time (seconds), solvent volume (mL), and extraction number were optimized. Regarding the extraction efficiency, the optimal MAE conditions included microwave power of 141 W, extraction time of 83 s, solvent volume of 9.8 ml, and extraction number of four times. Under these optimal conditions, approximately 594 µg of astaxanthin were extracted from the Haematococcuspluvialis, which closely matched the predicted content of 592 µg. Additionally, the extracts obtained under these optimal conditions exhibited a strong ability to inhibit the peroxidation of linoleic acid, a strong radical inhibition property against DPPH, and also a strong reducing power [32]. Hooshmand et al. (2018) in a study optimized the extraction of carotenoid pigments from wastes of Portunuspelagicus and Penaeussemisulcatus by ultrasound and microwave and analyzed the amount of extracted pigment. The studied factors included the extraction number, the extraction time, the power of ultrasound and microwave devices, and the ratio of solvent to waste. To extract carotenoid pigment using ultrasound waves from crab waste, they extracted 3 times, with a waste-to-solvent ratio of 1:15, power of 90 w, and time of 30 s. Finally, the amount of pigment was 1407.12 ± 0.584 µg/g. extraction was done from shrimp waste once with waste to solvent ratio of 1:10, power of 150 w, and time of 105 s and the amount of pigment 1257.43 ± 0.023 µg/g was obtained [33].

2,2-diphenyl-1-picrylhydrazyl (DPPH) is a hydrophobic radical that has a peak absorption at a wavelength of 517 nm. In the DPPH assay, antioxidant compounds containing hydroxyl groups cause the reduction of DPPH molecules by giving hydrogen atoms to DPPH radicals, resulting in a shift in the color of the reaction solution from deep purple to light yellow. Consequently, the absorption at the 517 nm wavelength decreases [34]. The absorption at the 517 nm wavelength reflects the quantity of DPPH that remains. The results showed that vegetable oil showed the highest antioxidant capacity among different solvent methods which agreed with the results of Kang & Sim (2008)[35]. In the explanation, it can be said that since the astaxanthin pigment is fat-soluble and is better preserved in an oily solvent, it is protected against environmental oxygen and its antioxidant properties are preserved more than the other two solvents. However, despite the extraction of more astaxanthin pigment, the ionic liquid solvent shows lower antioxidant properties than the oil solvent due to partial oxidation or possible subsequent damage during the storage period. Kang & Sim (2008) treated H. pluvialis cells with common vegetable oils and the astaxanthin oil yields reach more than 88% [35]. Dong et al. (2014) also evaluated four different methods for the extraction of astaxanthin from H. pluvialis. Methods include: hydrochloric acid pretreatment followed by acetone extraction (HCl-ACE), hexane/isopropanol (6 : 4, v/v) mixture solvents extraction (HEX-IPA), methanol extraction followed by acetone extraction (MET-ACE, 2-step extraction), and soy-oil extraction, were intensively. The results showed that the HCl-ACE method was able to obtain the highest yield of oil (33.3 ± 1.1%) and the amount of astaxanthin (19.8 ± 1.1%). The DPPH radical scavenging activity of the extract obtained with HCl-ACE was 73.2 ± 0.1%, which is the highest among the four methods. The reducing power of the extract obtained by four extraction methods was also investigated. It was concluded that the proposed extraction method of HCl-ACE in this work provides the possibility of efficient extraction of astaxanthin with high antioxidant properties [36]. to the results of extracted astaxanthin in both ultrasound and microwave methods, it had the ability to inhibit DPPH free radicals, however, due to the sensitivity of astaxanthin pigment to heat and the destructive effect of long-term microwave radiation on astaxanthin pigment, Astaxanthin extracted by microwave method had lower antioxidant activity compared to astaxanthin extracted by ultrasound method. Therefore, in the microwave method, the shorter the extraction time and the lower the power of the device, the better the pigments will be preserved and the antioxidant activity will be higher. Our results were consistent with the results of Roy et al. (2021) [37]. They extracted astaxanthin from shrimp waste using natural deep eutectic solvents with the help of ultrasound and studied its application in bioactive films. The extracted astaxanthin extract clearly showed strong antioxidant activities of ABTSþ and DPPHþ. Sowmya & Sachindra (2012) reported that ASX has strong antioxidant activity and even a small amount can show its activity in the final product [38].

Astaxanthin is classified in the xanthophyll group due to the presence of carbon, hydrogen and oxygen atoms in its structure. Astaxanthin contains carbon-carbon chains with conjugated bonds in its molecular structure. This structure effectively has the ability to remove active oxygen, the ability to inhibit free radicals and has antioxidant properties. In recent years, various methods have been used to extract astaxanthin, including ultrasound and microwave methods, which can lead to a reduction in the volume of the solvent used and an increase in extraction efficiency. Due to the problems caused by the separation of solvents from products, astaxanthin can be effectively extracted using green solvents such as ionic liquids. Therefore, the aim of this study was to extract astaxanthin from F. merguiensis shrimp and P. maeoticus Gammarus samples using ultrasonic and microwave methods using three solvents including organic solvent, vegetable oil and ionic microemulsion. The results showed that the ionic microemulsion solvent can perform better in the extraction of astaxanthin due to its hydrophobic and electrostatic interactions with astaxanthin. On the other hand, based on the results, extraction by ultrasonic method preserves the structure and antioxidant properties of astaxanthin due to the lower temperature during extraction, and astaxanthin extracted by ultrasonic method is preserved in a better way. However, the amount of extracted astaxanthin, the total concentration of extracted carotenoid and the extraction efficiency were higher in the microwave method than in the ultrasound method.

Author Contribution

A.Wrote the main manuscriptB.SupervisedC.Performed statistical D.prepared the formulasE.gave idea about the topicand all authors reviewed the manuscript.

Acknowledgements

This study did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Gao, J., et al., Enhanced extraction of astaxanthin using aqueous biphasic systems composed of ionic liquids and potassium phosphate. Food chemistry, 2020. 309: p. 125672. https://doi.org/10.1016/j.foodchem.2019.125672.
Saini, R.K. and Y.-S. Keum, Carotenoid extraction methods: A review of recent developments. Food chemistry, 2018. 240: p. 90–103. https://doi.org/10.1016/j.foodchem.2017.07.099.
Roy, V.C., et al., Recovery and bio-potentialities of astaxanthin-rich oil from shrimp (Penaeus monodon) waste and mackerel (Scomberomous niphonius) skin using concurrent supercritical CO2 extraction. The Journal of Supercritical Fluids, 2020. 159: p. 104773..https://doi.org/10.1016/j.jclepro.2020.125417.
Liu, X., et al., Recent advances in health benefits and bioavailability of dietary astaxanthin and its isomers. Food Chemistry, 2023. 404: p. 134605. https://doi.org/10.1016/j.foodchem.2022.134605.
Patil, A.D., P.J. Kasabe, and P.B. Dandge, Pharmaceutical and nutraceutical potential of natural bioactive pigment: astaxanthin. Natural products and bioprospecting, 2022. 12(1): p. 25. https://doi.org/10.1016/j.foodchem.2022.134605.
Kim, B., et al., Cell disruption and astaxanthin extraction from Haematococcus pluvialis: Recent advances. Bioresource Technology, 2022. 343: p. 126124. https://doi.org/10.1016/j.biortech.2021.126124.
Chen, Y., et al., Advances of astaxanthin-based delivery systems for precision nutrition. Trends in Food Science & Technology, 2022. https://doi.org/10.1016/j.tifs.2022.07.007.
Yao, Q., et al., A natural strategy for astaxanthin stabilization and color regulation: Interaction with proteins. Food Chemistry, 2023. 402: p. 134343. https://doi.org/10.1016/j.foodchem.2022.134343.
Hatta, F.A.M. and R. Othman, Carotenoids as potential biocolorants: A case study of astaxanthin recovered from shrimp waste, in Carotenoids: Properties, processing and applications. 2020, Elsevier. p. 289–325. https://doi.org/10.1016/B978-0-12-817067-0.00009-9.
Hu, J., et al., Extraction and purification of astaxanthin from shrimp shells and the effects of different treatments on its content. Revista Brasileira de Farmacognosia, 2019. 29: p. 24–29. https://doi.org/10.1016/j.bjp.2018.11.004.
Jeyachandran, S., et al., Identification and characterization of bioactive pigment carotenoids from shrimps and their biofilm inhibition. Journal of Food Processing and Preservation, 2020. 44(10): p. e14728. https://doi.org/10.1111/jfpp.14728.
Cheong, J. and M. Muskhazli, Turning leftover to treasure: An overview of astaxanthin from shrimp shell wastes. Global Perspectives on Astaxanthin, 2021: p. 253–279. https://doi.org/10.1016/B978-0-12-823304-7.00022-2.
Wang, W., et al., Gill transcriptomes reveal involvement of cytoskeleton remodeling and immune defense in ammonia stress response in the banana shrimp Fenneropenaeus merguiensis. Fish & shellfish immunology, 2017. 71: p. 319–328. https://doi.org/10.1016/j.fsi.2017.10.028.
Ertl, N.G., et al., Molecular characterisation of colour formation in the prawn Fenneropenaeus merguiensis. PLoS One, 2013. 8(2): p. e56920. https://doi.org/10.1371/journal.pone.0056920.
Ahmadi, M., et al., Caspian Sea gamarus (Pontogammarus maeoticus) as a carotenoid source for muscle pigmentation of rainbow trout (Oncorhynchus mykiss). Iranian Journal of Fisheries Sciences, 2005. 5(1): p. 1–12. 20.1001.1.15622916.2005.5.1.1.1.
Yu, W. and J. Liu, Astaxanthin isomers: Selective distribution and isomerization in aquatic animals. Aquaculture, 2020. 520: p. 734915. https://doi.org/10.1016/j.aquaculture.2019.734915.
Li, J., et al., High pressure extraction of astaxanthin from shrimp waste (Penaeus Vannamei Boone): effect on yield and antioxidant activity. Journal of Food Process Engineering, 2017. 40(2): p. e12353.https://doi.org/10.1111/jfpe.12353.
Parjikolaei, B.R., et al., Process design and economic evaluation of green extraction methods for recovery of astaxanthin from shrimp waste. Chemical Engineering Research and Design, 2017. 117: p. 73–82. https://doi.org/10.1016/j.cherd.2016.10.015.
Shen, Q., et al., Ultrasonic-assisted extraction of zeaxanthin from Lycium barbarum L. with composite solvent containing ionic liquid: Experimental and theoretical research. Journal of Molecular Liquids, 2022. 347: p. 118265. https://doi.org/10.1016/j.molliq.2021.118265.
Zhang, Y., et al., Optimization of ionic liquid-based microwave-assisted extraction of isoflavones from Radix puerariae by response surface methodology. Separation and Purification Technology, 2014. 129: p. 71–79. https://doi.org/10.1016/j.seppur.2014.03.022.
Ventura, S.P., et al., Ionic-liquid-mediated extraction and separation processes for bioactive compounds: past, present, and future trends. Chemical Reviews, 2017. 117(10): p. 6984–7052. https://doi.org/10.1021/acs.chemrev.6b00550.
Bi, W., et al., Task-specific ionic liquid-assisted extraction and separation of astaxanthin from shrimp waste. Journal of Chromatography B, 2010. 878(24): p. 2243–2248. https://doi.org/10.1016/j.jchromb.2010.06.034.
Gao, J., et al., Recovery of astaxanthin from shrimp (Penaeus vannamei) waste by ultrasonic-assisted extraction using ionic liquid-in-water microemulsions. Food chemistry, 2020. 325: p. 126850. https://doi.org/10.1016/j.foodchem.2020.126850.
Zou, T.-B., et al., Response surface methodology for ultrasound-assisted extraction of astaxanthin from Haematococcus pluvialis. Marine drugs, 2013. 11(5): p. 1644–1655. https://doi.org/10.3390/md11051644.
Zhao, L., et al., Optimization of microwave-assisted extraction of astaxanthin from Haematococcus pluvialis by response surface methodology and antioxidant activities of the extracts. Separation Science and Technology, 2009. 44(1): p. 243–262. https://doi.org/10.1080/01496390802282321.
Fan, Y., et al., Biocompatible protic ionic liquids-based microwave-assisted liquid-solid extraction of astaxanthin from Haematococcus pluvialis. Industrial Crops and Products, 2019. 141: p. 111809. https://doi.org/10.1016/j.indcrop.2019.111809.
Haque, F., et al., Intensified green production of astaxanthin from Haematococcus pluvialis. Food and bioproducts processing, 2016. 99: p. 1–11. https://doi.org/10.1016/j.fbp.2016.03.002.
Khoo, K.S., et al., Integrated ultrasound-assisted liquid biphasic flotation for efficient extraction of astaxanthin from Haematococcus pluvialis. Ultrasonics Sonochemistry, 2020. 67: p. 105052. https://doi.org/10.1016/j.ultsonch.2020.105052.
Ruen-ngam, D., A. Shotipruk, and P. Pavasant, Comparison of Extraction Methods for Recovery of Astaxanthin from Haematococcus pluvialis. Separation Science and Technology, 2010. 46(1): p. 64–70. https://doi.org/10.1080/01496395.2010.493546.
Zhao, X., et al., Effect of extraction and drying methods on antioxidant activity of astaxanthin from Haematococcus pluvialis. Food and Bioproducts Processing, 2016. 99: p. 197–203. https://doi.org/10.1016/j.fbp.2016.05.007.
Hu, J., et al., Extraction and purification of astaxanthin from shrimp shells and the effects of different treatments on its content. Revista Brasileira de Farmacognosia, 2019. 29(1): p. 24–29. https://doi.org/10.1016/j.bjp.2018.11.004.
Zhao, T., et al., Research progress on extraction, biological activities and delivery systems of natural astaxanthin. Trends in Food Science & Technology, 2019. 91: p. 354–361. https://doi.org/10.1016/j.tifs.2019.07.014.
Hooshmand, H., et al., The optimization of extraction of carotenoids pigments from blue crab (Portunus pelagicus) and shrimp (Penaeus semisulcatus) wastes using ultrasound and microwave. Journal of Marine Science and Technology, 2021. 20(2): p. 72–93. https://doi.org/10.22113/jmst.2018.105737.2084.
Chandra Roy, V., et al., Extraction of astaxanthin using ultrasound-assisted natural deep eutectic solvents from shrimp wastes and its application in bioactive films. Journal of Cleaner Production, 2021. 284: p. 125417. https://doi.org/10.1016/j.jclepro.2020.125417.
Kang, C.D. and S.J. Sim, Direct extraction of astaxanthin from Haematococcus culture using vegetable oils. Biotechnology Letters, 2008. 30(3): p. 441–444. https://doi.org/10.1007/s10529-007-9578-0.
Dong, S., et al., Four different methods comparison for extraction of astaxanthin from green alga Haematococcus pluvialis. The Scientific World Journal, 2014. 2014. https://doi.org/10.1155/2014/694305.
Roy, V.C., et al., Extraction of astaxanthin using ultrasound-assisted natural deep eutectic solvents from shrimp wastes and its application in bioactive films. Journal of Cleaner Production, 2021. 284: p. 125417. https://doi.org/10.1016/j.jclepro.2020.125417.
Sowmya, R. and N. Sachindra, Evaluation of antioxidant activity of carotenoid extract from shrimp processing byproducts by in vitro assays and in membrane model system. Food Chemistry, 2012. 134(1): p. 308–314. https://doi.org/10.1016/j.foodchem.2012.02.147.

Table 1. The independent factors and combination of independent factors for optimization theultrasound-assisted extraction of astaxanthinwith different solvents fromF. merguiensis shrimp wastes.

Run	Independent variables			Total carotenoid content (µg/g DW)			Astaxanthin Concentration (mg/mL)			Astaxanthin recovery yield (%)
Run	X₁	X₂	X₃	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion

1	5	200	5	46.936	40.745	76.142	37.539	38.491	69.142	83.33	93.66	91.33
2	20	60	30	28.476	17.682	49.682	21.555	16.205	48.047	78	91	96.33
3	5	130	17.5	57.332	35.015	78.92	45.158	34.11	72.761	81.33	97	91.66
4	20	130	17.5	35.983	18.666	48.46	26.761	16.936	47.11	75.33	92	96.66
5	5	60	30	59.586	43.253	79.777	45.888	41.713	76.301	78.66	96	95.33
6	5	200	30	64.142	45.364	90.459	53.444	43.628	85.475	85.66	96	93.66
7	12.5	130	17.5	53.444	24.047	65.443	37.793	19.634	62.031	71.33	81.33	94
8	12.5	130	17.5	35.047	28.856	74.761	26.285	27.507	72.412	81	94.33	96
9	12.5	60	17.5	52.078	14.65	56.555	43.571	13.968	53.713	83.66	94.33	94.33
10	12.5	130	30	51.31	26.333	66.682	41.793	24.872	63.936	84.66	93.66	95.33
11	20	60	5	31.92	8.682	50.746	25.555	7.364	50.333	80.66	84.66	98.33
12	12.5	130	5	44.412	24.079	65.031	36.618	22.444	62.65	84.66	93.66	96
13	20	200	5	36.745	12.84	44.904	25.793	10.253	41.237	69.66	79	90.66
14	12.5	130	17.5	49.047	31.92	65.65	33.317	30.539	62.269	71	95.66	94
15	12.5	130	17.5	54.444	16.793	64.73	38.872	15.888	62.189	73.33	94	95.33
16	5	60	5	53.618	31.65	78.047	42.73	30.507	73.38	83.66	96	93.66
17	12.5	200	17.5	30.84	27.856	58.523	21.888	26.095	56.301	77	92.33	95.66
18	12.5	130	17.5	57.063	24.015	67.031	41.729	19.221	64.348	74.33	79.66	95.33
19	12.5	130	17.5	53.317	37.945	70.586	39.682	36.603	67.364	77.33	96	94.66
20	20	200	30	45.825	28.729	47.364	33.031	27.221	45.714	72.33	94.33	96

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively.

Table 2. The independent factors and combination of independent factors for optimization themicrowave-assisted extraction of astaxanthin with different solvents from F. merguiensis shrimp wastes.

Run	Independent variables			Total carotenoid content (µg/g DW)			Astaxanthin Concentration (mg/mL)			Astaxanthin recovery yield (%)
Run	X₁	X₂	X₃	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion
1	20	300	5	33.517	25.158	46.872	20.968	23.221	44.951	62	91.66	95.33
2	20	200	57.5	41.777	18.936	61.269	27.841	16.761	59.374	66.33	87.66	96.66
3	12.5	200	57.5	43.596	31.231	74.507	31.158	27.904	71.983	70.66	85.66	95.66
4	12.5	200	57.5	59.238	26.65	72.983	46.015	23.476	69.666	79	86	95
5	20	100	110	40.834	17.602	49.39	39.808	16.285	47.713	97	92	96
6	12.5	200	110	66.739	45.555	80.412	51.494	41.503	74.031	78	90.33	92
7	12.5	200	5	56.229	28.587	67.332	39.618	26.348	64.698	69.66	92	95.33
8	5	300	5	52.523	46.126	59.206	51.945	41.968	57.079	98.33	90.33	95.66
9	12.5	200	57.5	66.205	43.04	78.11	46.171	38.396	74.317	69.33	88.66	94.66
10	5	200	57.5	25.634	42.913	67.936	24.523	39.698	65.507	94.66	92	96
11	5	100	5	62.327	50.698	82.428	45.516	39.825	79.017	75.33	79	95.33
12	12.5	100	57.5	57.501	36.841	71.999	38.904	32.237	69.38	67.33	86.66	96
13	12.5	200	57.5	69.253	37.04	87.396	62.802	33.634	71.714	91	90.33	82.66
14	20	300	110	49.43	18.555	64.342	43.618	16.476	62.729	90	87.33	96.66
15	12.5	200	57.5	47.812	34.671	68.812	40.094	30.936	65.999	86.33	88.66	95.33
16	12.5	300	57.5	54.57	30.532	75.206	52.618	27.809	72.697	96	90.33	96.33
17	5	300	110	35.088	44.252	66.65	33.618	40.796	61.681	95.33	91.66	92
18	5	100	110	41.92	45.872	87.967	40.364	41.961	84.348	96.33	91	95.33
19	12.5	200	57.5	48.411	42.666	73.378	39.999	29.279	59.095	83.33	73	76.66
20	20	100	5	38.079	20.913	51.872	24.65	19.015	49.999	64.33	90.66	96

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively.

Table 3. The independent factors and combination of independent factors for optimization theultrasound-assisted extraction of astaxanthin with different solvents from P. maeoticus Gammarus.

Run	Independent variables			Total carotenoid content (µg/g DW)			Astaxanthin Concentration (mg/mL)			Astaxanthin recovery yield (%)
Run	X₁	X₂	X₃	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion
1	5	200	5	24.698	4.475	44.142	21.729	2.825	39.983	85.33	59.66	90
2	20	60	30	12.237	1.936	27.364	9.062	1.412	26.555	73.33	70.66	96.66
3	5	130	17.5	33.501	2.269	57.285	27.269	1.698	47.126	81.66	72.33	84.66
4	20	130	17.5	14.475	4.491	33.682	10.571	4.237	31.745	72.33	94	94
5	5	60	30	22.809	4.888	76.269	19.285	4.317	68.142	83	84.33	89.33
6	5	200	30	25.158	7.11	68.11	18.11	6.618	58.364	73	92.33	86.33
7	12.5	130	17.5	16.111	6.047	54.507	12.349	4.793	47.825	76.66	74	87
8	12.5	130	17.5	15.364	3.936	54.57	12.65	2.936	52.46	83	70.33	95.66
9	12.5	60	17.5	18.618	1.825	52.777	14.936	1.539	49.682	79.33	83.66	93.66
10	12.5	130	30	23.11	5.523	52.84	17.888	5.142	49.491	78	92.66	93.33
11	20	60	5	9.999	1.872	36.729	7.777	1.333	34.841	75.66	70	94.33
12	12.5	130	5	13.221	3.253	51.428	10.507	1.523	49.364	79.66	47.33	95
13	20	200	5	9.745	1.459	37.301	6.967	1.269	33.571	71.33	86	89.33
14	12.5	130	17.5	20.824	4.364	43.19	17.729	3.841	40.904	83.66	86.33	94.33
15	12.5	130	17.5	21.158	3.094	51.317	16.189	2.602	49.015	76	84	95
16	5	60	5	30.396	2.993	51.332	28.587	2.476	48.682	93.66	81.66	94.66
17	12.5	200	17.5	15.936	3.174	48.19	13.269	1.698	46.221	83	53	95.33
18	12.5	130	17.5	25.285	3.967	52.825	20.301	3.317	47.634	80	83.33	89.66
19	12.5	130	17.5	22.031	4.729	47.968	17.158	4.078	45.174	76	81.33	93.66
20	20	200	30	14.301	6.57	40.968	11.174	4.761	38.92	72.66	71.66	94.33

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively.

Table 4. The independent factors and combination of independent factors for optimization themicrowave-assisted extraction of astaxanthin with different solvents from P. maeoticus Gammarus.

Run	Independent variables			Total carotenoid content (µg/g DW)			Astaxanthin Concentration (mg/mL)			Astaxanthin recovery yield (%)
Run	X₁	X₂	X₃	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion	Organic Solvent	Vegetable oil	Ionic microemulsion

1	20	300	5	13.897	6.252	28.475	10.936	5.587	27.856	77	89	97.33
2	20	200	57.5	14.983	7.841	32.237	10.444	7.507	31.46	69	95.33	97
3	12.5	200	57.5	20.682	8.777	45.968	15.126	8.317	45.063	73	94	97.33
4	12.5	200	57.5	14.682	6.009	56.475	9.983	4.205	49.301	67	67.66	88
5	20	100	110	12.857	2.968	64.887	8.666	2.444	45.714	67	81	70
6	12.5	200	110	18.401	7.444	68.602	12.777	6.285	66.65	68.66	83	96.66
7	12.5	200	5	13.396	9.986	47.921	8.771	8.663	47.031	65	87.66	97.33
8	5	300	5	16.396	4.12	81.824	14.269	3.317	54.983	86.33	80.66	66.66
9	12.5	200	57.5	15.174	3.079	52.38	11.443	2.523	51.412	74.66	80.66	98
10	5	200	57.5	15.358	7.65	48.713	13.348	6.761	47.825	85.66	86	97.66
11	5	100	5	26.094	5.92	60.459	20.19	5.552	58.406	77	93	96.66
12	12.5	100	57.5	16.396	5.57	54.888	11.825	4.586	53.809	71.66	81.66	97.66
13	12.5	200	57.5	16.983	8.253	74.748	11.571	7.507	71.38	68	88.33	95.33
14	20	300	110	14.539	4.348	60.412	10.079	3.824	47.555	69	81.66	78.66
15	12.5	200	57.5	16.285	10.158	59.031	11.714	7.983	44.951	71.33	75	77.66
16	12.5	300	57.5	16.358	7.444	89.396	11.38	6.65	66.111	69	88.66	72
17	5	300	110	25.729	5.824	54.936	17.555	4.735	52.285	68	81.33	95
18	5	100	110	22.428	7.159	45.586	15.999	6.314	44.253	70.66	89	96.66
19	12.5	200	57.5	16.712	6.935	42.841	11.777	6.507	40.745	69.66	93.33	94.66
20	20	100	5	12.174	4.587	31.317	8.809	4.396	30.634	71.66	93.66	97

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively.

Table 5.Analysis of variance for the ultrasound-assisted extraction of astaxanthin with different solvents from F. merguiensis shrimp wastes.

Total carotenoid content
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	1181.66	1542.45	2667.65	3	3	3	393.89	514.15	889.22	6.87	17.86	39.45	0.0035	< 0.0001	< 0.0001
X₁	1054.01	1197.45	2630.53	1	1	1	1054.01	1197.45	2630.53	18.39	41.60	116.70	0.0006	< 0.0001	< 0.0001
X₂	0.1416	156.95	0.6682	1	1	1	0.1416	156.95	0.6682	0.0025	5.45	0.0296	0.9610	0.0329	0.8655
X₃	127.51	188.05	36.46	1	1	1	127.51	188.05	36.46	2.23	6.53	1.62	0.1552	0.0211	0.2216
Residual	916.87	460.51	360.65	16	16	16	57.30	28.78	22.54
Lack of Fit	600.80	191.66	284.57	11	11	11	54.62	17.42	25.87	0.8640	0.3241	1.70	0.6118	0.9445	0.2905
Pure Error	316.07	268.84	76.08	5	5	5	63.21	53.77	15.22
Cor Total	2098.53	2002.96	3028.31	19	19	19
Astaxanthin Concentration
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	928.85	1548.20	2144.63	3	3	3	309.62	516.07	714.88	8.68	16.68	28.33	0.0012	< 0.0001	< 0.0001
X₁	847.58	1220.36	2091.44	1	1	1	847.58	1220.36	2091.44	23.77	39.44	82.89	0.0002	< 0.0001	< 0.0001
X₂	5.78	129.10	1.52	1	1	1	5.78	129.10	1.52	0.1621	4.17	0.0604	0.6925	0.0579	0.8089
X₃	75.49	198.74	51.67	1	1	1	75.49	198.74	51.67	2.12	6.42	2.05	0.1650	0.0221	0.1717
Residual	570.64	495.02	403.72	16	16	16	35.66	30.94	25.23
Lack of Fit	411.68	178.27	318.65	11	11	11	37.43	16.21	28.97	1.18	0.2558	1.70	0.4570	0.9722	0.2898
Pure Error	158.95	316.75	85.06	5	5	5	31.79	63.35	17.01
Cor Total	1499.49	2043.22	2548.35	19	19	19
Astaxanthin recovery yield
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	251.89	204.00	31.06	6	3	3	41.98	68.00	10.35	2.16	2.62	4.55	0.1147	0.0867	0.0173
X₁	134.40	141.90	15.23	1	1	1	134.40	141.90	15.23	6.93	5.46	6.69	0.0207	0.0327	0.0198
X₂	27.76	4.45	11.38	1	1	1	27.76	4.45	11.38	1.43	0.1713	5.01	0.2530	0.6845	0.0398
X₃	0.7076	57.65	4.45	1	1	1	0.7076	57.65	4.45	0.0365	2.22	1.96	0.8515	0.1557	0.1810
Residual	68.09	415.61	36.39	1	16	16	68.09	25.98	2.27	3.51			0.0837
Lack of Fit	0.8978	130.90	33.14	1	11	11	0.8978	11.90	3.01	0.0463	0.2090	4.63	0.8330	0.9856	0.0517
Pure Error	20.03	284.71	3.26	1	5	5	20.03	56.94	0.6513
Cor Total	252.24	619.61	67.45	13	19	19

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively. OS: organic solvent; VO: vegetable oil; IME: ionic microemulsion.

Table 6.Analysis of variance for the microwave-assisted extraction of astaxanthin with different solvents from F. merguiensis shrimp wastes.

Total carotenoid content
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	50.83	1661.64	1084.96	3	3	3	16.94	553.88	361.65	0.0999	18.40	3.93	0.9589	< 0.0001	0.0280
X₁	19.20	1656.29	817.98	1	1	1	19.20	1656.29	817.98	0.1132	55.03	8.90	0.7409	< 0.0001	0.0088
X₂	24.13	5.33	98.47	1	1	1	24.13	5.33	98.47	0.1423	0.1772	1.07	0.7110	0.6794	0.3161
X₃	7.51	0.0125	168.52	1	1	1	7.51	0.0125	168.52	0.0443	0.0004	1.83	0.8360	0.9840	0.1946
Residual	2713.35	481.58	1470.77	16	16	16	169.58	30.10	91.92
Lack of Fit	2144.96	274.65	1266.69	11	11	11	195.00	24.97	115.15	1.72	0.6033	2.82	0.2869	0.7749	0.1310
Pure Error	568.40	206.93	204.08	5	5	5	113.68	41.39	40.82
Cor Total	2764.18	2143.22	2555.74	19	19	19
Astaxanthin Concentration
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	239.70	1269.90	905.58	3	3	3	79.90	423.30	301.86	0.6598	21.09	4.40	0.5887	< 0.0001	0.0195
X₁	152.73	1265.40	686.68	1	1	1	152.73	1265.40	686.68	1.26	63.05	10.00	0.2780	< 0.0001	0.0060
X₂	18.29	0.0897	98.09	1	1	1	18.29	0.0897	98.09	0.1511	0.0045	1.43	0.7026	0.9475	0.2494
X₃	68.67	4.41	120.81	1	1	1	68.67	4.41	120.81	0.5671	0.2200	1.76	0.4624	0.6454	0.2033
Residual	1937.53	321.10	1098.47	16	16	16	121.10	20.07	68.65
Lack of Fit	1379.89	191.24	946.63	11	11	11	125.44	17.39	86.06	1.12	0.6694	2.83	0.4796	0.7315	0.1300
Pure Error	557.63	129.86	151.84	5	5	5	111.53	25.97	30.37
Cor Total	2177.22	1591.01	2004.06	19	19	19
Astaxanthin recovery yield
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	1573.10	24.72	7.93	3	3	3	524.37	8.24	2.64	5.58	0.3253	0.0882	0.0081	0.8071	0.9655
X₁	645.13	2.83	4.01	1	1	1	645.13	2.83	4.01	6.87	0.1117	0.1338	0.0185	0.7425	0.7193
X₂	170.90	14.38	0.7182	1	1	1	170.90	14.38	0.7182	1.82	0.5674	0.0240	0.1961	0.4622	0.8789
X₃	757.07	7.52	3.20	1	1	1	757.07	7.52	3.20	8.06	0.2967	0.1070	0.0118	0.5935	0.7479
Residual	1502.55	405.35	479.26	16	16	16	93.91	25.33	29.95
Lack of Fit	1128.33	205.60	140.26	11	11	11	102.58	18.69	12.75	1.37	0.4679	0.1881	0.3842	0.8635	0.9900
Pure Error	374.22	199.75	338.99	5	5	5	74.84	39.95	67.80
Cor Total	3075.65	430.07	487.18	19	19	19

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively. OS: organic solvent; VO: vegetable oil; IME: ionic microemulsion.

Table 7.Analysis of variance for the ultrasound-assisted extraction of astaxanthin with different solvents from P. maeoticus Gammarus.

Total carotenoid content
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	585.55	25.86	1668.78	3	3	3	195.18	8.62	556.26	13.67	5.59	11.97	0.0001	0.0081	0.0002
X₁	574.64	2.92	1466.38	1	1	1	574.64	2.92	1466.38	40.24	1.89	31.55	< 0.0001	0.1876	< 0.0001
X₂	1.78	8.60	3.32	1	1	1	1.78	8.60	3.32	0.1248	5.57	0.0714	0.7285	0.0313	0.7928
X₃	9.13	14.34	199.09	1	1	1	9.13	14.34	199.09	0.6394	9.29	4.28	0.4356	0.0077	0.0550
Residual	228.49	24.69	743.75	16	16	16	14.28	1.54	46.48
Lack of Fit	157.90	19.77	645.53	11	11	11	14.35	1.80	58.68	1.02	1.83	2.99	0.5303	0.2624	0.1185
Pure Error	70.59	4.92	98.23	5	5	5	14.12	0.9838	19.65
Cor Total	814.04	50.55	2412.53	19	19	19
Astaxanthin Concentration
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	489.09	22.58	1068.89	3	3	3	163.03	7.53	356.30	14.32	5.28	8.91	< 0.0001	0.0101	0.0011
X₁	482.04	2.42	934.41	1	1	1	482.04	2.42	934.41	42.34	1.70	23.37	< 0.0001	0.2110	0.0002
X₂	7.05	3.71	11.76	1	1	1	7.05	3.71	11.76	0.6195	2.60	0.2941	0.4427	0.1262	0.5951
X₃	0.0002	16.45	122.72	1	1	1	0.0002	16.45	122.72	0.0000	11.53	3.07	0.9965	0.0037	0.0989
Residual	182.15	22.83	639.72	16	16	16	11.38	1.43	39.98
Lack of Fit	134.75	19.60	564.44	11	11	11	12.25	1.78	51.31	1.29	2.76	3.41	0.4118	0.1360	0.0933
Pure Error	47.39	3.23	75.28	5	5	5	9.48	0.6453	15.06
Cor Total	671.24	45.41	1708.60	19	19	19
Astaxanthin recovery yield
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	368.02	525.68	74.88	3	3	3	122.67	175.23	24.96	9.25	1.08	2.55	0.0009	0.3846	0.0924
X₁	263.58	0.4040	56.03	1	1	1	263.58	0.4040	56.03	19.87	0.0025	5.72	0.0004	0.9608	0.0294
X₂	38.65	76.51	17.74	1	1	1	38.65	76.51	17.74	2.91	0.4728	1.81	0.1071	0.5016	0.1972
X₃	65.79	448.77	1.12	1	1	1	65.79	448.77	1.12	4.96	2.77	0.1139	0.0406	0.1153	0.7402
Residual	212.22	2589.13	156.76	16	16	16	13.26	161.82	9.80
Lack of Fit	150.32	2390.77	97.53	11	11	11	13.67	217.34	8.87	1.10	5.48	0.7484	0.4890	0.0366	0.6810
Pure Error	61.90	198.36	59.23	5	5	5	12.38	39.67	11.85
Cor Total	580.24	3114.81	231.64	19	19	19

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively. OS: organic solvent; VO: vegetable oil; IME: ionic microemulsion.

Table 8.Analysis of variance for the microwave-assisted extraction of astaxanthin with different solvents from P. maeoticus Gammarus.

Total carotenoid content
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	156.35	3.48	1083.10	3	3	3	52.12	1.16	361.03	6.23	0.2445	1.53	0.0052	0.8640	0.2443
X₁	141.04	2.19	550.42	1	1	1	141.04	2.19	550.42	16.86	0.4610	2.34	0.0008	0.5069	0.1458
X₂	0.9181	0.3183	335.31	1	1	1	0.9181	0.3183	335.31	0.1097	0.0671	1.42	0.7447	0.7990	0.2501
X₃	14.39	0.9747	197.38	1	1	1	14.39	0.9747	197.38	1.72	0.2054	0.8384	0.2082	0.6565	0.3735
Residual	133.86	75.93	3766.89	16	16	16	8.37	4.75	235.43
Lack of Fit	111.36	45.11	3122.55	11	11	11	10.12	4.10	283.87	2.25	0.6654	2.20	0.1912	0.7341	0.1978
Pure Error	22.49	30.82	644.34	5	5	5	4.50	6.16	128.87
Cor Total	290.20	79.41	4849.99	19	19	19
Astaxanthin Concentration
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	105.75	2.45	722.01	3	3	3	35.25	0.8173	240.67	8.37	0.2032	2.16	0.0014	0.8926	0.1322
X₁	105.15	0.8532	555.52	1	1	1	105.15	0.8532	555.52	24.97	0.2122	4.99	0.0001	0.6513	0.0400
X₂	0.1613	0.0674	25.52	1	1	1	0.1613	0.0674	25.52	0.0383	0.0168	0.2294	0.8473	0.8986	0.6384
X₃	0.4414	1.53	140.98	1	1	1	0.4414	1.53	140.98	0.1048	0.3807	1.27	0.7503	0.5459	0.2768
Residual	67.37	64.35	1779.67	16	16	16	4.21	4.02	111.23
Lack of Fit	52.93	37.39	1185.92	11	11	11	4.81	3.40	107.81	1.67	0.6304	0.9079	0.2987	0.7571	0.5871
Pure Error	14.44	26.96	593.75	5	5	5	2.89	5.39	118.75
Cor Total	173.13	66.80	2501.68	19	19	19
Astaxanthin recovery yield
Factor	Sum of squares			Degrees of freedom (df)			Mean sum of squares			F-value			P-value
Factor	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME	OS	VO	IME
Model	241.78	118.64	281.98	3	3	3	80.59	39.55	93.99	3.35	0.7619	0.7842	0.0456	0.5318	0.5200
X₁	115.53	11.36	16.00	1	1	1	115.53	11.36	16.00	4.80	0.2189	0.1335	0.0437	0.6462	0.7196
X₂	12.88	28.93	233.58	1	1	1	12.88	28.93	233.58	0.5349	0.5574	1.95	0.4751	0.4661	0.1818
X₃	113.37	78.34	32.40	1	1	1	113.37	78.34	32.40	4.71	1.51	0.2703	0.0454	0.2370	0.6102
Residual	385.32	830.52	1917.74	16	16	16	24.08	51.91	119.86
Lack of Fit	341.94	269.77	1613.70	11	11	11	31.09	24.52	146.70	3.58	0.2187	2.41	0.0850	0.9832	0.1708
Pure Error	43.38	560.75	304.04	5	5	5	8.68	112.15	60.81
Cor Total	627.10	949.16	2199.72	19	19	19

X₁, X₂, and X₃ are independent variables that correspond to the ratio of solvent to sample, power of the device, and extraction time, respectively. OS: organic solvent; VO: vegetable oil; IME: ionic microemulsion.

No competing interests reported.

Green Astaxanthin Extraction: Microwave and Ultrasound Pretreatment

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1. Materials

2.2. Preparation of samples

2.3. Preparation of solvents

2.4. Characterization of microemulsion

2.5. Experimental design and Optimization

2.6. Ultrasound-assisted extraction

2.7. Microwave-assisted extraction

2.8. Analysis

2.8.1. Determination of total carotenoid content (total astaxanthin)

2.8.2. Spectrophotometric determination of astaxanthin

2.8.3. Determination of astaxanthin recovery yield

2.8.4. Determination of antioxidant activity

2.9. Statistical analysis

3. Results

3.1. Physical properties of microemulsion

3.2. Optimizing the extraction of astaxanthin carotenoid pigment by ultrason-

3.3. Optimizing the extraction of astaxanthin carotenoid pigment by microwave-assisted extraction

3.4. DPPH antioxidant properties assay test

4. Discussion

5. Conclusion

Declarations

Author Contribution

Acknowledgements

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1