Effects of different proteases enzymatic extraction on the lipid yield and quality of Antarctic krill oil

Abstract This study was investigated the effects of six proteases (papain, compound proteinase, acidic protease, neutrase, pancreatin, and alcalase) on the lipid yield and quality of krill oil. The result shown that the krill oil extracted by alcalase and compound proteinase led to comparatively higher lipid yields (5.29% and 4.90%, respectively), Content of tocopherols and vitamin A, the content of omega‐3 polyunsaturated fatty acids (PUFAs) and phospholipids extracted by alcalase was relatively higher. Control and alcalase had comparatively higher concentration of astaxanthin. On the whole, compared with the extraction of solvent, enzymatic hydrolysis could improve the quality and the lipid yield of krill oil. Therefore, enzymatic hydrolysis could be used as a better method to extract krill oil.


| Lipid extraction
Papain, compound proteinase, acidic protease, neutrase, pancreatin, and alcalase were used as extraction proteases. 50 g of krill was homogenized with 50 ml distilled water, adjusted to the corresponding condition (papain 50°C pH 7.0, compound proteinase 50°C pH 7.0, acid protease 50°C pH 3.0, neutrase 50°C pH 7.0, pancreatin 50°C pH 7.0, and alcalase 55°C pH 8.0) in a water bath, added 0.4 g protease, and hydrolyzated 3 hr with stirring every 30min. After enzymatic hydrolysis extracted krill oil using 30 ml anhydrous ethanol, 30 minutes later extracted using 60 mL petroleum ether (30 ~ 60°C) for 30min, repeated the solvent extraction once and made a control that was not added protease at room temperature. The upper organic layer was centrifuged at 9.794 m/s 2 for 10 min at 4°C, and then, the solvent was removed in a rotary evaporator at 60°C.

| Analysis of lipid yield
Weighed the initial Antarctic krill (wet base) and the extracted krill oil. The lipid yield is defined as follows: where m is the weight of krill oil (g) and y is the mass of initial Antarctic krill (g).

| Analysis of phospholipid content in krill oil
The phospholipid content was determined by the method of Avalli and Contarini (2005) with slight modifications by a high-performance liquid chromatography (HPLC) (W2690/5, Waters, USA) equipped with a diode array detector (DAD) (Waters, USA). In brief, 0.1 g krill oil was dissolved in 1 ml chloroform:methanol (2:1, v:v), ten microliters of the product was injected into HPLC for analysis.

| Analysis of astaxanthin content in krill oil
Using a high-performance liquid chromatography (HPLC) (W2690/5, Waters, USA) equipped with a diode array detector (DAD) (Waters, USA) and a C18 column (5μm, 4.6 × 250 mm; Shanghai ANPEL Science Instrument Co., Ltd, Shanghai, China) with a mobile phase, containing dichloromethane:acetonitrile:me thanol (20:70:10, v/v/v) under isocratic condition with a flow rate of 1.0 ml/min to analyze astaxanthin, this method was determined by Sun et al. (2018) with slight modifications. Briefly, 0.5 g of krill oil mixed with 5 ml dichloromethane and then added 0.1 mol/L sodium hydroxide (NaOH) in methanol at 4°C for 9 hr in darkness for complete saponification. The determined wavelengths were 476 nm for astaxanthin and were identified and quantified by comparing with the standards (Shanghai Macklin Biochemical Co., Ltd, Shanghai, China).

Lipid yield (%)
m y = ×100 2.6 | Fatty acids composition analysis FAME was prepared and analyzed according to the previously developed procedure (Xie et al., 2018) with small modifications to the process of transesterification and oven temperature program. Briefly, 0.1 g krill oil mixed with 5 ml of 0.1 mol/L sodium hydroxide (NaOH) in methanol was transesterified with stirring in a 80°C water bath for 15 min and added 3 ml 14% boron trifluoride-methanol solution for further transesterification at 80°C for 5 min. After cooled to room temperature, 2 ml n-hexane and 5 ml saturated sodium chloride solution were added, respectively. The upper organic layer was filtered through a 0.22-um membrane and then stored at −20°C for further analysis.
The analysis of FAME samples was performed by a gas chromatog- of the corresponding FAME mixture standards. The fatty acid contents were expressed as the weight (g/100g) of the total fatty acids.

| Analysis of tocopherols and vitamin A contents and composition in krill oil
Using a high-performance liquid chromatography (HPLC) (Agilent 1260, Shanghai Zhiyan Scientific Instrument Co., Ltd. ，China) equipped with an ultraviolet detector (UV detector) (Agilent 1260 ，Shanghai Zhiyan Scientific Instrument Co., Ltd. ，China) to analyze tocopherols and vitamin A, this method was determined by Xie et al.
(2017) with slight modifications: In brief, saponification reaction of the krill oil l sample (0.5 g) was achieved using 15 mg of ascorbic acid and 5 ml of 2 mol/L KOH in ethanol at 60°C for 60min. After cooling to room temperature, 5 ml of 1.5 mg/100 ml butylated hydroxytoluene (BHT) in n-hexane was added to extract the unsaponifiables three times, combined the mixture, and evaporated the n-hexane to achieve extracts. The extracts were dissolved in 10 ml of n-hexane.
20 μl of the product was injected into HPLC, and the C18 column was purchased from Shanghai ANPEL Science Instrument Co., Ltd.
(Shanghai, China). The determined wavelengths of tocopherols and vitamin A were 295 nm and 325 nm, respectively. The two compounds were identified and quantified by comparison with standard products.

| Analysis of Iodine value and Acid value in krill oil
Iodine value is the measure of the degree of unsaturation of fat and oil, which indicates the amount of absorbed iodine. The determination of iodine value was tested according to the procedure described by Barlow, Bimbo, Miller, Thorisson, and Walters (1997).
Acid value is also called acid number, is expressed as the number of milligrams of potassium hydroxide (KOH) neutralized by the free acid present in 1 g of the substance, and determination of acid value is the same as method described by Cong et al. (2007).

| Lipid yield %
The lipid yields obtained by various proteases are presented in Figure 1. It could be observed that the lipid yields varied from 3.23%to 5.29% and they were all higher than not added protease of 2.67%.

| Phospholipid analysis
The total phospholipid content is shown in Figure 2 presented as wt % of krill oil. Phospholipid is an important lipid class in krill oil. In the F I G U R E 1 Lipid yield of different proteases for extraction from Antarctic krill study, the content of phospholipid in krill oil in the range of 33.49%-50.19% and extracted by different proteases was different, content of phospholipid extracted by alcalase was the highest, which was 50.19%, followed by neutrase, which was 49.45%. Compared with, influenced by, the type of extraction solvent varied from 16.5% to 34.5% (Sun et al., 2018), the content of phospholipids in krill oil extracted by enzymatic hydrolysis is higher.

| Astaxanthin analysis
Astaxanthin in Antarctic krill oil is currently recognized as the most active and safe bioactive substance. It is the most important carotenoid pigment in Antarctic krill oil, and it is also the natural strongest antioxidant and free radical scavenging in nature Pigment, which has many physiological functions such as antioxidation and tumor prevention. (Anderson, 2005;Kurashige, Okimasu, Inoue, & Utsumi, 1990;Maleyeff & Kaminsky, 2003;Maoka, Katsuyama, Kaneko, & Matsuno, 1985). Study has shown that the content of astaxanthin was in the range of 6.94-21.59 ppm (Kim et al., 2014) and astaxanthin content in krill tissue was reported in 1.5-2.0 mg/100g (Tou, Jaczynski, & Chen, 2010).
The astaxanthin content of the krill oil extracted by various proteases varied from 272.75 to 553.45mg/kg, as shown in Figure 3.
The content of astaxanthin in the Antarctic krill oil extracted by different proteases was different. The highest concentration of astaxanthin extracted by control and alcalase was 553.45 and 520.75 mg/kg, respectively, followed by the papain, which was 450.50 mg/kg, and there was no significant difference (p < 0.05).
Compared with the content of 93.87-222.64 mg/kg extracted by solvents (Xie et al., 2018), the astaxanthin content was higher by enzymatic hydrolysis.

| Fatty acids composition analysis
Contents of fatty acids in krill oil are listed in Table 1, C14:0, C15:1, C16:1, C18:1, C20:5 (EPA), and C22:6 (DHA). EPA and DHA are the main fatty acids in Antarctic krill oil. According to the other studies and findings described (Kolakowska, Kolakowski, & Szczygielski, 1994), the n-3 PUFA, EPA, and DHA are especially abundant in krill oil, which was due to Antarctic krill consuming single-cell marine microalgae. Table 1 shows the content of fatty acids extracted by different proteases was different, and this phenomenon may be related to its extraction principle. Compared with the research of Sidhu (2010) of 23.4%, the content of PUFA (25.03-34.49 g/100g) in krill oil extracted by enzymatic hydrolysis is relatively higher, and compared with the extraction of organic solvents (24.24%-33.05%) (Xie et al., 2017), the content of n-3 PUFA (24.38-31.42 g/100g) in krill oil extracted by enzymatic hydrolysis is relatively higher and the content of PUFA and MUFA is also higher. In addition to control and compound proteinase, the contents of fatty acids and n-3 PUFA were positively associated with lipid yield of krill oil, so we can conclude that the content of n-3 PUFA in the krill oil is related to the yield and enzymatic hydrolysis can better maintain the nutrition of krill oil. At the same time, it is also shown that besides the protease type, the extraction temperature and pH also have certain influence on the fatty acid composition of Antarctic krill oil, the raw materials we use are not dried, which better protects the unsaturated fatty acids from oxidation.

| Tocopherols and vitamin A analysis
According to the reports by Suzuki and Shibata (1990), krill was rich in tocopherol and vitamin A. Two kinds of tocopherol and  38.17 mg/100 g) and γ-tocopherol (0.54-1.11 mg/100 g), and δ-tocopherol has not been detected.
In the present study, the contents of vitamin A and tocopherol obtained by enzymatic hydrolysis of alcalase were higher, which was 95.32 µg/100g and 39.28 mg/100g, respectively, lower than 29.39 mg/100 g of tocopherols and higher than 34.32 mg/100 g of vitamin A of the research of Xie et al. (2018), and the contents of vitamin A and tocopherols were positively associated with lipid yield; these results provide date support for the selection of protease to hydrolyze Antarctic krill. It is proved that alcalase can be used as the best protease for extracting Antarctic krill oil compared with other proteases.

| Iodine value and Acid value analysis
Iodine value (IV) is the measure of the degree of unsaturation of fat and oil, which indicates the amount of absorbed iodine. It is an important parameter in oil industry, which can be used as guidance for oil processing. The more the fatty acid is unsaturated, the greater the iodine value is. (Haryati, Man, Ghazali, Asbi, & Buana, 1998). As shown in Table 3, the content of unsaturated fatty acids in krill oil hydrolyzed by alcalase was the highest, followed by control, acidic protease, and papain. The effects of Values are means ± standard deviation. Different superscript letters in a row indicate significant differences for individual fatty acid (p < 0.05).