Lipids from Cephalothorax of Pacific White Shrimp (Litopenaeusvannamei) using Ultrasonic Assisted Extraction Process :Pre-treatment, Enhanced Oxidative Stability and Applications

Abstract

Effect of different solvents along with ultrasonic assisted extraction (UAE) process on the extraction yield of lipids from cephalothorax of Pacific white shrimp (Litopenaeus vannamei) was investigated. Among all solvents used, the mixture of hexane:isopropanol (1:1) provided the highest yield (3.91 g/100g sample) and carotenoid content (1.97 mg/g lipid). When ultrasonication with different amplitudes (50-90%) was used for 25 min, the highest yield was obtained at 80% amplitude (p<0.05). With UAE at the same ultrasonication time, continuous mode rendered the higher yield than pulse mode (p<0.05). Lipid extracted with hexane:isopropanol mixture with UAE process had the yield of 8.39 g/100g sample. Phospholipids were major constituents of lipids extracted by solvent extraction. However, more free fatty acids, mono and diglycerides were found in lipids extracted by UAE process, indicating increased hydrolysis. UAE process resulted in higher oxidation of lipids as evidenced by increased peroxide values (PVs) and thiobarbituric acid reactive substances (TBARS). Those alterations were more pronounced in lipids extracted using UAE with continuous mode than pulsed mode as confirmed by Fourier transform infrared (FTIR) spectra. Impacts of different pre-treatments of cephalothorax before UAE on yield and characteristics of lipids were studied. Autolysis at 50 °C for 3 h in the absence and presence of 0.1% tannic acid (TA), a lipase inhibitor, was implemented. Pre-heating of cephalothorax containing 0.1% TA at 95 °C for different times (15-45 min) was also carried out. When lipids were extracted at amplitude of 80% for 25 min in continuous mode, samples with pre-heating rendered the highest lipid yield (13.3g-14.1g/100g sample). Pre-heating along with TA addition resulted in suppression of lipid oxidation. Free fatty acid (FFA) content was also found to be lower, whereas the control (without pre-treatment) had higher FFA. FTIR spectra confirmed lower oxidation in lipid from pre-heated samples added with TA. Lipid contained astaxanthin, astaxanthin monoester, astaxanthin diester, canthaxanthin and ẞ-carotene. After pulsed electric field (PEF) pre-treatment at different electric field strengths (4, 8, 12 and 16 kVcm-1) and pulse numbers (120, 160, 200 and 240) was applied on cephalothorax, PEF treated samples were subsequently subjected to lipid extraction UAE (amplitude of 80%) for 25 min in continuous mode. PEF pre-treated samples subjected to UAE rendered the highest lipid yield (10.44 g/100g sample). PEF pre-treatment resulted in reduced lipid oxidation. Lipid from PEF pre-treated samples extracted using UAE had higher content of polyunsaturated fatty acids (PUFAs) as well as carotenoids, including astaxanthin, etc. Impacts of different pre-treatment conditions and atmosphere on yield and oxidative stability of lipids using UAE were studied. Cephalothorax was subjected to vacuum-microwave (VM) heating (at 95 °C) prior to UAE using a mixture of isopropanol/n-hexane (1:1) as solvent. Nitrogen gas was flushed at two flow rates; low (2.15 L/min) and high (4.35 L/min) into the system during ultrasonication. Vacuum- microwave heating resulted in the increase of lipid yield and the highest yield was observed in the samples extracted by a combination of VM and UAE. Addition of TA at 0.1% in combination with VM, followed by nitrogen flushing resulted in the increased oxidative stability of lipids. Furthermore, astaxanthin content in the lipid was found to be increased by aforementioned treatments. Shrimp oil extracted from cephalothorax added with 0.1% TA and preheated at 95 °C for 5 min using an innovative UAE under nitrogen atmosphere, was encapsulated in nanoliposomes, prepared using ultrasonication (US) and microfluidization (MF). Nanoliposomes prepared by US and MF were characterized based on particle size, structure and stability. The particle size of US nanoliposomes ranged between 40 and 284 nm, while MF nanoliposomes ranged from 214 to 928 nm. US nanoliposomes exhibited better centrifugal stability than MF counterparts (p<0.05). Nanoencapsulation efficiency (NEE) of US nanoliposomes was higher (93.64%) than that of MF (75.18) and remained constant over the storage of 8 weeks at 30 °C. Nanoliposomes showed higher oxidative stability during the storage than unencapsulated oil (p<0.05) with higher retention of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), particularly in US nanoliposomes. Overall, encapsulation of shrimp oil in nanoliposomes could prevent oxidation of oil during storage and mask the undesirable fishy odor. Shrimp oil nanoliposomes (SONL) prepared using ethanol injection process followed by ultrasonication, were fortified into skim milk at various levels (2 - 10%, v/v), followed by pasteurization at 63 °C for 30 min. Skim milk showed lowered whiteness but increased redness and yellowness as added SONL levels increased (p<0.05). Viscosity of fortified samples was also augmented with increasing levels of SONL (p<0.05). Fortified milk skim samples had no perceivable fishy odor and were organoleptically acceptable. When skim milk fortified with 10% SONL was stored up to 15 days at 4 °C, microbial load was less than 2.54 log CFU/ml. pH and acidity values were also within the acceptable limits. Shrimp oil in SONL did not undergo oxidation during the extended storage. Fatty acid profile of shrimp oil revealed no loss of polyunsaturated fatty acids taken place during storage of fortified milk. Therefore, nanoliposomes could be an effective carrier for shrimp oil to be fortified in skim milk. Impact of B-glucan on masking bitterness of skim milk fortified 10% SONL was examined. B-glucan was added at various levels (0.05–0.2%). With the addition of SONL, fortified skim milk appeared more reddish in color due to the presence of astaxanthin. Addition of ẞ-glucan resulted in the increase in viscosity of the fortified milk by forming network of junction zones. During the storage of skim milk fortified with SONL and 0.1% ẞ-glucan at 4 °C for 15 days, no major quality changes took place. Simulated in vitro digestion studies revealed that 45.41% EPA and 48.86% DHA from shrimp oil were bioaccessible for absorption in the gut after digestion. Shrimp oil was encapsulated into nanoliposomes and dried using freeze drying and spray drying methods. Carboxy methyl cellulose (CMC) and fumed silica (SiO2), at different proportions were used as wall material and anti-caking agents to improve the flowability and reconstitution properties of the dried powders. Spray-dried powder was spherical with average particle size of 5.33±2.55 μm, while the freeze-dried powder was irregular in shape with average size of 243.6±256.7 μm. Spray dried powder had better flowability, compared to freeze-dried, which was more porous with much lower bulk density. Encapsulation efficiency and solubility of spray-dried SONL powder was higher than freeze-dried powder (p<0.05). However, the wettability of freeze-dried powder was higher with shorter reconstitution time. Freeze-dried powder exhibited lower oxidation of total encapsulated oil and better retention of n-3 fatty acids (p<0.05). Therefore, UAE could be effectively used to increase the extraction yield of lipids and carotenoids from Pacific white shrimp cephalothorax with appropriate treatments. Moreover, the extracted lipids could be encapsulated in nanoliposomes to prevent oxidation and mask the fishy odor of shrimp oil fortified in various foods. Dry nanoliposomes could also be produced with free-flowing behavior.