Abstract:
The present invention provides a method for oil extraction from dehydrated  Euphausia superbas,  which is related to the field of food biotechnology. The method combines a low temperature and low oxygen heat pump dehydration system with a microwave-assisted frozen-blasting dehydration system for  Euphausia superba  dehydration, resulting in formation of a large number of micro porous structures in dehydrated  Euphausia superbas  that is good for subsequent oil extraction. The dehydration and extraction process can be separated in the present invention. The low temperature and low oxygen heat pump dehydration system may be installed in shrimp boats to dehydrate  Euphausia superbas  before transportation so as to increase the effective payloads of shrimp boats. The extraction process of subcritical fluid assisted with ultrasonic operation is carried out under low temperature and low oxygen conditions, therefore the oxidation of active ingredients is avoided to a large extent. Compared with existing methods, the present invention not only possess excellent dehydration efficiency but also uses less time and energy.

Description:
CROSS-REFERENCES AND RELATED APPLICATIONS 
       [0001]    This application is a continuation of the international application PCT/CN2015/076056, with an international filing date of Apr. 8, 2015, which is herein incorporated by reference in its entirety. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    Field of the Invention 
         [0003]    The present invention relates to the field of food biotechnology, and more particularly relates to a method for extracting oil from dehydrated  Euphausia superba.    
         [0004]    Description of the Related Art 
         [0005]      Euphausia superba  (Antarctic krill) is a pelagic shrimplike invertebrate that lives in the Antarctic waters.  Euphausia superbas  live in large schools and feed on minute phytoplankton. It is the largest single biological resource in the world. The water content of fresh  Euphausia superba  is up to 80%. Currently, most of  Euphausia superba  are frozen for storage, transport and further process. In this way, the cost of transportation is very huge and the effective transportation payload is relatively low. 
         [0006]      Euphausia superba  is dehydrated before transportation and oil extraction. The process of dehydration is either under high temperature or high energy-consumption. The traditional solvent extraction of krill oil is accompanied by problems such as high temperature and incomplete desolvation. Supercritical CO 2  extraction is an effective method for oil extraction, however, the equipment for supercritical CO 2  extraction is pretty expensive and complex to use. Since supercritical CO 2  extraction is carried out under a high pressure, there are certain security risks. Other methods for preparing krill oil from  Euphausia superba  powder, fresh  Euphausia superba  or frozen  Euphausia superba  have the following disadvantages: (1) The drying process is high energy-consuming, low efficiency and environment-unfriendly. (2) The process of dehydration or oil extraction is carried out at high temperatures, which results in the oxidation of active ingredients. 
         [0007]    To solve the existing problems, the present invention provides a method for dehydrating and extracting oil from Antarctic krill under relatively low temperature in a closed anaerobic environment, thus minimizing the oxidation of active ingredients and eliminating environmental problems caused by solvent volatilization. The oil extraction is performed under an ultrasound-assisted subcritical fluid extraction condition. It uses a short-term ultrasonic approach to generate a “vacuolar effect” in the extraction system so as to increase the contact of the substrate and the solvent. Since subcritical fluid is in gas phase under ambient conditions, high temperature is not needed for desolvation, and the solvent residue problem is minimized. 
       DETAILED DESCRIPTION 
       [0008]    The first goal of the present invention is to provide a method for  Euphausia superba  dehydration. This method combines a low temperature and low oxygen heat pump dehydration system with a microwave-assisted frozen-blasting dehydration system so that the dehydrated  Euphausia superbas  form a micro porous structure that is good for subsequent oil extraction. The conventional dehydration method uses high temperatures (e.g. 80-100° C.) to remove water from  Euphausia superbas  which can lead to the oxidation of active ingredients. To solve this problem, the present invention performs the dehydration process under a lower temperature (e.g. 50-60° C.) in a closed anaerobic system with a heat pump, which can greatly reduce the oxidation of active ingredients during the dehydration. The dehydrated  Euphausia superbas  are further dehydrated with a microwave-assisted frozen-blasting dehydration system in which the dehydrated frozen  Euphausia superbas  are heated in microwave under a low pressure (e.g. &lt;20 Pa) and a low temperature (e.g. −45° C. to −50° C.) to directly convert ice to water vapor. This process produces dehydrated krill with micro porous structures good for oil extraction. Compared with lyophilization method, the present invention not only possesses excellent dehydration efficiency but also uses less time and energy. 
         [0009]    In one embodiment of the present invention, the  Euphausia superbas  are fresh or frozen. 
         [0010]    In one embodiment of the present invention, the low temperature and low oxygen heat pump dehydration system is a drying system that efficiently utilizes thermal energy to achieve krill dehydration under low temperature and low oxygen conditions. After treated with the low temperature and low oxygen heat pump dehydration system, the water content of krill is decreased from 80±1% to 30%-35%. The low temperature and low oxygen heat pump dehydration system can be installed in shrimp boats to help increase the effective payload of shrimp boats. 
         [0011]    In one embodiment of the present invention, the temperature of the low temperature and low oxygen heat pump dehydration system is maintained at 50-60° C. and filled with nitrogen to exclude air. 
         [0012]    In one embodiment of the present invention, the pressure of the microwave-assisted frozen-blasting dehydration system is adjusted to lower than 20 Pa and its temperature to −45 to −50° C.; and the power of the microwave is 0.2±0.05 w/g. After treating with the microwave-assisted frozen-blasting dehydration, the water content of krill is further decreased to 8-9%. 
         [0013]    The second goal of the present invention is to provide a method for extracting oil from dehydrated  Euphausia superbas.  Firstly,  Euphausia superba  is dehydrated by a low temperature and low oxygen heat pump dehydration system and a microwave-assisted frozen-blasting dehydration system; secondly, dehydrated krill is crushed into krill powder; thirdly, the krill powder is used for oil extraction by an ultrasound-assisted subcritical fluid extraction method. 
         [0014]    In one embodiment of the present invention, the dehydrated krill are crushed to 30-60 mesh. 
         [0015]    In one embodiment of the present invention, an ultrasound-assisted subcritical fluid extraction method is used to extract oil from dehydrated krill powder. The crushed krill is extracted in a subcritical extraction fluid under a high pressure with a simultaneous ultrasonic treatment. The extraction agent used for oil extraction is one or more reagents selected from butane, methyl ether, and 1,1,1,2-tetrafluoroethane, which are in a subcritical fluid state under a critical temperature and a critical pressure; the material-to-liquid ratio is 1:3-1:5(g/ml); the extraction temperature is 40-50° C.; the extraction pressure is 1.1-1.3 MPa; the ultrasonic frequency used during extraction is 25-35 KHz. Single extraction process lasts 15-30 min and the extraction process is repeated 3-5 times. The subcritical extraction solvent become gas and is removed under a normal or reduced pressure, and the remaining extracts are then collected and centrifuged to remove the impurities. 
         [0016]    The  Euphausia superba  oil prepared by the present invention is rich in unsaturated fatty acids and the DHA\EPA content of total fatty acids is more than 30%. The phospholipid content of  Euphausia superba  oil prepared by the present invention is more than 40%. 
         [0017]    The present invention uses a low temperature and low oxygen heat pump dehydration system and a microwave-assisted frozen-blasting dehydration system for  Euphausia superba  dehydration. The Low temperature and low oxygen heat pump dehydration system has the advantage of saving time and energy when treating high moisture materials. At the same time, lipase and amylase can also be inactivated during this process, which is beneficial for subsequent krill oil extraction and protein utilization of the extracts. The advantage of the present invention is low-temperature operation, saving energy and high effeciency extraction. 
         [0018]    In addition, dehydration and extraction process can be separated and carried out independently. The first stage of dehydration can be carried out in the boat if a low temperature and low oxygen heat pump dehydration system is installed in the shrimp boat. During the first stage, thermal energy is used efficiently to reduce the water content of the krill to 30-35%. Thus, the total weight of the krill material is reduced by about 70%. By this way, the present method is not only improved cargo capacity but also reduces transportation costs. The dehydrated  Euphausia superbas  can be frozen and stored in the boat after initial dehydration, and treated by a microwave-assisted frozen-blasting dehydration system until the ship is landed. In present invention, the pre-cooling procedure are omitted. The process of microwave-assisted frozen-blasting dehydration not only reduces water content but also generates a product with massive micro porous structures which is good for subsequent oil extraction. 
         [0019]    The oil extraction process using ultrasound-assisted subcritical fluid is carried out at a low temperature and low oxygen environment, therefore oxidation of active ingredients is greatly minimized Subcritical fluid is in gaseous state under ambient conditions, therefore less energy is needed for the process of desolvation and the problem of residual solvent is effectively solved. The present invention can shorten operation time and significantly save operation costs while maintaining the quality of the final oil product. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0020]      FIG. 1 . A scanning Electron Microscope image of krill dehydrated by a low temperature and low oxygen heat pump dehydration system. 
           [0021]      FIG. 2 . A scanning Electron Microscope image of krill dehydrated by a microwave-assisted frozen-blasting dehydration system. 
           [0022]      FIG. 3 . A schematic diagram of the subcritical fluid extraction.  1 , bather;  2 , extractor;  3 , separator;  4 , collector;  5 , heat exchanger pump;  6 , water tank;  7 , buffer tank;  8 , solvent pot;  9 , compressor;  10 , Condenser. 
       
    
    
     EXAMPLES 
     Materials and Methods 
       [0023]    The water content of  Euphausia superba  was evaluated according to the standard method of GB 5009.3-2010. The acid value was evaluated according to the standard method of GBT5530-2005 ISO660-1996. The phospholipid was evaluated according to the standard method of GB-T 5537-2008. 
         [0024]    The DHA/EPA of  Euphausia superba  oil was evaluated by gas chromatography (GC) according to the standard method of GBT 17377-2008. 
       Example 1 
       [0025]    Firstly, thawed  Euphausia superbas  were treated by a low temperature and low oxygen heat pump dehydration process. This process was carried out under nitrogen protection and the krill was spread with a thickness of no more than 2 centimeters. The temperature was controlled at 55° C. and the speed of hot wind was controlled at 3 m/s. The process lasted until that water content of  Euphausia superba  was reduced to 33±0.1%. Secondly, the krill was treated by a microwave-assisted frozen-blasting dehydration process. During the dehydration, the pressure was maintained at lower than 20 Pa and the microwave operation started when the temperature was reduced to lower than −45 ° C. The microwave power was set to 0.2 w/g. After dehydration, the water content of  Euphausia superba  was reduced to 8.5±0.1%. Then the dehydrated krill were crushed to 30 mesh. The krill oil was extracted by an ultrasound-assisted subcritical fluid method. The krill powder was extracted with a subcritical butane fluid (1 g krill/4 ml solvent) under ultrasonic operation to extract krill oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was kept at 45° C. Under this temperature and pressure, the extraction agent (e.g. butane) is in subcritical liquid phase. The ultrasonic frequency was 25 KHz during the extraction. The extraction process lasted 20 min. After that, the extraction solvent was removed under reduced pressure. A schematic diagram of the subcritical fluid extraction is shown in  FIG. 3 . The krill powder was added to and sealed in the extractor  2 . The subcritical butane fluid under a high pressure was added to the extractor  2  to extract the krill oil, and an ultrasound treatment was applied during the extraction. After extraction, the subcritical butane solution is moved to the separator  3 . The pressure of the separator  3  was reduced to ambient or lower pressures so that the butane is turned into gas phase and separated from the krill oil. The krill oil was collected via the collector  4  and the butane gas is transferred to and compressed in the compressor  9  and stored in the solvent pot  8 . 
         [0026]    The extraction was repeated 4 times and the extracts were collected and centrifuged to obtain the krill oil. The extraction rate of krill oil was 21.32% (dry basis). The acid value and the phospholipid content of the product were 8.4 and 43.10%, respectively. The DHA/EPA content of total fatty acids was 33.85%. As shown in  FIGS. 1 and 2 , a large number of micro porous structures appeared after the low temperature and low oxygen heat pump dehydration and the microwave-assisted frozen-blasting dehydration treatment. 
       Example 2 
       [0027]    Firstly, thawed  Euphausia superbas  were treated by a low temperature and low oxygen heat pump dehydration process. This process was carried out under nitrogen protection and the krill was spread with a thickness of no more than 2 centimeters. The temperature was maintained at 50° C. and the speed of hot wind through the materials was controlled at 3 m/s. The process lasted until that the water content of  Euphausia superba  was reduced to 33±0.1%. Secondly, the krill was treated by microwave-assisted frozen-blasting dehydration system. The pressure was maintained lower than 20 Pa and the microwave operation started when the temperature was reduced to lower than −45° C. The microwave power was set to 0.2 w/g. After dehydration, the water content was reduced to 8.5±0.1%. Then the dehydrated krill were crushed to 30 mesh. The krill powder was extracted with a subcritical butane fluid (1 g krill/4 ml solvent) under ultrasonic operation to extract krill oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was kept at 45° C. and the ultrasonic frequency was 25 KHz. The extraction process lasted 20 min. After that, the extraction solvent was removed under reduced pressure. The extraction was repeated 3 times and extracts were collected and filtered to obtain the krill oil. 
         [0028]    The extraction rate of  Euphausia superba  oil was 21.02% (dry basis). The acid value and the phospholipid content were 8.1 and 43.02%, respectively. The DHA/EPA content of total fatty acids was 33.48%. 
       Example 3 
       [0029]    Firstly, thawed  Euphausia superbas  were treated by a low temperature and low oxygen heat pump dehydration process. This process was carried out under nitrogen protection and the krill was spread with a thickness of no more than 2 centimeters. The temperature was maintained at 60° C. and the speed of hot wind through the materials was controlled at 3 m/s. The process lasted until that the water content of  Euphausia superba  was reduced to 33±0.1%. Secondly, the krill was treated by microwave-assisted frozen-blasting dehydration system. The pressure was maintained at lower than 20 Pa and the microwave operation started work when the temperature was reduced to lower than −45 ° C. The microwave power was set to 0.2 w/g. After dehydration, the water content was reduced to 8.5±0.1%. Then the dehydrated krill were crushed to 30 mesh. The krill powder was extracted with a subcritical butane fluid (1 g krill/4 ml solvent) under ultrasonic operation to extract krill oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was kept at 45° C. and the ultrasonic frequency was 25 KHz. The extraction process lasted 20 min. After that, the extraction solvent was removed under reduced pressure. The extraction was repeated 5 times and the extracts were collected and filtered to obtain  Euphausia superba  oil. 
         [0030]    The extraction rate of  Euphausia superba  oil was 21.62% (dry basis). The acid value and the phospholipid content were 9.5 and 43.16%, respectively. The DHA/EPA content of total fatty acids was 33.16%. 
       Example 4 
     Freeze-Drying Combined with Subcritical Extraction 
       [0031]    The frozen  Euphausia superbas  were freeze-dried to have a water content at 8.5±0.1% and then crushed to 30 mesh. The krill oil was extracted by an ultrasound-assisted subcritical fluid method. The krill powder was extracted with subcritical butane fluid (1 g/4 ml) under an ultrasonic operation to extract oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was 45° C. and the ultrasonic frequency was 25 KHz. The extraction lasted 20 min. After that, the extraction solvent was removed under reduced pressure. The extraction was repeated 4 times and extracts were collected and filtered to obtain  Euphausia superba  oil. 
         [0032]    The extraction rate of  Euphausia superba  oil was 22.10% (dry basis). The acid value and the phospholipid content were 7.2 and 43.52%, respectively. The DHA/EPA content of total fatty acids was 34.94%. 
       Example 5 
     Hot Air Drying Combined with Subcritical Extraction 
       [0033]    Thawed  Euphausia superba  was dried by hot air at 90° C. to reduce water content to 8.5±0.1%. Then the materials were crushed to 30 mesh. The krill powder was extracted with subcritical butane fluid (1 g/4 ml) under an ultrasonic operation to extract oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was 45° C. and the ultrasonic frequency was 25 KHz. The extraction lasted 20 min. After that, the extraction solvent was removed under reduced pressure. The extraction was repeated 4 times. The extracts are collected and filtered to obtain  Euphausia superba  oil. 
         [0034]    The extraction rate of  Euphausia superba  oil was 16.2% (dry basis). The acid value and the phospholipid content were 19.2 and 42.36%, respectively. The DHA/EPA content of total fatty acids was 31.94%. 
       Example 6 
     Hot Air Drying Combined with Solvent Extraction 
       [0035]    Thawed  Euphausia superba  was dried by hot air at 90° C. to reduce the water content to 8.5±0.1%. Then the materials were crushed to 30 mesh. 1 g Krill powder was mixed with 4 mL n-Hexane to extract oil. During the extraction process, temperature was kept at 55° C. Single extraction process lasted 2 h and repeated 3 times. Extracts were collected and filtered to obtain  Euphausia superba  oil. 
         [0036]    The extraction rate of  Euphausia superba  oil was 15.5% (dry basis). The phospholipid content and the acid value were 42.26% and 21.5, respectively. The DHA/EPA content of total fatty acids was 30.02%. 
       Example 7 
     Dehydration Combined with Subcritical Extraction 
       [0037]    Thawed  Euphausia superbas  were treated by a low temperature and low oxygen heat pump dehydration process. This process was carried out under nitrogen protection and the krill was spread with a thickness of no more than 2 centimeters. The temperature was maintained at 55° C. and the speed of hot wind through the materials was controlled at 3 m/s. The process lasted until that water content of  Euphausia superba  reduced to 35±0.1%. Then the krill was treated by a microwave-assisted frozen-blasting dehydration system. The pressure was maintained lower than 20 Pa, and microwave operation started when the temperature was reduced to lower than −45° C. The microwave power was set as 0.2 w/g. After dehydration, the water content was reduced to 8.5±0.1%. Then the materials were crushed to 30 mesh. 1 g krill powder was mixed with 4 mL subcritical extraction solvent and was treated with an ultrasound to extract oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was 45° C. and the ultrasonic frequency was 25 KHz. The extraction lasted 20 min. After that, the extraction solvent was removed by reducing pressure. The extraction was repeated 4 times and then extracts were collected and filtered to obtain  Euphausia superba  oil. 
         [0038]    The extraction rate of  Euphausia superba  oil was 21.52% (dry basis). The acid value and the phospholipid content were 8.3 and 43.20%, respectively. The DHA/EPA content of total fatty acids was 33.95%. 
       Example 8 
     Cryogenic Heat Dehydration Combined with Subcritical Extraction 
       [0039]    Thawed  Euphausia superbas  were treated by a low temperature and low oxygen heat pump dehydration process. This process was carried out under nitrogen protection and the krill was spread with a thickness of no more than 2 centimeters. The temperature was maintained at 55° C. and the speed of hot wind through the materials was controlled at 3 m/s. The process lasted until that water content of  Euphausia superba  was reduced to 8.5±0.1%. Then the materials were crushed to 30 mesh. 1 g krill powder was mixed with 4 mL subcritical butane solvent and was treated with an ultrasound to extract oil. During the extraction process, the extraction pressure was kept at 1.2±0.1 MPa, the temperature was 45° C. and the ultrasonic frequency was 25 KHz. The extraction lasted 20 min. After that, the extraction solvent was removed by reducing pressure. The extraction was repeated 4 times and extracts were collected and filtered to obtain  Euphausia superba  oil. 
         [0040]    The extraction rate of  Euphausia superba  oil was 17.42% (dry basis). The acid value and the phospholipid content were 12.3 and 42.78%, respectively. The DHA/EPA content of total fatty acids was 32.05%. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The parameter of each example 
               
             
          
           
               
                   
                   
                 Extraction 
                   
                   
                 DHA/EPA 
               
               
                   
                 Dehydration  
                 rate(dry 
                 Acid 
                 Phospholipid 
                 content of 
               
               
                   
                 time 
                 basis) 
                 value 
                 content 
                 fatty acids 
               
               
                   
               
             
          
           
               
                 Example 
                 4-7 
                 h 
                 21.32% 
                 8.4 
                 43.10% 
                 33.85% 
               
               
                 1 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 7-10 
                 h 
                 21.02% 
                 8.1 
                 43.02% 
                 33.48% 
               
               
                 2 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 3-6 
                 h 
                 21.62% 
                 9.5 
                 43.16% 
                 33.16% 
               
               
                 3 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 25-30 
                 h 
                 22.10% 
                 7.2 
                 43.52% 
                 34.94% 
               
               
                 4 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 2-4 
                 h 
                  16.2% 
                 19.2 
                 42.36% 
                 31.94% 
               
               
                 5 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 2-4 
                 h 
                  15.5% 
                 21.5 
                 42.26% 
                 30.02% 
               
               
                 6 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 7-11 
                 h 
                 21.52% 
                 8.3 
                 43.20% 
                 33.95% 
               
               
                 7 
                   
                   
                   
                   
                   
                   
               
               
                 Example 
                 3-6 
                 h 
                 17.42% 
                 12.3 
                 42.78% 
                 32.05% 
               
               
                 8 
               
               
                   
               
             
          
         
       
     
         [0041]    In example 1-3, combining double dehydration system with subcritical extraction, the extraction rate, acid value and phospholipid content of  Euphausia superba  oil were comparatively ideal. The extraction time of example 2 was less than example 1 and 3, therefore the extraction rate was slightly lower. In example 2, the temperature of heat pump was lower than example 1, thus the dehydration time needed is longer. In example 3, the temperature of heat pump was increased in order to decrease dehydration time, but the acid value was slightly higher than example 1. In example 7, the water content of  Euphausia superbas  was 35% after treated by the low temperature and low oxygen heat pump dehydration system. It made the dehydration time of the second process of microwave-assisted frozen-blasting longer, so that the whole dehydration time was increased. 
         [0042]    In example 4, extraction rate and acid value were slightly increased compared to example 1, but pre-treatment time was so long that much more energy was consumed. In example 5-6, the krill was not dehydrated by the double dehydration system but hot air. The extraction rate in examples 5-6 was 5-6% lower than that of examples 1-4. The method of hot air drying used in example 5 shortened dehydration time, but led to high acid value. In example 6, solvent extraction could not avoid the high acid value caused by high temperature. In example 8, krill was dried by hot air which decreased dehydration time but resulted in a lower extraction rate. 
         [0043]    It is found that high water content can negatively affect extraction efficiency while too low water content of the dehydrated krill material does not benefit the oil extraction, but it costs more time and energy. It is also found that material-to-liquid ratio has an important effect on the extraction rate. If the material-to-liquid ratio is too low, the extraction is incomplete and the extraction rate is lower with the same number of extractions. 
         [0044]    While the present invention has been described in some detail for purposes of clarity and understanding, one skilled in the art will appreciate that various changes in form and detail can be made without departing from the true scope of the invention. All figures, tables, appendices, patents, patent applications and publications, referred to above, are hereby incorporated by reference.