Abstract:
A process for the recovery of phytonutrients such as carotenes, phospholipids and ubiquinones from palm oil esters is disclosed. This process comprises the steps of vacuum distillation, treatment and purification of concentrate containing these phytonutrients. The alkyl esters is subjected to at least one stage vacuum distillation at temperature from 80° C. to 220° C. and pressure less than 40 mTorr. The carotenes concentrate is subjected to various physical and chemical treatments to yield higher carotenes concentration enriched with ubiquinones in indigenous diacylglycerols.

Description:
FIELD OF INVENTION  
       [0001]     This invention relates to a process of recovery of phytonutrients such as carotenes, phospholipids and ubiquinones using vacuum distillation, various physical and chemical treatments and purification of the phytonutrients containing natural esterified oils and fats and has particular but not exclusive application to their recovery from palm oil.  
       BACKGROUND ART  
       [0002]     Carotenoids are the natural pigments, which impart a rich orange-red colour in plants and animals. Carotenoids are found in abundance (˜600 types) in nature. These include beta-carotene and alpha-carotene, which can be converted into Vitamin A (retinal) in the body. Other non-vitamin A carotenoids includes lycopene and phytoene. All these are present in crude palm oil. In fact, crude palm oil is one of the richest natural plant sources with carotenes with concentration of 500-700 ppm. Carotenoids have a number of important physiological properties. For example lycopene suppresses the growth of various cancer lines. These include the lung and liver cancer as well as colon tumours.  
         [0003]     Ubiquinone (Coenzyme Q10) is a naturally occurring coenzyme found in palm oil. The concentration of ubiquinone in crude palm oil is determined in the range of 10-100 ppm (Hazura et al. 1990). Ubiquinone is found mostly in the inner mitochondrial membrane, especially in the heart, liver, kidney and pancreas. It plays an important role in the mitochondrial electron transport chain and is also a powerful antioxidant and free radical&#39;s scavenger, and it is believed to possess membrane-stabilising properties. Since its discovery, ubiquinone has been used to aid in the treatment of many cardiovascular diseases such as congestive heart failure, cardiac arrhythmias and hypertension.  
         [0004]     Phospholipids are essential for cell membrane repair, optimum mental function (it provides vital neurotransmitter precursor) and lipid metabolism. Phospholipids (phosphatides) are indispensable components of cell membranes and are also natural emulsifiers, helping fats dissolve in water. They support a healthy cardiovascular system and have been used as a fat emulsifier in preventing arteriosclerosis, cardiovascular disease, brain function, and proper nerve function and maintain proper electrical energy and nutrients transfer across the cell membrane.  
         [0005]     A number of patents have been filed on the recovery of carotenes from palm oil. These include U.S. Pat. No. 5,157,132, GB2160874, U.S. Pat. No. 6,072,092 and EP0349138. The recovery processes employ esterification/transesterification, molecular distillation, adsorbent at some stages. The current process is an advanced process integrating steps of at least one stage vacuum distillation, various physical and chemical treatment and purification to the phytonutrients concentrates. The integrated process yields higher carotenes concentration enriched with ubiquinones in indigenous diacylglycerols; and phospholipids enriched fraction.  
       SUMMARY OF INVENTION  
       [0006]     This present invention relates to a process for the recovery of carotene concentrates such as carotenes, ubiquinones, and phospholipids from natural esterified oils and fats has in particular but not exclusive to crude palm oil and palm oil products.  
         [0007]     This process involves the integration steps of (i) at least one stage vacuum distillation at temperatures ranging 80° C.-220° C. and pressure less than 40 mTorr; (ii) various physical and chemical treatment including filtration, solvent partitioning, saponification re-transesterification; and (iii) purification of phytonutrients containing concentrate.  
         [0008]     Esterification/transesterification of crude palm oil and degummed and bleached palm oil is carried out with alkyl alcohol in the presence of an alkaline catalyst under conditions sufficient to convert free fatty acids and acylglycerols into alkyl esters-rich layer is either subjected to another re-transesterification process or clean water wash for neutralisation. The esterified palm oil is subjected to one or multi-stage vacuum distillation.  
         [0009]     After first vacuum distillation, the carotenes enriched alkyl esters (residue) is subjected to the re-transesterification process. The process is carried out with alkyl alcohol with catalyst dissolving in alcohol or clean water under sufficient conditions to convert the traces of acylglycerols into alkyl esters and glycerol. The re-transesterified alkyl esters-rich layer is then subjected to second vacuum distillation for the production of carotenes concentrate.  
         [0010]     In some instances, the esterified and or re-esterified palm oil is subjected to one stage vacuum distillation, yielding a concentrate residue enriched in carotenes.  
         [0011]     The carotenes enriched alkyl esters layer from the first vacuum distillation is filtered or treated with hydrocarbon solvent to remove monoacylglycerols. The filtrate is subjected to second vacuum distillation for the production of carotenes concentrate.  
         [0012]     Mixture of carotenes concentrate could also be produced by second stage vacuum distillation alone under conditions without going through third stage vacuum distillation.  
         [0013]     A minimum amount of palm oil ethyl esters is added to the treated carotenes enriched alkyl esters (methyl esters in this case) prior to further vacuum distillation. Carotenes concentrate produced is enriched with ubiquinones in diacylglycerols with phospholipids. Treatment of carotenes concentrate is carried out using hydrophobic and hydrophilic solvents for further purification. The concentrate could be saponified to obtain desire concentration of carotenes fractions. Phospholipids are also recovered by membrane filtration of crude palm oil prior to conversion of oil into alkyl esters.  
         [0014]     This present invention has many advantages. It is an integrated process where carotenes are recovered from crude palm oil, and, degummed and bleached palm oil. Carotenes recovered from this process present in diacylglycerols which is an effective carrier and dietary oil. With the improved two stage vacuum distillation, various treatments can be incorporated between the distillation stages. For instance, indigenous monoacylglycerols can be removed from the residue of first vacuum distillation after ten times of concentration and recovered as a high purity co-product. Other valuble minor components, ubiquinone and phospholipids are being concentrated in carotenes concentrate during the process.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     EXAMPLE 1  
       [0015]     Crude palm oil (CPO) was esterified by using sodium hydroxide as catalyst with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to molecular distillation at temperature of 110° C., wiper speed of 250 rpm and pressure of 5 mTorr. Residue and distillate were collected for analysis of carotenes content. The carotenes concentration was 6.5% with recovery of 80.5%. Detail results are shown in the Table 1.  
       EXAMPLE 2  
       [0016]     Bleached and degummed palm oil (BDPO) was esterified by using sodium hydroxide with methanol to produce BDPO methyl ester (ME). Glycerol was drained and BDPOME was washed with hot distilled water. The neutralized BDPOME was subjected to molecular distillation at temperature of 130° C., wiper speed of 250 rpm and pressure of 5 mTorr. Residue and distillate were collected for analysis of carotenes content. The carotenes concentration was 12.9% with recovery of 92.5% was obtained. Detailed results are shown in the Table 2.  
       EXAMPLE 3  
       [0017]     Crude palm oil (CPO) was esterified by using sodium hydroxide with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to molecular distillation at temperature of 150° C., wiper speed of 300 rpm and pressure of 30 mTorr. All samples were analysed for carotenes content. The carotenes concentration was 5.9% with recovery of 79.9%. Detail results are shown in the Table 3.  
       EXAMPLE 4  
       [0018]     Bleached and degummed palm oil (BDPO) was esterified by using sodium hydroxide with methanol to produce BDPO methyl ester (ME). Glycerol was drained and BDPOME was washed with hot distilled water. The neutralized BDPOME was subjected to molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 5 mTorr. Residue was collected and analysed for carotenes content. The carotenes concentration was 8.5% with recovery of 91.7%. Detailed results are shown in the Table 4.  
       EXAMPLE 5  
       [0019]     Bleached and degummed palm oil (BDPO) was esterified by catalytic reaction with methanol to produce BDPO methyl ester (ME). Glycerol was drained and BDOME was washed with hot distilled water. The neutralised BDOME was subjected to 1 st  molecular distillation at temperature of 110° C., wiper speed of 250 rpm and pressure of 3 mTorr. Residue was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 3 mTorr. All samples were analysed for carotenes content. The carotenes concentration was 8.6% with recovery of 86%. Detail results are shown in the Table 5.  
       EXAMPLE 6  
       [0020]     Crude palm oil (CPO) was esterified by catalytic reaction with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to fast speed molecular distillation at temperature of 90° C., wiper speed of 250 rpm and pressure of 20 mTorr. Residue was re-transesterified to obtain higher degree of methyl esters conversion. The re-transesterification was carried out using sodium methylate as the catalyst. Treated sample was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 3 mTorr. The samples were analysed for carotenes and ubiquinone content. The carotenes concentration was 14.4% with recovery of 92.7% and ubiquinone concentration was 0.3% with recovery of 94.7%. Detail results are shown in the Table 6.  
       EXAMPLE 7  
       [0021]     Bleached and degummed palm oil (BDPO) was esterified by catalytic reaction with methanol to produce BDPO methyl ester (ME). Glycerol was drained and BDPOME was washed with hot distilled water. The neutralised BDPOME was subjected to fast speed molecular distillation at temperature of 90° C., wiper speed of 200 rpm and pressure of 20 mTorr. Residue was treated with hexane (1:1, v/v) and chilled to 0° C. for two hours. The mixture was filtered and pumped dried. Treated residue was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 5 mTorr. All samples were analysed for carotenes content. The carotenes concentration was 12.2% with recovery of 87.9%. Detailed results are shown in the Table 7.  
       EXAMPLE 8  
       [0022]     Crude palm oil (CPO) was esterified by catalytic reaction with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to fast speed molecular distillation at temperature of 90° C., wiper speed of 200 rpm and pressure of 20 mTorr. Residue was treated with hexane (1:1, v/v) and chilled to 0° C. for two hours. The mixture was filtered and washed with MeOH/H 2 O (5:2.5:0.5,v/v/v) for two times followed by vacuum pumped dried. Treated sample was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 5 mTorr. Al l samples were analysed for carotenes content. The carotenes concentration was 18.1 % with recovery of 87.9%. Detailed results are shown in the Table 8.  
       EXAMPLE 9  
       [0023]     Crude palm oil (CPO) was esterified by catalytic reaction with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to fast speed molecular distillation at temperature of 90° C., wiper speed of 200 rpm and pressure of 20 mTorr. Residue was treated with iso-octane (1:1, v/v) and chilled to 0° C. for two hours. The mixture was filtered and pumped dry. Treated sample was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 5 mTorr. All samples were analysed for carotenes content. The carotenes concentration was 11.0% with recovery of 88.3%. Detail results are shown in the Table 9.  
       EXAMPLE 10  
       [0024]     Crude palm oil (CPO) was esterified by catalytic reaction with methanol to produce CPO methyl ester (ME). Glycerol was drained and CPOME was washed with hot distilled water. The neutralised CPOME was subjected to fast speed molecular distillation at temperature of 90° C., wiper speed of 200 rpm and pressure of 20 mTorr. The residue was then subjected to re-esterification process, 50 g of the concentrate was re-transesterified with 1% alkaline catalyst (NaOH) dissolved in 20 ml methanol. The mixture was refluxed at 60-65° C. for 100 minutes. The sample of the re-esterification process was analysed for total carotenes, esters, acylglycerols and other minor components. The results of the analysis were shown in Table 10.  
       EXAMPLE 11  
       [0025]     The CPOME produced subjected to similar process to that of Example 10. The product produced was then subjected to re-esterification process, 50 g of the concentrate was re-transesterified with 1% sodium hydroxide dissolved in 5 ml distilled water. The mixture was refluxed at 60-65° C. for 30 minutes. The sample of the re-esterification process was analysed for total carotenes, esters, acylglycerols and other minor components. The results of the analysis were shown in Table 11.  
       EXAMPLE 12  
       [0026]     Residue from fast speed molecular distillation of CPOME (Example 8) was added with 10% (v/v) CPO ethyl esters. The mixture was subjected to 2 nd  molecular distillation at temperature of 150° C., wiper speed of 250 rpm and pressure of 1 mTorr. The mass flow rate of the mixture in the distillation processes has increased 3 times of the normal flow rate without addition of ethyl esters. All samples were analysed for carotenes content. The carotenes concentration was 12.8% with recovery of 87.4%. Detailed results are shown in Table 12.  
       EXAMPLE 13  
       [0027]     5.0 g of carotenes concentrate was subjected to unsaponification with 7.0 ml of 10% potassium hydroxide in 30.0 ml of ethanol. The mixture was refluxed for ½ hour. The reacted mixture was transferred to a separating funnel and the unsaponifiable matters were extracted with 50 ml of hexane: distilled water (90:10, v/v) for 3 times. The extracts were neutralised with copious of 10% ethanol in distilled water. The neutralised extract was then vacuum pumped dry and analysed. The results of the analysis are shown in Table 13.  
       EXAMPLE 14  
       [0028]     Carotenes concentrate (from Example 8) was used as crude material in the treatment. 0.1 g of carotenes concentrate was added to 1 ml of Hexane and 3 ml of Methanol. The mixture was chilled to −10° C. for 1 hour. The top and bottom layers were separated and vacuum pumped dried. Samples were analysed for total carotenes content. The carotenes concentration was 30.1% with recovery of 69%. Detail results are shown in the Table 14.  
       EXAMPLE 15  
       [0029]     Carotenes concentrate (from Example 8) was used as crude material in the treatment, 0.16 g of carotenes concentrate was added to 5 ml of Hexane and 10 ml of Methanol. The mixture was chilled to −10° C. for 1 hour. The top and bottom layers were separated and vacuum pumped dry. Samples were analysed for total carotenes content. The carotenes concentration was 24.3% with recovery of 84.7%. Detail results are shown in the Table 15.  
       EXAMPLE 16  
       [0030]     Carotenes concentrate produced from examples 1, 3 and 4 were analysed for total phospholipids content. The results are shown in Table 16 with the concentration ranging from 0.60% to about 4.0%.  
       EXAMPLE 17  
       [0031]     2 litres of CPO was filtered with a membrane filter with a 0.05 μm pore size. This process was carried out to reduce impurities in the CPO. These include phospholipids, iron and copper. The CPO was subjected to the membrane system with the temperature of 60° C., pressure of 2 bar with 300 rpm. The filtrate was analysed for total phospholipids. It was found that the total phospholipids could be reduced to 46.40 ppm from 171.17 ppm found in CPO.  
       EXAMPLE 18  
       [0032]     500 g of neutralised palm oil (NPO) was esterified by sodium methylate with methanol to produce NPO methyl esters (ME). Glycerol was drained and the NPOME was divided into two portions for different neutralisation approaches. To the first part of NPOME, 10% of distilled water was used for each washing step until neutralised NPOME was obtained. To the second part of NPOME, hydrochloric acid was added into distilled water until pH 4-5. 10% of the acidified distilled water was then used for each washing step until NPOME was neutralised. The result shows that the acidified distilled water is better than normal distilled water for neutralization of NPOME produced by reducing the total amount of distilled water used by 40%. All minor components such as carotenes, vitamin E, phytosterols and squalene were preserved well in acidified water washing. The results are shown in Table 18.  
                                                                 TABLE 1                           (Single Stage Distillation - No treatment)       Condition: 110° C., 250 rpm, 0.93 ml/min, 5 mTorr                Carotene                ppm   mg   % Recovery                            Feed: CPO Methyl Esters   571.0   246.7   100.0           Carotenes Concentrate   65232.6   198.6   80.5                      
 
         [0033]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 (Single Stage Distillation - No treatment) 
               
               
                 Condition: 130° C., 250 rpm, 0.93 ml/min, 5 mTorr 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 Feed: BDPO Methyl Esters 
                 571.0 
                 246.7 
                 100.0 
               
               
                 Carotenes Concentrate 
                 129159.0 
                 228.2 
                 92.5 
               
               
                   
               
             
          
         
       
     
         [0034]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                   
               
               
                 (Single Stage Distillation - No treatment) 
               
               
                 1 st  Distillation: 150° C., 300 rpm, 0.93 ml/min, 30 mTorr 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                   
                 Feed: CPO Methyl Esters 
                 698.0 
                 603.1 
                 100.0 
               
               
                   
                 Carotenes Concentrate 
                 58695.0 
                 481.8 
                 79.9 
               
               
                   
                   
               
             
          
         
       
     
         [0035]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                   
               
               
                 (Single Stage Distillation - No treatment) 
               
               
                 1 st  Distillation: 150° C., 250 rpm, 0.93 ml/min, 5 mTorr 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                   
                 Feed: BDPO Methyl Esters 
                 571.0 
                 246.7 
                 100.0 
               
               
                   
                 Carotenes Concentrate 
                 84819.0 
                 226.2 
                 91.7 
               
               
                   
                   
               
             
          
         
       
     
         [0036]    
       
         
               
             
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                   
               
               
                 (Two Stage Distillation - No treatment) 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 1 st  Distillation: 110° C., 250 rpm, 0.9 ml/min, 3 mTorr 
               
             
          
           
               
                 Feed: BDPO Methyl Estesr 
                 612.0 
                 3172.6 
                 100.0 
               
               
                 Carotenes enriched alkyl esters 
                 47174.0 
                 2959.1 
                 93.3 
               
             
          
           
               
                 2 nd  Distillation: 150° C., 250 rpm, 0.93 ml/min, 3 mTorr 
               
             
          
           
               
                 Carotenes enriched alkyl esters 
                 47174.0 
                 2794.4 
                 100.0 
               
               
                 Carotenes Concentrate 
                 86625.0 
                 2402.5 
                 86.0 
               
               
                   
               
             
          
         
       
     
         [0037]    
       
         
               
             
               
               
             
               
               
               
               
               
             
               
             
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
               
                   
               
               
                 (Two Stage Distillation - Re-transesterification of concentrate after 1 st  Distillation) 
               
               
                   
               
             
             
               
                 1 st  Distillation: 90° C., 200 rpm, 2.2 ml/min, 20 mTorr 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
               
                   
                 Feed: CPO Methyl Esters 
                 682.0 
                 4124.7 
                 100.0 
               
               
                   
                 Carotenes enriched alkyl esters 
                 6796.0 
                 4022.1 
                 97.5 
               
               
                   
                   
               
             
          
           
               
                 2 nd  Distillation: 150° C., 250 rpm, 0.93 ml/min, 3 mTorr 
               
             
          
           
               
                   
                 Carotene 
                 Ubiquinone 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
               
               
                   
                   
                   
                   
                 140 
                 60.5 
                 100.0 
               
               
                 Feed: Treated carotenes 
                 6790.0 
                 2933.3 
                 100.0 
               
               
                 enriched alkyl esters 
               
               
                 Carotenes Concentrate 
                 143123.0 
                 2720.5 
                 92.7 
                 3014.0 
                 57.3 
                 94.7 
               
               
                   
               
             
          
         
       
     
         [0038]    
       
         
               
             
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                   
               
               
                 (Two Stage Distillation - Treatment with Hexane) 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 1 st  Distillation: 90° C., 200 rpm, 2.2 ml/min, 5 mTorr 
               
             
          
           
               
                 Feed: BDPO Methyl Esters 
                 571.0 
                 2466.7 
                 100.0 
               
               
                 Carotenes enriched alkyl esters 
                 3949.4 
                 2149.7 
                 87.1 
               
             
          
           
               
                 2 nd  Distillation: 150° C., 250 rpm, 0.93 ml/min, 7 mTorr 
               
             
          
           
               
                 Feed: Treated carotenes enriched 
                 3949.4 
                 1023.7 
                 100.0 
               
               
                 alkyl esters 
               
               
                 Carotenes Concentrate 
                 121825.0 
                 900.2 
                 87.9 
               
               
                   
               
             
          
         
       
     
         [0039]    
       
         
               
             
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                   
               
               
                 (Two Stage Distillation - Treatment with Hexane 
               
               
                 and MeOH/H 2 0 Washing) 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 1 st  Distillation: 90° C., 200 rpm, 2.2 ml/min, 20 mTorr 
               
             
          
           
               
                 Feed: CPO Methyl Esters 
                 571.0 
                 2466.7 
                 100.0 
               
               
                 Carotenes enriched alkyl esters 
                 4991.5 
                 2630.7 
                 98.3 
               
             
          
           
               
                 2 nd  Distillation: 150° C., 250 rpm, 0.93 ml/min, 5 mTorr 
               
             
          
           
               
                 Feed: Carotenes enriched alkyl esters 
                 4991.5 
                 1293.8 
                 100.0 
               
               
                 Carotenes Concentrate 
                 181075.6 
                 1134.7 
                 87.7 
               
               
                   
               
             
          
         
       
     
         [0040]    
       
         
               
             
               
               
             
               
               
               
               
             
               
             
               
               
               
               
             
               
             
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                   
               
               
                 (Two Stage Distillation - Treatment with Iso-Octane) 
               
             
          
           
               
                   
                 Carotene 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 1 st  Distillation: 90° C., 200 rpm, 2.2 ml/min, 5 mTorr 
               
             
          
           
               
                 Feed: CPO Methyl Esters 
                 602.0 
                 2340.6 
                 100.0 
               
               
                 Carotenes enriched alkyl esters 
                 3720.0 
                 2105.2 
                 89.9 
               
             
          
           
               
                 2 nd  Distillation: 150° C., 250 rpm, 0.93 ml/min, 5 mTorr 
               
             
          
           
               
                 Feed: Treated carotenes enriched 
                 3720.0 
                 964.2 
                 100.0 
               
               
                 alkyl esters 
               
               
                 Carotenes Concentrate 
                 110481.0 
                 851.8 
                 88.3 
               
               
                   
               
             
          
         
       
     
         [0041]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                   
               
               
                 (Re-transesterification of carotenes enriched alkyl esters after 
               
               
                 first stage distillation-catalyst dissolved in methanol) 
               
             
          
           
               
                   
                 Percentage (%) 
               
             
          
           
               
                   
                 Esters 
                 MG 
                 DG 
                 TG 
                 Carotenes 
                 Others Minor Components 
               
               
                   
                   
               
             
          
           
               
                 CPO Methyl esters 
                 99.413 
                 0.296 
                 0.043 
                 N.D. 
                 0.071 
                 0.177 
               
               
                 Carotenes enriched alkyl esters 
                 96.730 
                 0.876 
                 0.509 
                 N.D. 
                 0.632 
                 1.253 
               
               
                 Treated carotenes enriched alkyl esters 
                 98.032 
                 0.274 
                 N.D. 
                 N.D. 
                 0.609 
                 1.085 
               
               
                   
               
               
                   Note:    
               
               
                   MG: Monoacylglycerol    
               
               
                   DG: Diacylglycerol    
               
               
                   TG: Triacylglycerol    
               
               
                   N.D.: Non-detectable    
               
               
                   CPO: Crude Palm Oil    
               
               
                   BDPO: Bleached and Degummed Palm Oil    
               
               
                   Other Minor Components: Squalene, Sterols, Tocols (tocopherols and tocotrienol)    
               
             
          
         
       
     
         [0042]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                   
               
               
                 (Re-transesterification of carotenes enriched alkyl esters after first 
               
               
                 stage distillation-catalyst dissolved in treated water) 
               
             
          
           
               
                   
                 Percentage (%) 
               
             
          
           
               
                   
                 Esters 
                 MG 
                 DG 
                 TG 
                 Carotenes 
                 Others Minor Components 
               
               
                   
                   
               
             
          
           
               
                 CPO Methyl esters 
                 99.413 
                 0.296 
                 0.043 
                 N.D. 
                 0.071 
                 0.177 
               
               
                 Carotenes enriched alkyl esters 
                 96.730 
                 0.876 
                 0.509 
                 N.D. 
                 0.6320.632 
                 1.253 
               
               
                 Treated carotenes enriched alkyl esters 
                 97.740 
                 0.263 
                 0.019 
                 N.D. 
                 0.622 
                 1.357 
               
               
                   
               
             
          
         
       
     
         [0043]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                   
               
               
                 (Two Stage Distillation-with addition of ethyl esters) 
               
               
                 2 nd  Distillation: 150° C., 250 rpm, 1 mTorr, 3 ml/min 
               
             
          
           
               
                   
                 Carotenes 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                 Feed: CPO Methyl Esters + 
                 4991.5 
                 998.3 
                 100 
               
               
                 10% Ethyl Esters 
               
               
                 Carotenes Concentrate 
                 128120 
                 872.7 
                 87.4 
               
               
                   
               
             
          
         
       
     
         [0044]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 13 
               
             
             
               
                   
               
               
                   
               
               
                 (Saponification of carotenes concentrate) 
               
             
          
           
               
                   
                 Percentage (%) 
               
             
          
           
               
                   
                 FFA 
                 Esters 
                 MG 
                 DG 
                 TG 
                 Carotenes 
                 Others 
               
               
                   
                   
               
             
          
           
               
                 Carotenes 
                 1.45 
                 1.11 
                 34.61 
                 18.26 
                 33.19 
                 2.10 
                 9.28 
               
               
                 Concentrate 
               
               
                 Unsaponified 
                 24.42 
                 0.00 
                 0.00 
                 13.92 
                 34.95 
                 11.58 
                 15.13 
               
               
                 Sample 
               
               
                   
               
             
          
         
       
     
         [0045]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 14 
               
             
             
               
                   
               
               
                   
               
               
                 (Partition of carotenes) 
               
             
          
           
               
                   
                 Carotenes 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                   
                 Carotenes Concentrate 
                 170982 
                 0.0172 
                 100.0 
               
               
                   
                 Hexane Layer 
                 301060 
                 0.0119 
                 69.0 
               
               
                   
                 Methanol Layer 
                  58556 
                 0.0036 
                 30.2 
               
               
                   
                   
               
             
          
         
       
     
         [0046]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 15 
               
             
             
               
                   
               
               
                   
               
               
                 (Partition of carotenes) 
               
             
          
           
               
                   
                 Carotenes 
               
             
          
           
               
                   
                 ppm 
                 mg 
                 % Recovery 
               
               
                   
                   
               
             
          
           
               
                   
                 Carotenes Concentrate 
                 170982 
                 0.0278 
                 100.0 
               
               
                   
                 Hexane Layer 
                 243538 
                 0.0235 
                 84.7 
               
               
                   
                 Methanol Layer 
                  41274 
                 0.0027 
                 11.6 
               
               
                   
                   
               
             
          
         
       
     
         [0047]    
       
         
               
               
             
               
               
               
             
           
               
                   
                 TABLE 16 
               
               
                   
                   
               
               
                   
                   
               
               
                   
                 Phospholipids (%) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Carotenes Concentrate (from Example 1) 
                 1.78 
               
               
                   
                 Carotenes Concentrate (from Example 3) 
                 3.83 
               
               
                   
                 Carotenes Concentrate (from Example 4) 
                 0.78 
               
               
                   
                   
               
             
          
         
       
     
         [0048]    
       
         
               
               
             
               
               
               
             
               
               
               
             
           
               
                   
                 TABLE 18 
               
             
             
               
                   
                   
               
               
                   
                   
               
               
                   
                 Concentration (ppm) 
               
             
          
           
               
                   
                 Carotenes 
                 Vitamin E 
               
               
                   
                   
               
             
          
           
               
                 Neutralised Palm Oil 
                 512 
                 950 
               
               
                 Acidified distilled water washed NPOME 
                 508 
                 908 
               
               
                 Normal distilled water washed NPOME 
                 500 
                 921