Patent Publication Number: US-2009227678-A1

Title: Process for obtaining lipid form cells

Description:
The present invention relates to a process for obtaining lipid from a composition comprising cells and water. The invention also relates to lipid obtainable by this process. 
     Lipids can be produced by micro-organisms, for instance in a fermentation process. They can also be present in plant cells. Lipids have many uses, and can for example be included into foodstuffs, feed, nutritional supplements and pharmaceuticals. 
     Obtaining lipid from cells often involves obtaining the lipid from a composition comprising the cells and water. The composition can for instance be a fermentation broth or a partly dewatered product obtained by separating part of the water from a fermentation broth, for instance by mechanical separation techniques. Various processes are known to allow recovery of lipid from a composition comprising cells and water. For instance, it is known to dry the composition by spray drying, and to recover the lipid from the dried product by extraction with an organic solvent. However, the quality of the lipid can be impaired under the conditions that prevail during spray drying. Moreover, the morphology of the dried product may render subsequent extraction less efficient. 
     WO-A-97036996 describes a process for obtaining lipid from microbial cells, which involves granulation of the cells into granules, preferably by extrusion. The granules are dried, and the lipid is obtained from the dried granules, for instance by extraction with an organic solvent. The extraction is efficient, the resulting lipid has a high quality, and the yield, i.e. the quantity of lipid recovered per quantity of lipid present in cells, is high. 
     In spite of the advantages of the process of WO-A-97036996, there is still a desire for further improvement. In particular, the process involves relatively many steps, such as granulation and drying of the granules. Moreover, the drying step requires energy. Therefore, there is a desire for a process requiring fewer steps, wherein energy can be saved, and which enables the lipid to be extracted in an efficient manner. 
     This goal is achieved according to the invention by providing a process for obtaining lipid from a composition comprising cells and water, said process comprising: 
     (a) contacting the composition with a desiccant; and 
     (b) recovering the lipid from the cells. 
     The process according to the invention enables lipid of high quality to be obtained with a high yield. According to the invention, drying the cells by heating prior to recovering the lipid from the cells is not needed. This is of particular advantage for heat sensitive lipid, for instance lipid comprising a polyunsaturated fatty acid (PUFA). The process according to the invention further has the advantage that formation of fines or dust—which are for instance formed by prior art processes that involve spray drying or milling—is avoided or minimized. Moreover, it is found that—when the lipid is recovered by extraction with a solvent—relatively small quantities of solvent suffice to obtain a relatively high yield. The invention also enables recovering the lipid by extraction in the presence of the desiccant, i.e. without the need of separating the desiccant from the cells. Hence, the extraction can be carried out in the presence of water adsorbed to or absorbed by the desiccant. This is surprising in that the prior art advocates to remove water in order to obtain high yields (see e.g. WO-A-97036996). 
     The process according to the invention comprises contacting the composition comprising cells and water with a desiccant. As a result, water is taken up from the composition by the desiccant. Taking up water may be by adsorption and/or absorption. The desiccant is not limited to a specific desiccant, and a wide variety of agents capable of taking up water from the composition by adsorption and/or absorption may be used as desiccant. Such agents are also referred to in the art as adsorbents and/or absorbents. 
     Examples of desiccants that may be used in the process are for instance silica (e.g. silica gel), activated carbon, activated alumina, a molecular sieve (e.g. a carbon molecular sieve or a zeolite), or a cross-linked polymer (e.g a poly-acrylate). Silica is preferred. Preferably, the desiccant is in particulate form. 
     Preferably, the desiccant has a high absorption capacity. The absorption capacity refers to the quantity of water that can be absorbed per quantity of desiccant. A relatively high absorption capacity has the advantage that optimal extraction yields can be obtained using relatively small quantities of desiccant. The desiccant may have an absorption capacity of at least 50 g of water per 100 g of desiccant, preferably at least 100 g of water per 100 g of desiccant. There is no specific upper limit for the absorption capacity. In practice, the absorption capacity for water is below 20,000 g of water per 100 g of desiccant, for instance below 10,000 g of water per 100 g of desiccant. As used herein the weight of the desiccant refers to the moisture-free weight of the desiccant. The absorption capacity may be determined by techniques known to the skilled person. A suitable method is by DBP absorption, for instance according to DIN 53601. 
     Preferably, the desiccant has a high specific surface area. A relatively high specific surface area has the advantage that optimal extraction yields can be obtained using relatively small quantities of desiccant. The desiccant may for instance have a specific surface area of at least 25 m 2 /g, preferably at least 50 m 2 /g, preferably at least 100 m 2 /g. There is no specific upper limit for the specific surface area of the desiccant. In practice, the specific surface area is below 2000 m 2 /g, for instance below 1000 m 2 /g. The specific surface area may be determined by techniques known to the skilled person, for instance according to ISO 5794/1, Annex D. 
     In a preferred embodiment, the desiccant is porous. This has the advantage that the surface area available for adsorption is relatively high. 
     In the process according to the invention, the composition to be contacted with the desiccant may comprise water in any suitable quantity. The water content of the composition to be contacted with the desiccant may for instance be at least 5 wt. %, for instance at least 10 wt. %, for instance at least 20 wt. %, for instance at least 30 wt. %. There is no specific upper limit for the water content of the composition to be contacted with the desiccant. The water content of the composition to be contacted with the desiccant may for instance be below 98 wt. %, preferably below 95 wt. %, preferably below 90 wt. %, preferably below 80 wt. %, preferably below 70 wt. %. A lower water content has the advantage that less desiccant may be used to obtain an optimal yield. 
     As used herein, the water content of the cells is given by w w /(w cells +w w )*100% wherein 
     w w =weight of the water in the composition 
     w cells =weight of dry matter of the cells in the composition 
     The skilled man will understand that the weight of dry matter of the cells (w cells ) refers to the weight of the moisture-free cells, but including the weight of the lipid. It will be appreciated that the weight of the water comprised in the composition (w w ) includes the total weight of water in the composition, including intracellular, intercellular and extracellular water. The values of w w  and w cells , and accordingly the water content of the composition can be determined by methods known to the skilled person, for instance by evaporating the water at a temperature of 105° C., and determining the weight of the evaporated water and the remaining cells. An infrared dry-matter balance may for instance be used. 
     In a preferred embodiment, the invention provides preferred quantities of desiccant to be contacted with the composition. It is found that the extraction yield increases with increasing quantity of desiccant that is contacted with the composition until an optimal yield is achieved. Based on this teaching provided by the invention, the skilled man can—by varying the quantity of desiccant—determine the optimal quantity of desiccant in any situation. 
     It is in particular found that the preferred quantities of desiccant are dependent on the water content of the composition and on the properties of the desiccant. In a preferred embodiment, w des /(w cells(a) +w w(a) )&gt;(1/x)*(1−w cells(a) /(0.8*(w cells(a) +w w(a) ))), wherein 
     w des =weight of desiccant to be contacted with the composition in (a)
 
w cells(a) =weight of dry matter of the cells in the composition to be contacted with the desiccant in (a)
 
w w(a) =weight of water in the composition to be contacted with the desiccant in (a)
 
x=absorption capacity of the desiccant.
 
     In another preferred embodiment, 
         w   des /( w   cells(a)   +w   w(a) )&gt;(1 /x )*(1 −w   cells(a) /(0.9*( w   cells(a)   +w   w(a) ))) 
     In a preferred embodiment, the process according to the invention comprises contacting the composition with a quantity of desiccant such that the yield is at least 70%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%. As used herein, the yield refers to the quantity of lipid recovered from the cells per quantity of lipid present in the cells. 
     The composition to be contacted with the desiccant may be any suitable composition comprising cells and water from which lipid can be obtained. The composition may for instance be a fermentation broth, or a partly dewatered product obtained by separating of water, for instance by mechanical separation, from a fermentation broth. The composition may for instance be a wet cake, a sludge, slurry, concentrate and/or cream. Although granulation is not needed according to the invention, the process according to the invention also offers advantages when the cells are in the form of a granulate, for instance an extrudate. 
     Contacting the composition with the desiccant may be effected in any suitable manner, preferably by mixing the composition with the desiccant to obtain a mixture comprising the cells and the desiccant. The lipid may be recovered from the mixture, for instance by extraction with a solvent. 
     In view of the above, in a preferred embodiment, the invention provides a process for obtaining lipid from a composition comprising cells and water, said process comprising (a) mixing the composition with a desiccant to give a mixture comprising the cells and the desiccant; and (b) recovering the lipid from the mixture. 
     In another aspect, the invention also provides a mixture comprising (i) cells comprising a lipid and (ii) desiccant. It will be appreciated that preferred aspects and/or embodiments of the process according to the invention apply to the mixture according to the invention. 
     In a preferred embodiment, the mixture further comprising water, wherein w des, mix /(w cells, mix +w w, mix )&gt;(1/x)*(1−w cells, mix /(0.8*(w cells, mix +w w, mix ))), preferably w des, mix /(w cells, mix +w w, mix )&gt;(1/x)*(1−w cells, mix /(0.9*(w cells, mix +w w, mix ))), wherein 
     w des, mix =weight of the desiccant in the mixture
 
w cells mix =weight of dry matter of the cells in the mixture.
 
w w, mix =weight of the water in mixture
 
x=absorption capacity of the desiccant.
 
     Mixing can be carried out in any suitable mixer, for instance in a high shear mixer, for instance an Orbit Screw Mixer, a Plow Mixer, a Paddle Mixer, a Tumble Blender with intensifier bars or a Diosna Mixer. 
     The lipid may be recovered from the cells resulting from the contacting in (a) in any suitable manner. The lipid may for instance be recovered from the cells, for instance by extraction with a solvent, after or during contacting the composition with the desiccant. 
     In a preferred embodiment, the lipid is recovered from the cells by extraction with a solvent. A wide variety of solvents may be used. The solvent may for instance be a C 1-10  alkyl ester (e.g. ethyl or butyl acetate), toluene, a C 1-3  alcohol (e.g. methanol, propanol), a C 3-6  alkanes (e.g. hexane) or a supercritical fluid (e.g. liquid CO 2  or supercritical propane). Mixtures of the solvents are also possible. In a preferred embodiment, the solvent is an organic solvent. The process according to the invention is especially advantageous, if the solvent is an apolar solvent. Most preferably, the solvent is hexane or supercritical CO 2 . 
     The cells or mixture comprising the cells may be contacted with the solvent in any suitable manner, for instance by percolation extraction, countercurrent extraction, or extraction in a mixer. Extraction in a mixer is preferred. In a preferred embodiment, between 2 to 40 weight parts of solvent are used per weight part of cells. 
     In an embodiment of the invention, at least part of the desiccant (e.g. at least 50%) is separated from the mixture prior to contacting the cells with the solvent. In a preferred embodiment, the mixture comprising the cells and at least part of the desiccant is contacted with the solvent. Accordingly, recovering the lipid by extraction with a solvent may be effected in the presence of (at least part of) desiccant. This has the advantage that a separation step to separate the desiccant from the cells is not needed. Surprisingly, the process according to the invention still results in an efficient extraction, even in the presence of water. In an embodiment, 
         w   w(b) /w cells(b) &gt;0.5* w   w(a), /w cells(a)    
     wherein
 
w w(b) =weight of water in mixture from which the lipid is recovered in (b)
 
w cells(b) =weight of dry matter of the cells in mixture from which the lipid is recovered in (b)
 
w w(a) =weight of water in the composition to be contacted with the desiccant in (a)
 
w cells(a) =weight of dry matter of the cells in the composition to be contacted with the desiccant in (a)
 
In another embodiment, w w(b) /w cells(b) &gt;0.7*w w(a) /w cells(a)  
 
In another embodiment, w w(b) /w cells(b) &gt;0.8*w w(a) /w cells(a)  
 
In another embodiment, w w(b) /w cells(b) &gt;0.9*w w(a) /w cells(a)  
 
     The process according to the invention may be used to obtain a wide variety of lipids from a wide variety of cells. The Lipid may for instance comprise one or more of the following compounds: lipstatin, statin, TAPS, pimaricine, nystatine, fat-soluble antibiotic (e.g. laidlomycin) fat-soluble anti-oxidant (e.g. co-enzyme Q10), cholesterol, phytosterol, desmosterol, tocotrienol, tocopherol, carotenoid, or xanthophylls, for instance beta-carotene, lutein, lycopene, astaxanthin, zeaxanthin, or canthaxanthin, fatty acids, such as conjungated linoleic acids or polyunsaturated fatty acids (PUFAs). In a preferred embodiment, the lipid comprises at least one of the compounds mentioned above at a concentration of at 5 wt. %, more preferably at least 10 wt. % (with respect to the weight of the lipid). 
     Lipid may be obtained comprising for example triglyceride, phospholipid, free fatty acid, fatty acid ester (e.g. methyl or ethyl ester) and/or combinations thereof. In a preferred embodiment, the lipid has a triglyceride content of at least 50%, more preferably at least 70%, optimally at least 90%. 
     In a particularly preferred embodiment of the invention, the lipid comprises a polyunsaturated fatty acid (PUFA), for instance a PUFA having at least 18 carbon atoms, for instance a C18, C20 or C22 PUFA. In a preferred embodiment, the PUFA is an omega-3 PUFA (Ω3) or an omega-6 PUFA (Ω6). Preferably, the PUFA has at least 3 double bonds. Preferred PUFAs are:
         docosahexaenoic acid (DHA, 22:6 Ω3);   γ-linolenic acid (GLA, 18:3 Ω6);   α-linolenic acid (ALA, 18:3 Ω3);   dihomo-γ-linolenic acid (DGLA, 20:3 Ω6);   arachidonic acid (ARA, 20:4 Ω6); and   eicosapentaenoic acid (EPA, 20:5 Ω3).       

     In a preferred embodiment, the lipid comprises at least one PUFA (for instance ARA or DHA) at a concentration of at least 5 wt. %, for instance at least 10 wt. %, for instance at least 20 wt. % (with respect to the weight of the lipid). 
     The PUFA may be in the form of a (mono-, di, or tri) glyceride, phospholipid, free fatty acid, fatty acid ester (e.g. methyl or ethyl ester) and/or combinations thereof. Preferably, a lipid is obtained wherein at least 50% of all PUFAs is in triglyceride form. 
     The lipid may be an oil or fat, for instance an oil comprising a PUFA. Preferred embodiments for the lipid apply to the oil or fat mutatis mutandis. 
     The cells may be any cells comprising a lipid. Typically, the cells have produced the lipid. The cells may be whole cells or ruptured cells. The cells may be of any suitable origin. The cells may for instance be plant cells, for instance cells from seeds or cells of a micro-organism (microbial cells). Examples of microbial cells are yeast cell, bacterial cells, fungal cells, and algal cells. Fungi are preferred, preferably of the order Mucorales, for example  Mortierella, Phycomyces, Blakeslea, Aspergillus, Thraustochytrium, Pythium  or  Entomophthora . The preferred source of arachidonic acid (ARA) is from  Mortierella alpina, Blakeslea trispora, Aspergillus terreus  or  Pythium insidiosum . Algae can be dinoflagellate and/or include  Porphyridium, Nitszchia , or  Crypthecodinium  e.g.  Crypthecodinium cohnii ). Yeasts include those of the genus  Pichia  or  Saccharomyces , such as  Pichia ciferii . Bacteria can be of the genus  Propionibacterium . Examples of plant cells comprising a lipid are cells from soy bean, rape seed, canola, sunflower, coconut, flax and palm seed. In an embodiment of the invention, the cells are plant cells comprising lipid which lipid comprises ARA. 
     In an embodiment of the invention, the cells are produced by fermentation. Suitable processes for fermentation are known to the skilled person, and are for instance disclosed in WO-A-9737032. Preferably, the cells produced by fermentation are heated or pasteurised. 
     In a preferred embodiment, the process according to the invention comprises one or more of the following steps prior to contacting the composition with the desiccant: (i) heating or pasteurising the cells; (ii) separating water from the cells by mechanical separation; (iii) washing the cells; and (iv) squeezing the cells. 
     Heating or pasteurizing may be effected at a temperature of from 65 to 120° C. It may inactivate or denature enzymes such as lipases and/or lipoxygenases. 
     Separating water from the cells by mechanical separation can advantageously be used to obtain the preferred values for the water content and/or dry matter content as disclosed hereinabove. Mechanical separation may for instance involve filtering, centrifuging, squeezing, sedimentation, or the use of a hydrocyclone. 
     The lipid may further be treated in any suitable manner. If the lipid is recovered by extraction with a solvent, the lipid may be obtained from the solvent by evaporation of the solvent. 
     The lipid obtained or obtainable by the process according to the invention may be subjected to further treatments, for instance to acid treatment (also referred to as degumming), alkali treatment (also referred to as neutralization), bleaching, deodorizing, cooling (also referred to as winterization). 
     The lipid obtained or obtainable by the process according to the invention has many uses. It may for instance be used for the preparation of a food product, for instance a human food product (e.g. infant formula), or an animal feed product. It may also be used for the preparation of a pharmaceutical product or a cosmetic product. Accordingly the invention also provides a food product (e.g. fortified food or a nutritional supplement), for instance a human food product (e.g. infant formula), or an animal feed product, a pharmaceutical product, a cosmetic product, comprising the lipid obtained or obtainable by the process according to the invention. 
     The invention will now be elucidated with reference to the following examples, without however being limited thereto. 
    
    
     EXAMPLES 
     Comparative Experiment A 
     A fermentation broth containing ARA was obtained by  Mortierella  alpina fermentation under the conditions as disclosed in WO-A-9736996, page 45, and was subsequently pasteurized at 65° C. for one hour. The broth was filtered using a belt filter, washed with water to remove residual medium components, and squeezed. The water content of the squeezed filter cake was 60.7 wt. %. The dry matter content (cells, including oil in the cells) was 39.3 wt. %. The squeezed filter cake was extruded as described in WO-A-9736996, followed by drying to a dry matter content of 94 wt. % using a fluid bed dryer. 
     Extraction was performed as follows. In a first extraction stage 100 grams of the dried product obtained as described above was added to 300 ml of laboratory-grade n-hexane, and magnetically stirred at room temperature (20° C.) for one hour. After the first extraction stage the liquid phase (comprising oil dissolved in hexane) was decanted from the solids (comprising cells). In a second extraction stage the remaining solids were added to 250 ml of n-hexane and stirred for 30 minutes. After the second extraction stage the liquid phase (comprising oil dissolved in hexane) was separated from the solids by vacuum filtration. The liquid phase from the first and second extraction stage were combined, after which the hexane was evaporated using a Rotavapor® obtained from Büchi, Switzerland. Operated at 68° C., and a pressure between 100 and 400 mbara for 30 minutes. 
     For the purpose of this patent application, the yield (of ARA-containing oil) of comparative experiment A is defined as 100%. The yields obtained in the following experiments are given vis-à-vis the yield of comparative experiment A. 
     Reference Experiment B 
     Comparative experiment A was repeated, with the difference that no extrusion or drying was performed. The mixture was extracted using the same procedure as described in comparative experiment A. The yield was 29.68%. 
     Example 1 
     Reference experiment B was repeated, with the difference that 82 weight parts of the squeezed filter cake were crumbled, and mixed with 18 weight parts of silica (SIPERNAT® 22 obtained from Degussa AG, Germany) in a Hobart high shear mixer. Extraction was performed in the same manner. The yield was 84.68%, which is an increase with 55% compared to the reference experiment wherein no desiccant was used. 
     Example 2 
     Example 1 was repeated with the difference that the quantity of silica was increased from 18 weight parts to 25 weight parts. This resulted in a further increase of the yield (with 4.8%) to a value of 89.48%. 
     Example 3 
     Example 2 was repeated with the difference that the quantity of silica was further increased from 25 weight parts to 35 weight parts. This resulted in a further increase of the yield with 7.53% to a value of 97.01%. 
     An overview of examples 1, 2 and 3 and experiments A and B is given in table 1. This table shows that the use of a desiccant results in an increase of the extraction yield, and that the extraction yield increases with increasing amount of desiccant used. This enables optimal extraction yields to be obtained by varying the quantity of silica used. The experiments show that virtually the same yield can be obtained by using a desiccant according to the invention, and that extrusion and drying is obviated. 
                     TABLE 1                  Yield obtained by process according to the       invention vs. yield of experiments A, B                                     Ex. No.   Cake (wt. %)   Silica (wt. %)   yield                                             Comp. A   Extrusion and drying   100                                     Ref. B.   100   0   29.68           Example 1   82   18   84.68           Example 2   75   25   89.48           Example 3   65   35   97.01                        
The lipid obtained by the process according to the invention and that obtained by the process involving extrusion and drying have virtually the same composition, see table 2.
 
     Example 4 
     A squeezed filter cake ( Mortierella alpina ) having a dry matter content of 45.6% was prepared as described in the previous examples. 50 g of this cake was mixed with 5 g of poly-acrylate super absorber (Luquasorb® FP 800, obtained from BASF, Germany). The mixture obtained was extracted with 250 ml of hexane under stirring. The quantity of extracted ARA-containing oil was determined after 7 hours of extraction (after evaporation of the hexane), and was found to be 7.8 g. 
     Example 5 
     Example 4 was repeated with the difference that 10 g of silica desiccant (SIPERNAT® 22 obtained from Degussa AG, Germany) was used instead of 5 g of poly-acrylate. After 7 hours of extraction, the quantity of extracted ARA-containing oil was 7.8 g. 
     Performing the extraction of examples 4 and 5 using dried extrudates as disclosed in WO-A-9736996 (in the absence of desiccant) results in between 7.65 and 8.1 g of extracted oil. 
     It is clearly seen that by using a desiccant according to the invention the same yield can be obtained without the need for granulation/extrusion and/or drying by evaporation. 
     
       
         
           
               
             
               
                 TABLE 2 
               
             
            
               
                   
               
               
                 Composition of lipid (oil) obtained by process 
               
               
                 according to the invention vs. experiments A, B 
               
            
           
           
               
               
               
            
               
                   
                 Invention (as 
                   
               
               
                   
                 examples 1-3, 
                 Comparative 
               
               
                   
                 but with 20 
                 experiment A 
               
               
                   
                 weight parts 
                 (extrusion + 
               
               
                   
                 silica) 
                 drying) 
               
               
                   
                   
               
            
           
           
               
               
               
               
            
               
                   
                 ARA (kg/kg) 
                 404.5 
                 421.5 
               
               
                   
                 ARA % of total fatty acids 
                 46.4 
                 46.8 
               
               
                   
                 Polymers 
                 0.6 
                 0.5 
               
               
                   
                 Tri-glycerides 
                 91.9 
                 92.2 
               
               
                   
                 di-glycerides 
                 5.5 
                 5.4 
               
               
                   
                 sterols 
                 2 
                 2 
               
               
                   
                 C14:0 
                 0.6 
                 0.6 
               
               
                   
                 C16:0 
                 13.1 
                 13.1 
               
               
                   
                 C17:0 
                 0.4 
                 0.4 
               
               
                   
                 C18:0 
                 9.3 
                 9.4 
               
               
                   
                 C18:1w9c 
                 8.5 
                 8.4 
               
               
                   
                 C18:1w7c 
                 0.5 
                 0.4 
               
               
                   
                 C18:2w6 
                 7.6 
                 7.4 
               
               
                   
                 C20:0 
                 0.9 
                 0.9 
               
               
                   
                 C18:3w6 
                 3.9 
                 3.8 
               
               
                   
                 C20:1w9 
                 0.3 
                 0.3 
               
               
                   
                 C20:2 
                 0.7 
                 0.7 
               
               
                   
                 C22:2 
                 1.6 
                 1.6 
               
               
                   
                 C20:3w6 
                 4.7 
                 4.7 
               
               
                   
                 C24:0 
                 1.5 
                 1.6