Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing oxygen-containing organic compound
Reexamination Certificate
1998-01-20
2001-01-30
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing oxygen-containing organic compound
C435S911000
Reexamination Certificate
active
06180376
ABSTRACT:
This invention relates to an extraction process.
Vegetable oils can be extracted from dried plant seeds using pressure. This in general leaves vegetable oil in the crushed plant tissue and this may be extracted using a water-immiscible organic solvent, for example hexane.
It is possible to extract microbial oils from whole microbes by hexane extraction. The microbial material must be dried usually to a water content of less than 5% (w/w) before the oil can be effectively extracted by hexane. Not only does this involve a considerable consumption of energy because, in general, microbial cells contain at least 70% for example 80 to 95% (w/w) water based on their non-oil content, but we have also found that sensitive oils for example oxygen or heat sensitive oils may undergo chemical reactions in the drying process.
In alternative methods of extracting oils from microorganisms, water-miscible solvents, for example isopropanol, may be used. Such extractions are in general less selective than those with non-polar solvents and a wide range of other cellular components may be extracted in addition to the oils, for example phospholipids and cell wall components. Evaporation of the water-miscible solvent leaves a residue which must therefore be purified further.
By “oils” is meant materials which are liquid at the extraction temperature and which are sparingly soluble in water.
In general oils recovered from microorganisms are triglycerides.
This invention comprises a process in which an oil is extracted from oil containing microorganisms which comprises disintegrating the microorganisms and contacting them in the presence of a water content of at least 70% by weight of that originally present in the cellular material and preferably in the presence of substantially all of the original water content of the microorganisms with a water immiscible solvent for the oil, separating the solvent from the microorganisms and recovering the oil from the solvent.
The invention also comprises a process for extracting a triglyceride from microbial matter containing it in which the triglyceride is released from the cells by pressure homogenisation and the released triglyceride is then extracted by contacting the microbial matter with a water-immiscible solvent for example an alkane suitably having 4 to 12 and preferably 5 to 8 carbon atoms, for example cyclohexane or preferably hexane. The invention avoids the requirement for dehydrating the microbial matter whilst permitting a good selectivity of extraction into the water-immiscible solvent to be secured.
It is preferred that the microorganism should be disintegrated in the presence of an aqueous culture medium in which it has been cultured, as this avoids the need for separating the microbial matter from the culture medium prior to the process. Typically the organism will constitute 5% to 20% by volume of the culture medium. The same solvent may be re-used two or more times for extracting successive quantities of disintegrated organism containing triglycerides, thus increasing the triglyceride content of the solvent. This leads to economies in recovering the triglyceride from the solvent by evaporation. Such processes may, if desired, be conducted by countercurrent extraction. Suitably, this may be carried out by using a solvent which is less dense than the phase containing the disintegrated microorganism and feeding it at a lower level than a phase which contains the disintegrated microorganism to a vessel in which extraction occurs. The vessel may be provided with baffles, restricting vertical flow, which define contact zones between the baffles and in which means is provided for stirring material in the contact zones.
If desired, separated solvent containing the triglyceride may be used together with fresh solvent to extract previously unextracted microorganisms. The solvent may be separated by centrifuging. The solvent may suitably be a hydrocarbon, for example an alkane which suitably has 4 to 12 and preferably 6 to 10 carbon atoms and is suitably hexane.
The organisms may be disintegrated by enzyme cell disruption using cell wall lytic enzymes, mechanical methods such as bead milling, colloid milling, disruption by pressure release, impinging jets, ultrasonication and preferably using high shear mixing and/or high pressure homogenisation. The disruption should be sufficient to disrupt the cell wall, thus enhancing the access of the solvent to the triglycerides contained within the cell. Any form of pressure homogeniser may be employed for this purpose.
In general in solvent extraction processes, small quantities of solvent are used compared with the material which is to be extracted. This has the advantage of achieving a high concentration of the desired solute in the solvent phase but when microbial materials are extracted in this way there is a tendency for organic matter present to act as an emulsifying agent, leading to the formation of a solvent-in-water emulsion of high stability. In order to reduce the stability of such emulsions, it may be necessary to add demulsifying agents thus enabling the emulsion to be separated suitably using high gravity conditions. However, the demulsifying agent may contaminate the product which it is desired to separate and involves additional cost. Contamination may be particularly undesirable if the demulsifier is toxic.
We have found that by operating in the presence of high concentrations of solvent sufficient to form a water-in-solvent dispersion, the dispersion is less stable and may be more readily separated in the absence of a demulsifying agent, for example by gravity settling or use of a centrifuge. It is desirable in this case to increase the triglyceride content of the solvent by extracting successive quantities of microbial material with it.
This invention therefore comprises a process as aforesaid in which the disintegrated microorganisms are contacted with a continuous phase of the solvent. It is desirable that the ratio of solvent phase to other matter present during the extraction step should be at least 1 to 1, preferably at least 1.5 to 1 and more preferably at least 2 to 1 and suitably at most 10 to 1 and preferably at most 1 to 1.
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Talaro et al. In: Foundations in Microbiology. W. C. Brown Publishers, 1993. p. 100.
Morris et al. Counter-current distribution. In:Separation Methods in biochemistry. Ed.Morris and Morris. Pitman Publishing. 1976. p. 639.
Database WPI, Section Ch, Week 8413, Derwent publications Ltd., Class D16,AN 84-077725, XP002018398 & JP,A,59 028 480, Feb. 15, 1984.
Patent Abstract of Japan, vol. 008, No. 114 (C-225), May 25, 1984 & JP,A,59 028480, Feb. 15, 1984.
Afremova Vera
Marx Irene
Pillsbury Madison & Sutro LLP
Zeneca Limited
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