Microbial Production of Fats and Oils
Single Cell Oil is the term applied to triglyceride fats generated by microbial means on non-fat substrate. Single Cell Oils are now formed by a range of microorganisms as a commercial source of Arachidonic acid and Docosahexaenoic acid which are used extensively as dietary supplements in infant formulas. The widespread use of microorganisms for food production has occurred in various civilizations, and the safe and sound consumption of microorganisms themselves, as well as the foods that they have produced, is now well established as a universal movement. The finest producers with maximum oil contents are different variety of yeast and fungi with several key algae too are capable of producing high levels of nutritionally important polyunsaturated fatty acids. Evaluation is done to note the prospective of bacteria, yeasts and molds for producing oils as supplements to existing supplies of plants seed oil. Even though bacteria may provide evidence to be of use for this area but the foremost considered for the growth of microbial oils are species of yeast and mold.
The single-celled entities that are edible oils extracted from micro-organisms which are at the base of the food chain are known as Single Cell Oils. Various diverse variety of yeasts and fungi with numerous key algae are the finest producers with the maximum oil contents. They are also known for producing high levels of nutritionally significant PUFA. Their potential to produce the Poly Unsaturated Fatty Acids (PUFA) has now stimulated the current interest in these Single Cell Oils. They are rich in highly desirable fatty acids that are essential for our well being. They are neither available from plants nor from animals. This symbolises a triacylglycerol type of oil, parallel to what is found in plant and animal edible oils and fats (Kyle et al., 1992, Ratledge, 1993, Boswell et al., 1996). Micro organisms have been receiving increasing attention as a source of novel lipids. Those gathering more than 20-25% of their biomass as oil may perhaps be termed as Oleaginous and their oils as single cell oils or unicellular or microbial oils (Fidler, N, et al. 1999). Microbial oils, also known as Single cell oils, have potential commercial application as nutraceuticals, pharmaceuticals, and feed ingredients for aquaculture and feedstock for producing biodiesel (Lewis et al, 2000). The major hindrance to its commercialisation is its high cost of production. The term Single Cell Oil was coined parallel to the term Single Cell Protein. The term Single Cell Protein was used in the 1960s that covered microbial biomass generated as a source of protein, which was mainly to be used as an animal feed material (Akoh, 2006). Single cell oils can be grown in the fermentors on an organic carbon source and, therefore, are a highly striking, renewable, and toxin free source of long- chain polyunsaturated fatty acids (O'Brien, 2009). The study of microbial lipids has a long history going back to the mid-1870s. Microbial oils were then being considered as sources of commodity oils and fats. They were made throughout most of the last century, with serious efforts being made in Germany during both world wars to develop processes that would provide useful amounts of fats and oils for a country which has been denied access to major supplies of such commodity (Gadd, et al. 2002). Micro organisms have been used as a source of foods and food ingredients by human beings since ancient times. We have been consuming a wide variety of fermented beverages, including beers and wines, and numerous fermented foods, such as cheese, yogurt, salami, etc., since almost the very beginnings of civilization. .In many civilizations, Micro organisms have been widely used for food production. The safe consumption of micro organisms, as well as the foods that they have produced, is now well established as a global activity. The advent of various Single Cell Protein processes was of major significance in the use of micro organisms for food (Wynn and Ratledge, 2006).
These Microbial lipids, which are also known as Single Cell Oils, have been the foremost part of biotechnological products for a number of years. However, before these Single Cell Oils could be completely considered as commercially viable products, they have to compete with the abundance of oils available from agricultural sources (Ratdlege, 1993). In fact, production cost of the Singe Cell Oils is much higher than the respective plant oils, so only Single Cell Oils which commanded a high price could be economically produced. The high-priced Single Cell Oils surround oils containing high amounts of Polyunsaturated fatty acids (PUFAs) such as arachidonic and docosahexanoic acids. They have high amount of dietary important nutrients and Gamma Linolenic Acid (GLA) with unique anticancer properties (Kenny et al., 2000; Das and Undurti, 2004). In order to eliminate the production cost, the current research on Single Cell Oils production is focussing on the use of selected industrial and agro-industrial by-products as substrates (Makri.A, Belllou.S, Fakas.S, Aggelis.G). The concept of using microorganisms as sources of oils and fats has a long history. Over a century, there has been a keen interest in exploiting microbial lipids as alternative sources of oils and fats for human consumption probably since the early years of 20th century. Various Researches by a number of groups in various countries on the prospects of using microorganisms as a source of oils and fats continued to escalate during the earlier part of the last century. They not only studied the process of lipid biosynthesis but also the factors influencing its accumulation (Ratledge, 2005). Earlier, the main focus in attempting to produce Single Cell Oils focused mainly on yeast which was then considered to be the most abundant producers of triacylglycerol oils. But these oils were almost similar to those that could be obtained from plant seeds. They were high in contents of oleic acid, linolenic acid and palm tic acid, but did not offer anything that could not be obtained much more cheaply from agriculture (Akoh, 2006). What lead to the transitions of microbial oils from being more or less academic curiosities 30 years ago to being important nutraceuticals for inclusion in infant formulas has been the clear evidence of the dietary significance of very long chain, polyunsaturated fatty acids coupled with the realization that there is no adequate or safe source of them from plants or animals. The diversity of microorganisms is so great that it can almost be assured that these current products will not be the last ones that will be launched in the 21st century as Single Cell Oils (Cohen & Ratledge, 2005).
Biochemistry of Lipid Accumulation in the Oleaginous Micro organisms:
Micro organisms, like all living cells, also contain lipids. Few species of these micro organisms can even produce abundant amount of lipids. Those microorganisms may be considered similar to the oil-bearing plant seeds, Oleaginous. With few exceptions, oleaginous micro organisms are eukaryotes, including algae, yeasts and moulds (Hammond and Glatz, 1988). Yeast and Fungi were the main oil-accumulating micro organisms as the bacteria production is much less as compared to the extractable edible oil. The oil produced by these microorganisms was, like plant oils, mainly composed of triacylglycerol having component fatty acids (FA) that were, in almost every case, similar to what had already been recognized in plant oils.
Micro organisms have been receiving increasing attention as a source of novel lipids. Those which gather more than 20-25% of their biomass as oil may be termed as Oleaginous. Giving a specific definition to oleaginous micro organisms poses some difficulty (Fidler, N. et al, 1999). Oleaginous organisms differ from non-oleaginous organisms as the oleaginous organisms are able to continuously convert carbon after reducing the amount of nitrogen present starting from the expansion medium into intracellular lipid. The existing cells grow large as their lipid droplets continue to grow when the protein, nucleic acid synthesis and cell division have stopped. Both oleaginous and non-oleaginous continue to take up glucose during nitrogen starvation, but oleaginous organisms metabolize the glucose, thus increasing the ATP: AMP ratio in the cell (Cheetham. P.S.J and King, R.D. 1987). Oleaginous micro organisms have been gaining wider interest because microbial lipids and plant oils have some common important characteristics with regard to fatty acid distribution, triacylglycerol types, and secondary metabolites. Thus, oleaginous microorganisms are considered as an alternative source of lipids, because of their enormous growth rates on a variety of substrates, their ability to synthesize an array of products, and their amenability to genetic manipulation (Neidelman, 1997).
The diversification of the range of fatty acids that microorganisms produce to those from plant sources is extensive. However, if we restrict ourselves to the oleaginous organisms, that are those which gather additionally more than 20-25% of their biomass as oil, then the range of fatty acids is much more restricted and is the same as those found in plant and animal oils and fats (Shetty, et al. 2006). The main condition which had to be satisfied was that the organisms had to be grown so that its multiplication was eventually curtailed by the exhaustion of a nutrient other than carbon from the medium. The organism would then, without further cell proliferation, convert the excess carbohydrate substrate into lipid. This led to the two-stage profile for lipid accumulation in batch culture becoming established:
The above diagram shows that after reducing the amount of nitrogen present from the growth medium, the lipid accumulation in a microbial cell begins. Thus to make sure that nitrogen is exhausted while the other nutrients remain in excess the medium has to be formulated with a high Carbon: Nitrogen ratio. The process of lipid accumulation continues until the cells reach a personal limit of obesity. Some cells may continue to produce oil until they are physically unable to accumulate any more as shown in the next , while others though oleaginous, stop lipid accumulation at some apparent limit that may vary from 20 -25% up to 60% or so.
Correlation between the possession of the enzyme citrate ATP-citrate lyase and the ability of yeast to accumulate more than 20% of its biomass as lipid was observed. The importance of the enzyme is that it serves to produce acetyl-CoA (from citrate), that is known to be the substrate for the fatty acid biosynthesis (Boulton and Ratledge 1981).
Citrate + ATP + CoA → acetyl-CoA + oxaloacetate + ADP + Pi
Acetyl-CoA cannot be produced in the cytoplasm from pyruvate. Oleaginous microorganisms accumulate citrate in the mitochondria which is then transported into the cytoplasm and there cleaved by ATP-citrate lyase. Non oleaginous organisms do not possess the citrate- cleaving enzyme and must rely on less effective means of producing acetyl-CoA in the cytoplasm. The possession of ATP-citrate lyase may be thus considered as a key to oleaginicity (Ratledge, 1986).
(Lipid biosynthesis in oleaginous micro organisms) (Ratledge and Wynn, 2002)
Glucose is considered to be the most common substrate for Single Cell Oil production, but other carbon sources, such as glycerol, could also be used for assimilation and conversion into microbial lipids. Oleaginous micro organisms accumulate lipid in nitrogen limited conditions when a suitable carbon substrate is found in excess in the growth environment. Also, the exhaustion of nitrogen, in the growth medium triggers Lipid accumulation. In nitrogen limited conditions the AMP concentration in the cell is diminished and the activity of isocitrate dehydrogenase within the mitochondrion slows or even stops. Although TCA is disrupted, assimilation of the carbon substrate remains high leading to the accumulation of citric acid which is finally excreted into the cytosol. There, citric acid is cleaved by the ATP: citrate lyase (enzyme that is present only in oleaginous species) and acts as acetyl-CoA donor in the biosynthetic pathway of storage triacylglycerol. Oleaginous micro organisms accumulate lipids mainly in the form of triacylglycerol with esterified straight chain fatty acids containing 0 to 3 double bonds. Unsaturated fatty acids are preferentially incorporated into the central carbon atom of glycerol. The concerted action of two complex enzyme systems,
1. Acetyl- CoA carboxylase and
2. Fatty acid synthetase
Helps synthesise the saturated and the unsaturated fatty acids from acetyl-CoA (Boulton and Ratledge, 1985).
Lipid biosynthesis is affected to different degrees depending on the strain by many factors, including:-
1. The growth medium and its composition,
4. Oxygen level, and
5. Growth rate.
Although pH effects may be important depending on the strain, most organisms are able to grow when combined with the effects of temperature over a wide range of pH values. The increase in the degree of lipid unsaturation, on the lipid quality factor, has generally been shown with an increase in:
1. Unsaturation of fatty acyl-supplement in the medium
2. The saturation level of n-alkenes serving as the carbon source
3. Decrease in the growth temperature
4. Higher pH values
General Ideology, Processing, Removal and Refinement
The Latent Biotechnology Single Cell Oil Method should aim at the production of speciality oils and fats that would be esteemed at considerably higher prices. The biochemical effectiveness of converting sugar into triacylglycerol indicates that three tons of sugar substrate will be needed to produce one ton of microbial oil. A further one to two tons of sugar will also be needed to provide the remainder of the (oil free) biomass. As sugar costs about $300 per ton at world prices, the costs of substrate alone will be about $1500 to produce one ton of Single Cell Oil. Additionally costs will be incurred in the extraction and refining that must be done to produce a high quality, food grade oil. Hence even the cheapest Single Cell Oil is not likely to cost less than $5000 per ton and, if a slow growing micro organism is used with particular growth requirements or with a low oil yield, then these costs could easily multiply further (Whitworth, D.A., C. Ratledge. 1974). Regardless of the high cost of the fermentation technology required to produce Single Cell Oils, these oils have become a commercial reality and have advantages over oils from traditional sources. The Single Cell Oil production process involves:-
1. Growing of a suitable microbial culture, through shake-flask culture,
2. then placing into small (10–100 L) stirred fermentors, and
3. Finally placing in a large seed fermentor that is usually about 10% of the volume of the final fermentor to be used.
The use of a large inoculum capacity means that the expansion time of the organism in the final fermentor is minimized; that is, the productivity measured as the oil produced per unit time per unit volume of fermentor is maximized.
Fermentation-During the fermentation process, all the major parameters which are temperature, pH and dissolved oxygen are measured carefully and taken care of. Temperature control is very critical in case if fermentation is exothermic, so for this reason fermentations done at 30˚C or more are preferred. Sterility of the fermentation system is also very important thus keeping a check at the final fermentation step. During the final hours of fermentation, the amount of residual substrate may be exhausted which stimulates the cells to use the lipid that they have just accumulated. This will not only diminish the final oil yield but also the switch from the cells using glucose to using oil as a substrate induces the formation of lipases. The presence of lipases can be detrimental to the final quality of the oil. In general, aim of this process is to produce clear, bright oil with a minimum of coloring; though some yellowness is acceptable, dark, brown oil would not be regarded as desirable. In spite of being in practice, many microbial oils, due to the presence of natural antioxidants that have been co-extracted with the oil, are generally considered to be very stable to oxidation. The various characteristics of the oil need to be carefully evaluated, chief among them being the overall stability of the oil. Some food grade, antioxidant material can be added to guarantee a high strength of the oil,
Single Oil Production Process:
A). Process for GLA Production:
γ- Linolenic acid (GLA) (18:3, n-6) has a very long history of use, occurring as it occurs in the seed oil of the Evening Primrose (Oenothera Biennis). Evening primrose oil was considered to be useful in the treatment of multiple sclerosis. It was also known to be useful for the treatment of a number of other disorders and it is still sold as the nutraceutical for the relief of premenstrual tension. GLA oil has been recommended as a food supplement or health food. DHA (Docosahexaenoic acid) is an important structural component in tissue membranes of the human body. It is found in human brain and important structural component of neurological and retinal tissues (Crawford, 1993,Koletzko, 1990, Nettleton, 1993, Neuringer et al., 1988). γ- Linolenic acid (GLA) is found in a relatively small number of plant seed oils, principal among which is evening primrose oil in which it constitutes only about 8-10% of the total fatty acids (Laskin. I.A., et al, 2002). The first Single Cell Oil was thus produced using Mucor circinelloides grown in large-scale fermentors (55000 US gallons). The oil was sold under the name of Oil of Javanicus and also as GLA-Forte that was used by one retailer of the oil. It achieved some limited penetration in the food market. The most suitable species appeared to be mold strains of the order Mucaroles that produce up to 30% GLA in the oil. Strains producing high GLA levels generally have low oil content, whereas those with high oil content have low GLA levels. The carbohydrate substrates that is the glucose-based medium which are normally being used in the industrial processes, being expensive to an extent, was replaced with monocarboxylic acids, such as acetic acid obtained from waste materials from petrochemical process to minimise the cost. GLA oil has been recommended as a food supplement or health food (Neidleman, 1997).
B). Process for the production of Cocoa Butter equivalent fat:
Cocoa Butter is a simple, three-component system consisting of the following triglycerides:-
1. Palmitic-Oleic-Palmitic (POP),
2. Palmitic-Oleic-Stearic (POS), and
3. Stearic-Oleic-Stearic (SOS)
Proper blending of these three triglycerides in a proper proportion would result in the formation of cocoa- butter (O'Brien, D.R, 2009). Initially, the price of cocoa butter reached as high as more than $8000 per ton. This made the possibility of producing a facsimile fat — known as a Cocoa Butter Equivalent (CBE) fat — using yeast technology somewhat attractive. Yeasts unlike many molds and fungi, tend to produce only limited amount of PUFA and some strains can have relatively high content of stearic acid (18:0). Unfortunately, most microbial oils are low in their contents of stearic acid and therefore modification of the composition of the yeast fat was necessary if a CBE was to be produced. This was initially attained using an inhibitor of the Δ9-desaturase that converts stearic acid into oleic acid. It was found that the inhibitor used, sterculic acid was more expensive to use than the final price of the product. The simplest and perhaps the most booming approach to achieving an increase in the stearate content of the yeast fat was that which was to grow the yeast with a deficiency of oxygen . This required trials to be done in fermentors in order to decrease the ventilation rate sufficiently to cause the desaturases to stop functioning. Ultimately this process was unsuccessful as the process still deemed to be uneconomical (Ratledge and Cohen, 2005; Shetty et al,. 2006).
C). Significance of Polyunsaturated fatty acids and their processing:
PUFA (Polyunsaturated fatty acids) are considerably in demand essentially as dietary supplements and are regarded as nutraceutical materials.
PUFA (Polyunsaturated fatty acids) fall into two main categories:-
1. The n-6 (Arachidonic acid ARA) and
2. The n-3 (Docosahexaenoic acid DHA) series,
These depend on the position of the final double bond in the acyl chain relative to the terminal methyl group as shown in the picture below.
The n-6 (Arachidonic acid ARA):-
Arachidonic Acid is a long chain polyunsaturated fatty acid with twenty carbon atoms and four double bonds. Its systematic name is (all-cis)- 5,8,11,14-eicosatetraenoic acid(ETA). A significant body of evidence shows that the neural development in the growing infant is due to the benefit from the provision of this fatty acid combined with docosahexaenoic acid (DHA). This oil has been target for biotechnologists for many years. A proprietary strain of Mortierella alpine is cultivated in large fermentors. To achieve the highest possible cell density, both nitrogen and carbon are fed into the fermentor during the fermentation. Once high cell density is obtained the N feed is discontinued, but the C feed is still maintained. The resulting oil is yellow in colour due to presence of some carotenoids, it has brilliant oil characteristics. As this oil is prone to oxidation, additional antioxidants are added during processing to ensure complete protection against oxidation. It is known that plants do not contain significant levels of LCPUFA and in the case of Arachidonic acid even animal sources are not available but most of the Arachidonic acid comes from the animal derived foods (Cohen & Ratledge, 2005).
The n-3 (Docosahexaenoic acid DHA):-
DHASCO is free flowing oil that contains a standardized 40 wt% DHA. Two heterotrophic eukaryotes were identified and developed as production organisms for DHA-rich SCO; both are microalgae belonging to the order thraustochytriales. DHA microalgae represent an attractive alternative for the production of high quality DHA oils. So as to identify these PUFA producing microorganisms a pcr- based screening system has been developed which is based on the detection of DHA related genes by using degenerated oligonucleotides derived from conserved regions of PUFA synthesizing polyketide synthases. For the efficient production of edible oil rich in DHA, an appropriate downstream process, extraction procedure and a refining protocol have been developed. The polyunsaturated fatty acid of omega-3 class, DHA has the potential of becoming one of the most important health ingredients. It is known to be important for the normal neurological development and vision and also has beneficial effects on diverse diseases such as cancer and cardiovascular diseases. Some clinical studies have shown that it leads to decrease of risk of sudden death and triglyceride level. The main source of DHA in our diet is fatty cold water fish such as salmon and tuna. (DHA is of such importance to the World Health Organization, British Nutritional Foundation, and the International Society for the study of Fatty Acids has recommended that Long Chain Polyunsaturated Fatty Acids should be included in all infant formula.). Both of them produce an oil rich in DHA. The quality of oil is vigilantly protected during production and processing which is maintained by adding 250ppm each of ascorbyl palmitate and tocopherols (they act as antioxidants)in the final product and by storing the oil at low temperature in N2 purged containers. The strength of oil is such that it has a shelf life of two years from the date when it is shipped (Cohen & Ratledge 2005). The SCO used for neonatal nutritional supplementation constitutes greater than 95% of the global market for DHA-rich SCO. DHA is one of the most carefully tested oils in terms of safety and efficacy.
Significance of Single Cell Oil:
Even though the first Single Cell Oil appeared on the market in 1985 it was not until the twenty-first century that Single Cell Oils have really become a successful commercial article of trade. The base of this success lies on the enclosure of a mix of both ARA-rich Single Cell Oil, and DHA-rich Single Cell Oil in infant formulae in many countries in Europe, Australia, and the Far East. However, the market was somewhat limited due to the enclosure of the ARA-rich and DHA-rich SCO blend in only formula designed for premature babies, and to the initial refusal of the FDA to allow inclusion of the DHA/ARA-SCO in infant formulae. A step forward was taken when the FDA finally gave GRAS status to the DHA/ARA-SCO for its inclusion in infant formulae in the USA. As a result of this success, market is now limited by the supply, rather than demand, of both DHA-rich, and ARA-rich SCOs. Production and consumption of ARA-rich SCO, and DHA-rich SCO should continue to expand for few more years based solely on the increased use of these products in infant formula. It should be noted that although the current use of SCO is rapidly expanding, this expansion represents, to a very large degree, the success of a single product, a DHA/ ARA-SCO blend (Formulaid) for one specific application (infant formula). New products and new markets must be developed to sustain the growth of SCO production, if it continues to grow beyond the next 5 to10 years (Ratledge. C, 1976).
Advantages and Disadvantages of Single Cell Oil:
Advantages of Single Cell Oils:
1). they have a simple fatty acid profile.
2). both quality and quantity of the product can be guaranteed.
3). they are not affected by the geographical or enviornmetal factors.
4). GLA is active ingredient in evening primrose oil which is used as a remedy for pre-menstural syndrome and eczema.
5). Rapid increase in cells in a short time and independence from climatic conditions.
6). SCO act as a novel source of polyunsaturated fatty acids, such as DHA and AA for nutritional supplementation.
Disadvantages of Single Cell Oils:-
1). they have Limited production capacity.
2). Potential adverse public perception.
3). the DHA-rich SCO are known to be associated with growth retardation, as a result of which,
It has not been regarded as safe form the food and drug agency.
Nutritional Aspects of Single Cell Oils:
One of the dynamic forces known for the development of Single Cell oil containing Long-Chain Polyunsaturated Fatty Acids (LCPUFA) was the presence in human milk of two particular LCPUFA, Docosahexaenoic acid and Arachidonia acid. Once it was recognized that these PUFAs played an important role in the brain, attempts were made to provide these PUFAs naturally from fish oils and egg phospholipids. DHA is a long-chain polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, the first one located at the third carbon atom from the methyl terminus. It is an important structural component in tissue membrane of the human body and important structural component of neurological and retinal tissues (Crawford, 1993, Koletzko, 1990, nettleton, 1993). Studies conducted on SCO in animals have been concerned with the efficiency of these oils in supplying tissues with PUFA, especially in relation to brain PUFA and brain function. The commercial development of SCO and their FDA approval has allowed the addition of LCPUFA from SCO sources to infant formulas. It has thus enabled the composition of infant formulas to approach that of human milk. (Andrew Sinclair)
Safety Evaluation of Single Cell Oils:
The first commercial Single Cell Oils (Oil of Javanicus) was not being produced commercially until 1985 even after regulations and regulatory bodies relating to food safety were in place. This was because, the security of these food products had to be determined and proven to the regulatory authorities and the general public. The advent of Single Cell Oils on the food prospect is a reasonably recent event. The security of Oil of Javanicus was relatively easy to validate as both the producing organism (Mucor circinelloides) and the active ingredient (γ-linolenic acid) have long been part of the human diet and were therefore deemed safe by historical association with food products. Various Safety evaluation studies of
ARASCO (fungal oil highly enriched in AA),
DHASCO (algal oil highly enriched in DHA) and
Formulaid (a combination of ARASCO and DHASCO)
were performed in rats. The results showed that the tested Single Cell Oils have no demonstrable toxicity and that administration of these Single Cell Oils is no less safe than administration of sunflower oil.
There are two routes for the safety evaluation and obtain a clearance for a food ingredient. A food additive petition process requires premarket review and approval by FDA, where in case of GRAS (Generally Regarded as Safe) a manufacturer can determine that a substance is GRAS if there is scientific consensus among qualified experts about its safety under the intended condition of use.
The main difference between food additive petition and a GRAS notification is that a food additive places the responsibility of declaring that a substance is safe and approved under the conditions of use with the regulatory agency whereas in GRAS places the responsibility of demonstrating that a substance is GRAS and is therefore safe for use (Zeller Sam, 2003). For some years the inclusion of high levels of ARA in the human diet was considered undesirable, due to potential adverse effects on blood clotting. This apparent lack of toxicity in animal models prompted a major study at the Western Human Nutrition Research Center, San Francisco, USA, of the effect of dietary supplementation with ARASCO. During an intensive and prolonged study (in which human volunteers lived in a “metabolic ward” to decrease the effect of external influences) the inclusion of ARA did not cause any significant adverse effects on any of the test individuals. Factors examined included blood coagulation, immune response, and the production of arachidonic acid derived signal molecules. The conclusion of the study was that the inclusion of ARASCO did not have any toxic effects on adult humans and that this oil should be considered safe as a human food ingredient. Evaluation of the overally safety of the SCO components involves the review of the safety of oil. SCO are typically comprised of fatty acids esterified to glycerol and may contain minor amounts of other lipid classes. FA present have been described as a component of normal human diet and the sterols are commonly found in traditional food sources including animal fat, vegetable oil and human milk.
The extensive safety evaluation of both DHA-rich SCO and ARA-rich SCO was a necessary prerequisite to the release of these “novel” foods on to the open market, particularly as their major application was infant nutrition. However, as the intrinsic safety of microbe-derived oils has now been demonstrated, it seems likely that the next generation of SCOs will not have to undergo the same high level of toxicological testing. As microbial oils have been shown to be no more toxic than oils from traditional sources it should be sufficient in the future to demonstrate the lack of pathogenicity and toxigenicity of the producing organism. This should decrease the development costs of future products significantly.
Competition of Single Cell Oil from Genetically Modified Plant Oils:
Despite the fact that upcoming prospects for the continued production of various SCO look enormously strong, there is the definite prospect that one or more of the current SCO may be produced in plants at some future stage. Hereditary strategy of plants for improved characteristics has long been ongoing, and many companies are engaged in attempting to clone key genes into agronomically important plants to convert the existing fatty acids of the oilseeds into AA or DHA. But as none of these fatty acids occurs in an agriculture crop, it is necessary for genes coding for various fatty acid desaturases and elongases to be taken from a microorganism and inserted into a plant's DNA. These then have to be made to work and the resultant proteins have to be active catalytically and they have to do the same work that they did in the original microorganism but they have to work only during accumulation of oil in the seed. If novel polyunsaturated fatty acids be produced all the way through the complete plant i.e. in leaves, stems and roots then the plant would most likely be unable to grow properly. Thus massive problems have to be overcome for the flourishing genetic engineering of very low polyunsaturated fatty acids into plants. For geneticists, the simplest key is to try to clone an entire gene sequence from a microorganism which will then be able to code for the entire set of proteins needed for the synthesis of new fatty acids (Cohen & Ratledge 2005).
Future developments of Single Cell Oils:
According to the research done by Colin Ratledge on the topic- Single Cell Oils- have they a biotechnological future he stated that Microorganisms have been known to bethe producers of edible oils for long, opportunities for their biotechnological exploitation are limited to the highest-value commodities. A recent attempt to develop a yeast oil cocoa-butter equivalent has not succeeded, not because of the inability to produce the correct formulation of fatty acids, but because of the falling price of cocoa butter on the world market. Better prospects appear to exist for producing polyunsaturated fatty acids (PUFAs) of the n-6 and n-3 series, using bacteria, fungi or algae. Many microbial PUFA-oils are characterized by the absence of other PUFAs, making purification of individual fatty acids an easier task than it is from other sources. Certain microorganisms may also produce prostaglandin precursors, or even prostaglandins themselves, as well as cerebroside lipids and other unusual lipids that are not normally regarded as being of microbial origin.
The commercial development of SCO has gone through a series of ups and down since the first recognition of their potential many years ago. The story of success of DHASCO and ARASCO in infant formulas was due to the fact that at that time there was no other commercial alternative available for the industry. As we know that the industry is reluctant to change, so it is unlikely that alternatives will replace DHASCO and ARASCO in the coming future until and unless there is a substantial reason for change. The scale up of these two oils has validated the concept proposed. It is because of this background of SCO that one can look forward to a bright future of SCO (Cohen and Ratledge, 2005). Single Cell oils have been regarded as a source of omega-3 fatty acids by (Armenta, R.E and Barrow, C.J, Ocean nutrition Canada Ltd). Omega-3 fatty acids mainly docosahexaenoic acid and eicosapentaenoic acid, have been linked to several beneficial health effects (i.e. mitigation effects of hypertension, stroke, diabetes, osteoporosis, depression, schizophrenia, asthma, macular degeneration, rheumatoid arthritis, etc). “The chief source of omega-3 fatty acids is fish oil; lately however, fish oil market prices have increased significantly. This has prompted a significant amount of research work on the use of single-cell oils as a source of omega-3 fatty acids. Some of the microbes reported to produce edible oil that contains omega-3 fatty acids are from the genusMortierella, Schizochytrium, ThraustochytriumandUlkenia. An advantage of a single cell oil is that it usually contains a significant amount of natural antioxidants (i.e. carotenoids and tocopherols), which can protect omega-3 fatty acids from oxidation, hence making this oil less prone to oxidation than oils derived from plants and marine animals. Production yields of single cell oils and of omega-3 fatty acids vary with the microbe used, and with the fermentative growing conditions and extractive procedures employed to recover the oil. This paper presents an overview of recent advances, reported within the last five years, in the production of single cell oils rich in omega-3 fatty acids.
The production of Single Cells Oils - that is edible oils from microorganisms - is now occurring to produce high value oils rich in arachidonic acid (ARA) and docosahexaenoic acid (DHA). The existing processes are using Mortierella alpina, for the production of ARA, and two different organisms for the production of DHA. These oils are used in infant nutrition and also as nutriceuticals and food additives appropriate for adults. Potential therapeutic applications are very promising. All processes run using large-scale fermenters, up to 200,000 litres. The extremely dynamic nature of the microbial cells means that to ensure stabilization precautions must be taken before the cells are harvested from the fermenters. Once stabilised, the cells are harvested and then dried with or without conditionning as a preparation for solvent extraction. The conventional solvent extraction, as is used in oil extraction from plant seeds, is suitable for the extraction of SCOs. However, more specialized equipment and conditions are used due to oil sensitivity to oxidation and because the volumes of SCOs are comparatively low. All SCOs produced to date have proved to have remarkable inherent stabilities. (Fichtali J, Maktec Biosciences Corporation). Single cell oils (SCOs) are now formed by various microbes as industrial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA). Both of them are now widely used as nutritional enhancers in infant formulas. An understanding of the fundamental biochemistry and genetics of oil accumulation in such microorganisms is therefore indispensable if lipid yields are to be enhanced. Also an understanding of the biosynthetic pathways involved in the production of these polyunsaturated fatty acids (PUFAs) is also highly desirable as a prerequisite to increasing their content in the oils. An account is provided of the biosynthetic machinery that is necessary to achieve oil accumulation in an oleaginous species where it can account for lipid build up in excess of 70% of the cell biomass.
1). Boswell, K., Koskelo, E.K., Carl, L., Glaza, S., Hensen, D.J., Williams, K.D., Kyle, D.J., Preclinical Evaluation of single cell oils that are highly enriched with arachidonic acid. Food Chemic. Toxicol., 34(1996), 585-593.
2). Kyle, D.J., Sicotte, V.J., Singer, J.J., Reeb, S.E., Bioproduction of docosahexaenoic acid by microalgae In industrial application of single cell oils. Champaign, American Oil Chemists Society, 1992, 287-300.
3). Ratledge, C., Single Cell Oils- have they a biotechnological future? Trends-Biotechnology, 11(1993), 278-284.
4). Cohen, Z., and Ratledge, C., 2005. Single Cell Oils, AOCS Press, Champaign, Illinois.
5). Fidler, N., Koletzko, B., and Sauerwald, T.U., Single Cell Oil Production and Application, 1999, Page 1.
6). Lewis, T., Nichols, P.D., and McMeekin, T.A., Evaluation of extraction methods for recovery of fatty acids from lipid producing microheterotrophs, J. Microbiol. Meth. 43(2000), pp 107-116.
7). Akoh, C.C., 2006. Handbook of Functional Lipids, CRC Press. Taylor & Francis Group. Boca Raton, FL.
8). O' Brien, R.D., 2009. Fats and Oils- formulating and processing for applications, CRC Press. Taylor & Francis Group. Boca Raton, FL.
9). Gadd, G.M., Bennett, J.W., and Laskin, A.I, 2002. Advances in applied microbiology, Volume 51. Academic Press London.
10). Wynn, J.P., and Ratledge, C., Microbial Oils and Fats, In: Bailey's Encyclopedia of industrial oil and fat products, 6th ed., Shahidi, F., ed., Hoboken, NJ: J. In press.
11). Wynn Kenny et al., (2000). Int. J. Cancer 85: 643-648.
12). Das & Undurti, (2004). Prostaglandins, Leukotrienes, Essent. Fatty Acids 70: 539-552.
13). Makri, A., Bellou, S., Fakas, S., and Aggelis, G., Biotechnology of Single Cell Oils, Laboratory of microbiology, Division of Genetics, Cell and Development Biology, Department of biology, University of Patras, Greece.
16). Nettleton, J.A Are n-3 fatty acids essential nutrients for fetal and infant development? J. Am. Diet. Assoc., 93(1993), 58-64.
17).Crawford, M.A. The role of essential fatty acids in neural development: implications for perinatal nutrition. Am. J. Clin. Nitr., 57suppl(1993), 703S-710S.
18). Ratledge, C. Microbial production of oils and fats. In: Food from Waste, Birch, G.G., K.J. Palmer, J.T. Worgan, eds., London: Applied Science 1976, pp 98–113.
19). Shetty, K., Paliyath, G., Pometto, A., and Levin, R.E., 2006, Food Biotechnology. Second Edition. CRC Press.
20). Laskin, A.I., Bennett, J.W and Gadd, G.M., 2002, Advances in applied microbiology Volume 51. Academic Press, USA.
21). Neidleman, S.L, 1997. Advances in applied microbiology Volume 43. Academic Press, USA.
22). Neuringer. M., Anderson, G.J., Connor, W.E, The essential of n-3 fatty acids for the development and function of the retina and brain. Ann. Rev. Nutr., 8(1988), 517-541.
23). Whitworth, D.A., C. Ratledge. Microorganisms as a potential source of oils and fats. Proc. Biochem. 9(9):14–22, 1974.
24). Cheetham, P.S.J., and King, R.D. 1987. Food biotechnology. Volume 2. Elsevier science publishers ltd, England.
25). http://www.aocs.org/archives/am2004/session.cfm?session=BIO+5:+Single+Cell+Oils (Hammond & Glatz 1988).