FIELD OF INVENTION

[001] The present invention generally relates to a method for producing omega-3 unsaturated fatty acid, and, more particularly, to the production of docosahexaenoic acid (DHA) by batch fermentation.

BACKGROUND OF INVENTION

[002] Docosahexaenoic acid (DHA) is an omega-3 unsaturated fatty acid essential for normal growth. DHA is found in 3 phospholipids:

phosphatidylethanolamine, ethanolamine plasmalogens, and phosphatidylserine. It is found to be abundant in brain and retina, and is essential for optimum mental and visual activity. A deficiency of DHA in diet could result in deficiency in brain and visual functions. DHA is also found to inhibit the growth of cancer cells, reduce incidence of conditions such as rheumatoid arthritis, thrombosis and atherosclerosis. The consequences linked to the deficiency of DHA have increased the commercial value of DHA. Products having DHA-containing lipids are now available in market as dietary supplements.

[003] In humans, DHA is either known to be supplied in the diet or derived through Eicosapentaenoic acid (EPA). The well known sources of DHA are seafood and algae. Among fishes, cold water fishes are the best source of DHA. Generally, it is known that the colder the water, the greater the omega-3 content in fish oil. Besides fish oil, vegetable oil such as soy, canola, etc are also good sources of DHA. Eggs are also considered to be a good source of DHA.

[004] However, production of DHA from sources specific to sea food involves the following problems: unstable DHA phospholipids supply due to variable fish catch, uneven product quality due to seasonal and climate changes, and un-assured product safety due to marine contamination. Additionally, the problems include: resistance to consumption due to unpleasant fish odor that is specific to fish oil, and elevated refining costs. In eggs, from hens, phospholipids content is high at 30% of yolk lipids, but weight of the total lipids is low. The DHA content in ethanol extract of the yolk is merely about 12%.

[005] Another source of omega-3 unsaturated fatty acid, other than the aforementioned sources, which is capable of production of DHA in large quantity are microorganisms, particularly microorganisms capable of producing DHA.

[006] The production of DHA using microorganisms, particularly of genus Thraustochytrium and genus Schizochytrium, are already known. However, the processes provide the use of dextrose and starch as the carbon source which are expensive, and require continuous addition during fermentation process adopting fed batch fermentation. Thus, there is a need for a simple and commercially viable process for biological production of DHA.

OBJECT OF INVENTION

[007] The principal object of the invention is to provide a method for the production of omega-3 unsaturated fatty acid by batch fermentation.

BRIEF DESCRIPTION OF FIGURES

[008] This invention is illustrated in the accompanying drawings, throughout which like reference letters indicate corresponding parts in the various figures. The embodiments herein will be better understood from the following description with reference to the drawings, in which:

[009] FIG. 1 is an illustration of the Comparison Bar chart in ultimate potency using different carbon source, according to embodiments as disclosed herein; and

[0010] FIG.2 is an illustration of the Comparison Bar chart depicting the variation in the production of DHA with varying enzyme concentration, according to embodiments as disclosed herein.

DETAILED DESCRIPTION OF INVENTION

[0011] The embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying drawings and detailed in the following description. Descriptions of well-known components and processing techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0012] The embodiments disclosed herein provide a method for the production of omega-3 unsaturated fatty acid. The omega-3 unsaturated fatty acid, according to the disclosed embodiments, is docosahexaenoic acid (DHA). The embodiments disclosed herein achieve a simple and commercially viable process for the production of DHA by batch fermentation.

[0013] According to the embodiments disclosed herein, DHA is produced from microorganisms which are capable of producing DHA utilizing Dextrin white as the carbon source. The supply of Dextrin white may be continuous or interrupted. The list of microorganisms capable of producing DHA utilizing Dextrin white as carbon source include Thraustochytrium aureum, Thraustochytrium roseum, Schizochytrium limacinum and Crypthecodinium cohnii, or their strains, variants or mutants thereof

[0014] The method of producing DHA, according to the embodiments disclosed herein, is a batch fermentation method involving; introducing microorganisms to a fermentation medium comprising dextrin white, and inducing controlled hydrolysis during fermentation.

[0015] hi an embodiment, the microorganisms are Schizochytrium limacinum strain having characteristics of ATCC Accession No. 20888 and Schizochytrium limacinum strain having characteristics of ATCC Accession No. 20889.

[0016] The microorganisms are inoculated into a fermentation medium comprising dextrin white. The fermentation medium further includes ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate, and magnesium sulphate. The amount of dextrin white present in the fermentation medium may be such that it enables the production of DHA. The amount of dextrin white present in the fermentation medium may be in the range of 20% to 30% of the batch volume.

[0017] The batch fermentation conditions are maintained appropriately such that it facilitates optimum DHA production. In an embodiment, the Impeller tip speed is maintained at around 2.0m/s with airflow of about 0.7 to 0.8 wm in order to maintain the dissolved oxygen level within the range of around 5% to 15% after 48 hours of inoculation. Further, beyond 48 hours the dissolved oxygen was maintained between 30-35%.

[0018] Further, controlled hydrolysis is induced during fermentation in order to yield DHA. The controlled hydrolysis is induced preferably between 55 to 70 log hours of fermentation. In an embodiment, amylolytic enzymes such as alpha amylase enzyme may be used to induce controlled hydrolysis. An example of the alpha amylase enzyme that is instrumental in inducing controlled hydrolysis is Biotempase L. Alternatively, other generally known techniques that are capable of hydrolyzing carbohydrates may also be used to induce controlled hydrolysis. The amylolytic enzymes may be added between 55 to 70 log hours of fermentation, to induce controlled hydrolysis. The concentration of amylolytic enzyme used to induce controlled hydrolysis may be in the range of 100 to 150 ppm, preferably 120 ppm. Alternatively, other enzyme solutions, that are able to hydrolyze carbohydrates, which are generally known in the art, may also be used to induce controlled hydrolysis.

[0019] The DHA produced by the method disclosed in the embodiments herein may further be recovered and/or purified by methods generally known in the art including filtration of the fermentation broth, extraction, concentration, neutralization, bleaching and deodorization.

[0020] Experiments providing the production of DHA utilizing various carbon sources are provided herein by way of examples only and should not be construed to limit the scope of the present invention.

[0021] ExampIe-1 - Control fed batch fermentation using Dextrose as the carbon source:

the art including filtration of the fermentation broth, extraction, concentration, neutralization, bleaching and deodorization.

[0020] Experiments providing the production of DHA utilizing various carbon sources are provided herein by way of examples only and should not be construed to limit the scope of the present invention.

[0021] Example-1 - Control fed batch fermentation using Dextrose as the carbon source:

[0022] 10% of laboratory inoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate and magnesium sulphate. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained within the range of 1.0 -3.8 m/s, preferably 2.0m/s, with airflow of 0.2 -1.3 wm, preferably 0.7-0.8 wm, in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours, the dissolved oxygen was maintained between 30-35%. 30% of the batch volume of Dextrose was sterilized separately and added continuously to the fermentor after 30 hours of inoculation till the end of the cycle in order to maintain the reducing sugar level between 0.2-0.3%.

[0023] The DHA content in the broth at the end of fermentation was 16.4

[0024] £xainpIe-2 -Control fed batch fermentation using hydrolyzed starch as carbon source.

[0025] 10% of laboratory innoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate and magnesium sulphate. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained at 2.0m/s with airflow of 0.7-0.8 wm in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours, the dissolved oxygen was maintained between 30-35%.

[0026] Enzyme hydrolyzed starch was used to maintain the reducing sugar level between 0.2-0.3%.

[0027] The DHA content in the broth at the end of fermentation was 11.2 g/L.

[0028] £xainple-3 - Batch fermentation using Dextrin White as carbon source.

[0029] 10% of laboratory innoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate, magnesium sulphate and Dextrin white. Dextrin white was used in an amount of 30% of the batch volume. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained at 2.0m/s with airflow of 0.7-0.8 wm in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours the dissolved oxygen was maintained between 30-35%.

[0030] The sterile enzyme solution, commercially known as Biotempase L was added between 55-70 log hours aseptically (150 ppm of the total batch volume) for controlled hydrolysis.

[0031] The DHA content obtained in the broth at the end of fermentation was 14.3 g/L.

[0032] The results of the experiments are tabulated below:

[0033] FIG. 1 is a Comparison Bar chart depicting the results of aforementioned experiments. It shows the variation in the production of DHA with varying carbon sources. The carbon sources that have been considered for comparison are Dextrose, Starch and Dextrin White. As observed in FIG. 1, the use of dextrin white as carbon source in Batch fermentation under controlled hydrolysis yields good results.

[0034] Experiments providing the production of DHA at varying enzyme concentrations are provided herein by way of examples only and should not be construed to limit the scope of the present invention.

[0035] Example-4 - DHA production by Batch fermentation using with lOOppm enzyme concentration

[0036] 10% of laboratory innoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate, magnesium sulphate and Dextrin white. Dextrin white was used in an amount of 30% of the batch volume. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained at 2.0m/s with airflow of 0.7-0.8 wm in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours the dissolved oxygen was maintained between 30-35%.

[0037] For controlled hydrolysis, the sterile enzyme solution (Biotempase L) was added between 55 to 70 log hours, aseptically. The concentration of enzyme solution used was 100 ppm of the total batch volume.

[0038] The DHA content obtained in the broth at the end of fermentation was 13.1 g/L.

[0039] Example-5 - DHA production by Batch fermentation using with 120ppm enzyme concentration

[0040] 10% of laboratory innoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate, magnesium sulphate and Dextrin white. Dextrin white was used in an amount of 30% of the batch volume. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained at 2.0m/s with airflow of 0.7-0.8 wm in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours the dissolved oxygen was maintained between 30-35%.

[0041] For controlled hydrolysis, the sterile enzyme solution (Biotempase L) was added between 55 to 70 log hours, aseptically. The concentration of enzyme solution used was 120 ppm of the total batch volume.

[0042] The DHA content obtained in the broth at the end of fermentation was 14.4 g/L.

[0043] Example-6 - DHA production by Batch fermentation using with 150ppm enzyme concentration

[0044] 10% of laboratory innoculum was transferred aseptically into a fermentation medium comprising of ingredients such as sodium chloride, soya flour, potassium di-hydrogen phosphate and magnesium sulphate. The media ingredients were sterilized and then cooled to ambient temperature before use. The Impeller tip speed was maintained at 2.0mys with airflow of 0.7-0.8 wm in order to maintain the dissolved oxygen level between 5-15% after 48 hours of inoculation. Beyond 48 hours the dissolved oxygen was maintained between 30-35%.

[0045] For controlled hydrolysis, the sterile enzyme solution (Biotempase L) was added between 55 to 70 log hours, aseptically. The concentration of enzyme solution used was 150 ppm of the total batch volume.

[0046] The DHA content obtained in the broth at the end of fermentation was 14.3 g/L.

[0047] The results of the experiments are tabulated below.

[0048] FIG. 2 is a Comparison Bar chart depicting the results of aforementioned experiments. It shows the variation in the production of DHA with varying enzyme solution concentration. The experiments illustrated in Example 4 through 6 were performed to arrive at the optimum concentration of enzyme solution (Biotempase L). As depicted in FIG. 2 the enzyme concentration of 120 ppm was observed to be the optimum for DHA production using dextrin white as carbon source.

[0049] The foregoing description of the specific embodiments will so fully reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments herein have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

CLAIMS

We Claim,

1. LA batch fermentation method for producing DHA comprising:

introducing microorganisms to a fermentation medium comprising dextrin white; and

inducing controlled hydrolysis during fermentation.

2. The batch fermentation method for producing DHA as claimed in claim 1, wherein said microorganism comprises of Schizochytrium limacinum, or their strains, variants or mutants thereof.

3. The batch fermentation method for producing DHA as claimed in claim 1, wherein said microorganisms are selected from the list consisting of Schizochytrium having the characteristics of ATCC Accession No. 20888, and Schizochytrium having the characteristics of ATCC Accession No. 20889.

4. The batch fermentation method for producing DHA as claimed in claim 1, wherein said fermentation medium further includes sodium chloride, soya flour, potassium di-hydrogen phosphate and magnesium sulphate

5. The batch fermentation method for producing DHA as claimed in claim 1, wherein said dextrin white is present in the fermentation medium in an amount in the range of 20% to 30% of the batch volume.

6. The batch fermentation method for producing DHA as claimed in claim 1, wherein said controlled hydrolysis is induced by addition of at least one amylolytic enzyme.

7. The batch fermentation method for producing DHA as claimed in claim 6, wherein said amylolytic enzyme is an alpha amylase enzyme.

8. The batch fermentation method for producing DHA as claimed in claim 6, wherein concentration of said amylolytic enzyme is in the range of 100 ppm to 150 ppm.

9. The batch fermentation method for producing DHA as claimed in claim 6, wherein said addition of at least one amylolytic enzyme is performed between 55 to 70 log hours of fermentation.