FIELD OF THE INVENTION
The present invention relates to the process for production of dextranase and more particularly relates to the process for indigenous production of dextranase using solid state fermentation technique.
BACKGROUND OF THE INVENTION
Dextranase enzyme has various applications. The search for organisms producing large amounts of an enzyme to break down dextran began in the 19th century. The dextranase is found both as intracellular and extracellular. Dextranase in pharmaceutical industry, in food industry and in sugar industry has wide applications. In the sugar industry, the production of the sugar is affected by various undesirable compounds synthesized by microorganisms from sucrose. The undesirable compounds e.g dextran results in the increasing viscosity of the flow and reducing industrial recovery, causes significant losses from sugar production. Not only is sugar lost due to dextran formation but dextran itself causes processing problems like increased juice viscosity, poor clarification and crystal elongation are all associated with the dextran. The use of dextranase enzyme is the most efficient method for hydrolyzing the dextran at sugar mills. The Dextranase improved the filtration of sugar products; increase the recovery rate of the sugar, lower viscosity. Some bacterial strains, filamentous fungi and a small number of yeasts have been shown to produce dextranase. The uses of these dextranases have limitations as the conditions of temperature and brix are high, however the treatment at this point (syrup) needs higher doses which is economically unfeasible. Some thermo tolerant dextranases identified by recombinant organisms by some foreign companies are much expensive, and do not have GRAS status and hence unviable for Indian sugar industry.
US3702805A discloses an improved process for the production of dextranase which comprises culturing a dextranase-producing microorganism selected from the genera
chaetomium, humicola, sporotrichum, anixiella, macrosporium, streptomyces, gibberella, gloeosporium and glomerella, particularly chaetomium spirale, chaetomium gracile, sporotrichum asteroides, or gibberella fujikuroi in a fermentation medium and recovering from said medium the dextranase which accumulates therein. Dextranase itself is known to be useful, for example as enzymatic preparations for prevention of tooth decay or scale deposit.
CN105713887A discloses methods, of dextranase freeze-dried powder and belongs to the field of biotechnologies. The method comprises steps as follows: strains are deactivated after fermental cultivation; filtering is performed through a perforated panel filter with the mesh size being 0.5 mm; an obtained liquid passes a belt-type filter and then is sent to an ultra-filtration membrane circulating tank, a clear liquid enters a nanofiltration storage tank, and then materials enter a nanofiltration liquid storage tank; the liquid passes a liquid filter with the mesh size being 0.45 mu m and a liquid filter with the mesh size being 0.22 mu m for sterile filtration and then enters a freeze-drying plate of a freeze dryer; the liquid is subjected to freeze-drying and sublimation drying treatment and mixed; the liquid is packaged by a vacuum packaging machine, and the dextranase freeze-dried powder is obtained. The total yield of enzyme activity units of dextranase is 80%-83%, the enzyme activity units of the dextranase freeze-dried powder are about 100,000-150,000, and large-scale industrial production of dextranase is realized.
CN105671104A discloses a method for preparing micromolecular dextran by degradation of Tdextran with immobilized dextranase. The method includes: preparing a fermentation medium, subjecting penicillium aculeatum and acetobacter to fermentation culture on the medium to obtain thalli, performing ultrasonication to obtain bacterial solution, mixing the bacterial solution with carrageenan to obtain a mixture, dripping the mixture into a calcium chloride solution to obtain dextranase immobilized cell microspheres, transferring the microspheres to a substrate solution, reacting in a shaker, precipitating with ethanol, washing and drying. By fermentation of the penicillium aculeatum and the acetobacter to
obtain the thalli containing the dextranase and adoption of the carrageenan for immobilization, high immobilization rate is achieved while activity and stability of the dextranase are improved; by adoption of the dextranase for degradation of dextran, molecular weight of the dextran can be reduced, by-products are reduced, and quality of the dextran is improved.
In the existing inventions the enzyme production from the various microbes are very complex and costly. The major problem with the dextranase is to produce stable and viable dextranase. All the existing invention used submerged fermentation for the production of the dextranase. The present invention overcomes all drawbacks of the existing inventions hence there is need of the present invention.
OBJECTIVE OF THE INVENTION
The main objective of the present invention is to design a process by SSF technique using economical agri-waste for the production enzyme dextranase.
The principal objective of the present invention is to optimize the parameters of solid state fermentation production, develop extraction and stabilization technique for enzyme dextranase.
Another objective of the present invention is to modulate a non-GMO strain to produce dextranase.
Yet another objective of the present invention is to produce thermally stable and viable enzyme.
Yet another objective of the present invention is to reduce the cost of operation in enzyme production.
Yet another objective of the present invention is to achieve higher production rate of enzyme.
Further objectives, advantages and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example and appropriate reference to accompanying drawings.
SUMMARY OF THE PRESENT INVENTION
The present invention relates to a process for production of dextranase using solid state fermentation technique. This invention is based on identification of suitable micro-organism strain for dextranase production using solid state fermentation technique and dextranase stabilization at ambient temperature and commercially viable production process are the key elements of invention. Pure component of fermentation media and submerged fermentation process imposes high cost of the product which in turn causes high cost to the industry. In present invention the economic production of dextranase achieved from mutation of micro-organism to the SSF production parameters using cheap easily available agri-waste as carbon and nitrogen sources. The resultant product is an endo-dextranase useful for hydrolysis of dextran in the sugar cane based industry. The process comprises: identification of strain, modulation of strain, development of cost effective production technique, extraction, concentration and stabilization of harvested enzyme and development of a suitable formulation system for industrial application. The strain is selected from the genii penicillium and the strain is modulated using U.V. radiation. The agricultural waste is used as a carbon and nitrogen source for the penicillium strain. The enzyme is extracted with buffer system. The extracted enzyme concentrated by ultra-filtration technique using hollow fiber.
One advantage of the present invention is that the present invention provides an effective process for the thermally stable and viable dextranase.
Yet another advantage of the present invention is that the present invention does not use GMO strain for the dextranase production.
Yet another advantage of the present invention is that the present invention uses agricultural waste to produce dextranase.
Yet another advantage of the present invention is that the present invention is economically suitable due to the low production cost.
Yet another advantage of the present invention is to increase the higher production of the enzyme.
Yet another advantage of the present invention is that the present invention provides economical and technical solutions for the removal of dextran during sugar production.
Yet another advantage of the present invention is that the operational and running cost of the present invention is very low as compared to the other invention or process.
Yet another advantage of the present invention is that the present invention achieves higher success rate or recovery rate with low operational cost.
Yet another advantage of the present invention is that the present invention uses solid state fermentation technique to produce thermally stable and economically viable dextranase.
Further objectives, advantages and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example and appropriate reference to accompanying drawings.
DETAILED DESCRIPTION OF THE INVENTION
While this invention is susceptible to embodiment in many different forms, as shown in the drawings and will herein be described in detail specific embodiments, with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, like reference numerals are used to describe the same, similar or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order of those skilled in the art to practice the invention.
Definition
The terms “a” or “an”, as used herein, are defined as one or as more than one. The term “plurality”, as used herein, is defined as two or as more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.
The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended. The term “comprising” is used interchangeably used by the terms “having” or “containing”.
Reference throughout this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the
appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps or acts are in some way inherently mutually exclusive.
As used herein, the term “Solid State Fermentation Technique” generally refers to, but not limited to, a biomolecule manufacturing process used in the food, pharmaceutical, cosmetic, fuel and textile industries. These biomolecules are mostly metabolites generated by microorganisms grown on a solid support selected for this purpose.
As used herein, the term “Dextranase” generally refers to, but not limited to, is an enzyme with systematic name 6-alpha-D-glucan 6-glucanohydrolase. Dextranase enzyme is the most efficient method for hydrolyzing the dextrans at sugar mills. Some bacterial strains, filamentous fungi and a small number of yeasts have been shown to produce dextranase. The fungal dextranase showed the highest reaction rate at low Brix, with pH and temperature close to 5.0 and 50 °C.
As used herein, the term “Dextran” generally refers to, but not limited to, a complex branched glucan composed of chains of varying length. The straight chain consists of α-1,6 glycosidic linkages between glucose molecules, while branches begin from α-1,3 linkages.
As used herein, the term “GMO” generally refers to, but not limited to, is any organism whose genetic material has been altered using genetic engineering techniques (i.e., a
genetically engineered organism/genetically engineered organism). GMOs are used to produce many medications and genetically modified foods and are widely used in scientific research and the production of other goods.
As used herein, the term “Pure culture” generally refers to, but not limited to, a laboratory culture containing a single species of organism. A pure culture is usually derived from a mixed culture (one containing many species) by transferring a small sample into new, sterile growth medium in such a manner as to disperse the individual cells across the medium surface or by thinning the sample many folds before inoculating the new medium.
As used herein, the term “nutrient medium” generally refers to, but not limited to, a liquid or gelatinous substance containing nutrients in which microorganisms, cells, or
tissues are cultivated for scientific purposes.
As used herein, the term “seed culture” generally refers to, but not limited to, the most common type of blood cell and the vertebrate's principal means of delivering oxygen (O2) to the body tissues—via blood flow through the circulatory system. RBCs take up oxygen in the lungs, or gills of fish, and release it into tissues while squeezing through the body's capillaries.
As used herein, the term “cell cycle” generally refers to, but not limited to, asexual reproduction, or cell division, of a bacterium into two daughter cells, in a process called binary fission. Providing no mutational event occurs, the resulting daughter cells are genetically identical to the original cell. The growth of bacteria (or other microorganisms, as protozoa, microalgae or yeasts) in batch culture can be modeled with four different phases: a lag phase (A), a log phase or exponential phase (B), a stationary phase (C), and a death phase (D).
As used herein, the term “centrifuge” generally refers to, but not limited to, a piece of equipment that puts an object in rotation around a fixed axis (spins it in a circle), applying a potentially strong force perpendicular to the axis of spin (outward). The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction.
As used herein, the term “enzyme” generally refers to, but not limited to, a substance produced by a living organism which acts as a catalyst to bring about a specific biochemical reaction.
As used herein, the term “buffer solution” generally refers to, but not limited to, an aqueous solution consisting of a mixture of a weak acid and its conjugate base, or vice versa. Its pH changes very little when a small amount of strong acid or base is added to it. Buffer solutions are used as a means of keeping pH at a nearly constant value in a wide variety of chemical applications.
In preferred embodiment, a process for indigenous production of dextranase using solid state fermentation technique, the process includes:
identification of strain, the identification of strain comprises:
pure culture of penicillium aculeatum obtained from the MTCCI;
modulation of strain, the modulation of strain comprises:
the culture of penicillium aculeatum modulated using U.V radiation and was maintained at 25 c on CYA medium,
the culture of penicillium aculeatum after modulation was used for optimizing the secretion of dextranase in mid log phase in shake flask using a fixed nutrient medium and varying the inducers, and
series of alpha 1, 6 and 1, 4 oligosaccharides were screened to determine the most effective inducer;
development of cost effective production technique, the development of cost effective production technique comprises:
optimization of concentration of inducer, carbon and nitrogen sources for the growth of seed culture,
setting of SSF parameters for optimized yield, and
identification of peak cycle of growth;
extraction, concentration and stabilization of harvested enzymes, the extraction, concentration and stabilization of harvested enzymes comprises:
extraction of enzyme from the fermented broth using various buffer system, dextranase extracted from the fermented bran with o.1 M phosphate buffer (pH 5.0 ) 90% and 10% sorbitol mixture, fermented bran with 5 times its weight, were mixed with buffer-sorbitol mixture and agitated for 30 minutes. Buffer-sorbitol mixture is then centrifuged,
the crude enzyme is then passed through hollow fiber membrane and the concentration of extracted enzyme is collected by ultrafiltration technique using hollow fiber and
polyols but not limited to, propylene glycol, glycerol PEG(polyethylene glycols) were used at different concentration for the stability of the concentrated enzyme;
development of a suitable formulation system for industrial application, the development of a suitable formulation system for industrial application comprises:
formulation stability confirmation,
laboratory scale dextran reduction and doses optimization, and
cost benefit analysis in context of industrial usage.
Further objectives, advantages and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains
may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.

CLAIM
1. A process for production of dextranase using solid state fermentation technique, the process comprises:
identification of strain, wherein the strain is selected from the genii penicillium;
modulation of strain, wherein the strain is modulated using U.V. radiation;
development of cost effective production technique,
optimization of concentration of inducer, a carbon and nitrogen source for growth of penicillium culture,
setting of solid state fermentation parameters for optimized yield, and
identification of peak cycle of growth;
extraction, concentration and stabilization of harvested enzyme,
buffer solution is used for the extraction of enzyme from the fermented broth,
enzyme concentration, and
polyols are used with different concentration for the stability of concentrated enzyme; and
development of a suitable formulation system for industrial application.
2. The process claimed in claim 1, wherein penicillium aculeatum is selected from the genii penicillium and used to produce dextranase.
3. The process claimed in claim 1, wherein the agricultural waste is used as a carbon and nitrogen source for the strain penicillium aculeatum.
4. The process claimed in claim 1, wherein the penicillium aculeatum strain is maintained at 25C on CYA medium after U.V modulation.
5. The process claimed in claim 1, wherein the enzyme is extracted with the buffer system.
6. The process claimed in claim 1, the extracted enzyme is concentrated by ultrafiltration technique using hollow fiber.
7. The process claimed in claim 1, enzyme concentration is achieved by ultrafiltration technique using hollow fiber.