ES2204316B1 - PROCEDURE FOR OBTAINING BIOCAPSULAR OF LEAVES, BIOCAPSULES AS WELL OBTAINED AND APPLICATIONS. - Google Patents

Abstract

Procedure for obtaining yeast biocapsules, biocapsules thus obtained and applications. Said process comprises the operation of causing a spontaneous co-immobilization between a filamentous fungus and a yeast, artificially creating the appropriate conditions to induce the corresponding symbiosis between the two, in the absence of physical-chemical supports. Said biocapsules are in the form of hollow spheres, whose wall has a smooth surface composed of mycelium of said fungus and cells of said trapped yeast, which limits an interior space partially occupied by free yeast cells and others associated with hyphae. The aforementioned biocapsules have application in industrial fermentation processes.

Description

Procedure for obtaining biocapsules of yeasts, biocapsules thus obtained and applications.

Technical Field of the Invention

The present invention fits within the Technical field of Biotechnology and Industrial Microbiology, and more specifically within the sector of the immobilization of biocatalysts for fermentative processes, or for processes of degradation or obtaining of chemical products.

More specifically, the present invention provides a procedure for obtaining biocapsules, which they present important technical advantages within the field of immobilization of biocatalysts and their applications.

State of the art prior to the invention

Molds and yeasts are fungi widely distributed in Nature. Molds or filamentous fungi are presented as long filaments of cells (hyphae) that form a mycelium, tangled mass more or less compact. Yeasts are unicellular fungi, usually oval or cylindrical. Molds such as Penicillium and Aspergillus are aerobic and due to their chemical activities they are very important in the industry (production of: antibiotics, cheeses, citric acid, gluconic acid, gallic acid and numerous enzymes that are applied to many industrial processes such as saccharification) . Saccharomyces cerevisiae is an optional anaerobic yeast and is of great economic importance because it is involved in alcoholic fermentation processes to obtain ethanol and alcoholic beverages. Certain breeds of S.cerevisiae can form a veil or flower on the surface of the wine once the alcoholic fermentation is finished and the fermentable sugars are exhausted, a form of spontaneous immobilization.

These are the flower yeasts (Martínez et al., 1997). For more information on filamentous fungi and yeasts for industrial use see Leveau and Bouix (2000).

According to Chibata (1978) the immobilization of enzymes is one in which "enzymes are found physically confined or located in a defined region of space maintaining its catalytic activity, and can be repeated and continuously used. "The term" immobilization " It is also applicable to cell organelles and cells both Microbial like plants or animals. The immobilization of cells has a number of advantages for industrial processes of fermentation such as: increased productivity, stability Cellular and reduced process costs due to the easy cell recovery and recycling (Groboillot et al., 1994). Tanaka and Kawamoto (1999) describe numerous methods of Immobilization and classify them into four categories:

one)

Binding methods of biocatalyst to a  carrier substrate or water-insoluble support, through joints ionic, covalent, physical adsorption or biospecific junctions. The most used materials are: water insoluble polysaccharides (cellulose, dextran and agarose derivatives), proteins (gelatin and albumin), synthetic polymers (polystyrene derivatives, ion exchange resins and polyurethane) and materials inorganic (clay, crystal, sand, ceramic and magnetite) these brackets can be used directly or after a activation.

2)

Cross-linking methods, through the use of biomultifunctional compounds (eg the most commonly used reagent is glutaraldehyde that crosslinks groups amino by Schiff base junctions, also used toluene and hexamethylene diisocyanate) that will lead to Water insoluble networks of biocatalysts.

3)

Methods of entrapment, through retention of the biocatalyst in a lattice produced by a matrix of one or more polymers (polyacrylamide gel, gel alginate, carrageenan gel, synthetic resins), or by encapsulation: in microcapsules of a synthetic polymer semipermeable, in liposomes, membranes or inverse micelles.

4)

A combination of the three previous methods:

Immobilizations of a single microorganism have been obtained naturally, by aggregating cells in small pellets or microspheres, such as flocculating yeasts, bacteria or fungal mycelia. (Webb et al., 1986; Fontana et al. 1991; Wieczorek and Michalski, 1994; Paiva et al., 1996). Non-hollow microspheres (150-200 um) have also been obtained using different polymers such as carrageenan, Sephade G-25-50, collagen, cellulose, etc. (Perusich et al., 1991; Szajani et al., 1996, Nigam, 2000). Microencapsulations have also been performed, especially for the immobilization of enzymes because a large number of enzymes can be trapped with synthetic polymers (cellulose acetate thalate) without denaturing or undergoing chemical modifications (Tzan et al., 1991; Gibbs et al., 1999). Other techniques have achieved the co-immobilization of Aspergillus awamori and Saccharomyces pastorianus using various cellulose supports (Fujii et al., 2001), for application in obtaining ethanol from starch, by simultaneous saccharification and fermentation. Yeasts S. cerevisiae and Candida shehatae have also been co-immobilized in a micropore membrane / agar layer structure, which has been applied in the continuous alcoholic fermentation of glucose / xylose mixture (Lebeau et al., 1998).

In the previous paragraphs it has been done reference to bibliographic citations in abbreviated form, so as not to make the exhibition too cumbersome. Next, a enumeration of said bibliographic citations indicating all the data necessary to be able to locate them easily, if it were precise.

Bibliography

Chibata I ( 1978 ). Immobilized Enzymes-Research and Development. John Wiley & Sons., Inc., New York.

Fontana , A .; Chraibi , M .; Guiraud , JP and Ghommidh , C. ( 1991 ). Diffusivity measurement in a flocculating yeast layer. Biotechn. Techniques 5: 133-138).

Fujii , N., Oki , T .; Sakurai , A .; Suye , S. and Sakakibara , M. ( 2001 ) Ethanol production from starch by immobilized Aspergillus awamori and Saccharomyces pastorianus using cellulose carriers. J. Ind. Microbiol. Biotechnol 27: 52-57.

Gibbs , BF Kermasha , S., Alli , I, and Mulligan . CN ( 1999 ). Encapsulation in the food industry; to review Int. J. Food Sci. Nutr . 50: 213-224.

Groboillot TO.; Boadi , DK; Poncelet , D. and Neufeld , RJ ( 1994 ). Immobilization of cells for application in the food industry. Crit. Rev. Biotechnol . 14: 75-107.

Lebeau , T .; Jouenne , T. and Junter , GA ( 1998 ). Continuous alcoholic fermentation of glucose / xylose mixtures by co-immobilized Saccharomyces cerevisiae and Candida shehatae . Appl. Microbiol Biotechnol . 50: 309-313.

Leveau , JY and Bouix , M. ( 2000 ). Industrial Microbiology The microorganisms of industrial interest. Editorial Acribia SA Zaragoza.

Martínez , P., Pérez . L. and Benítez . T. ( 1997 ). Evolution of flor yeast population during the biological aging of fine Sherry wine. Am. J. Enol. Vitic 48: 160-168.

Nigam , JN ( 2000 ), Continuous ethanol production from pineapple cannery waste using immobilized yeast cells. J. Biotechnol . 80: 189-193.

Paiva , CT; Sato , S .; Visconti , AE and Castro , LA ( 1996 ) Continuous alcoholic fermentation process in a tower reactor with recycling of flocculating yeast. Appl. Biochem Biotechnol 57-58: 535-541.

Perusich , CM; Goetghebeur , S. and Hu , WS ( 1991 ). Virus production in microsphere-induced aggregate culture of animal cells. Biotechnol Techniques 5: 145-148.

Szajani , B .; Buzas , S; Dallmann , K; Gimesi , I .; Krisch , J. and Toth , M. ( 1996 ). Continuous production of ethanol using yeast cells immobilized in preformed cellulose beads. Appl. Microbiol Biotechnol 46: 122-125.

Tanaka , A. and Kawamoto , T. ( 1999 ). Cell and enzyme immobilization. In Manual of Industrial Microbiology and Biotechnology. Second Edition (Eds AL Demain and JE Davies; RM Atlas, G. Cohen, Ch. L. Hershberger, Wei-Shou Hu, DH Sherman, RC Wilson, JH David Wu). ASM PRESS, Washington, DC pp. 94-102.

Tzan , YL; Lin , SY; Weng , CN and Lee , CJ ( 1991 ). Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate; the effect of swine serum on micrometric properties. Biotechnol Techniques 5: 139-144.

Webb , C. Black , GM and Atkinson , B. ( 1986 ). Process engineering aspects of immobilized cell systems. Published by The Institution of Chemical Engines. Pergamon Press Ltd., Headington Hii Hall Oxford, England.

Wieczorek , A. and Michalski , H. ( 1994 ). Continuous ethanol production by flocculating yeast in the fluidised bed bioreactor. FEMS Microbiol. Rev. 14: 69-74.

The applicant has directed his efforts researchers in the immobilization sector of biocatalysts that constitutes a field of biotechnology of great interest in its many industrial applications.

As a result of these efforts he has achieved tune up a new immobilization process of biocatalysts that provides important advantages in the field. This finding constitutes the basis of the present invention which exposes in detail in the following sections of this descriptive memory.

Detailed description of the invention

The present invention, as set forth in its statement refers to a procedure to obtain yeast biocapsules, to the biocapsules themselves thus obtained and to your applications

The originality of the present invention consists in provoking a spontaneous co-immobilization of two microorganisms: a filamentous fungus and a yeast, by means of a symbiotic induction, in the absence of chemical binding compounds and external supports, artificially creating the appropriate conditions to favor correspondent
symbiosis.

This co-immobilization is it achieves by adding to the culture medium an unusable nutrient by yeast and yes by the filamentous fungus in a buffered medium and agitated.

Through this technique, biocapsules are obtained hollow, whose wall is composed of mycelium and cells of trapped yeasts, probably linked by phenomena of biospecific adsorption. In this regard, the applicant has been able to check that flower yeasts adhere more efficiently that yeasts that do not possess this property, to provide hollow capsules of spherical shape and smooth surface, whose walls they limit an interior space partially occupied by cells of free yeasts and others associated with hyphae, forming clusters, leaving the culture medium transparent.

According to the above, it is noteworthy that the novelty of the present invention resides fundamentally in use a living organism as immobilizing substrate (the aforementioned filamentous fungus) instead of a support inert physicochemical, as in the art previous.

The yeast biocapsules of the present Invention are very useful in various industrial applications.

The co-immobilization operation which forms the basis of the present invention is characterized by the cultivation of filamentous fungi and yeasts in media buffered, in the absence of inert external physical supports, containing said nutrient media usable by fungi filamentous and not usable by yeasts.

Basically the technique developed by the This invention consists in creating the right conditions for that this symbiosis occurs. These conditions are fundamentally the following:

one)

A culture medium provided with a carbon source usable by the filamentous fungus and not usable by yeast. Between the Carbon sources that meet these requirements can be cited on gluconic acid, starch, cellulose, inulin and other molecules similar colloidal.

2)

A buffered culture medium such that the pH does not decrease during the process as a result of the excretion of acids by the filamentous fungus (such as citric acid, gluconic acid, etc.). It is very important to properly control the pH of the medium to prevent yeast cells from dying, in which case, the biocapsules formed would consist only of the fungus filamentous, which would result in lax and fibrous spheres not smooth. Specifically, said pH should be maintained between 3 and 7.

3)

A culture medium permanently agitated so that the filamentous fungus cells and biocapsules can be produced. Stirring can be carried out with an orbital shaker, in a fermenter with a stirring-aeration system, or with any other means that is suitable for agitation required The size of the sphere-shaped biocapsules depends mainly from external factors such as speed and time of agitation, being able to vary its diameter between a few millimeters and several centimeters Another important factor is the temperature of incubation.

Basically, as a summary of the above previously, obtaining yeast cell biocapsules it depends on internal factors, such as microorganisms of Employee heading and chemical composition of the medium; and of factors external that must be controlled, such as the temperature of incubation, stirring speed and stirring time.

Between internal and external factors it is done then an enumeration of those that are especially suitable for the execution of the invention:

Internal factors:

to)

Microorganisms

-

Breeds of Saccharomyces cerevisiae such as capensis, bayanus , etc.

-

Species of filamentous fungi Aspergillus, Penicillium , etc.

b)

Media composition

-

YNB ("yeast nitrogen base") without amino acids.

-

Source of carbon used by the filamentous fungus and not by yeast (starch, cellulose, inulin, gluconic acid, etc.).

-

Means, medium buffered

-

pH between 3 and 7

External factors:

-

Incubation temperature included between 20 and 30 ° C.

-

Stirring speed between 90-270 rpm.

-

Weather 24 or more hours of cultivation.

While the description made above it's obvious enough how for an expert in the field can appreciate the advantages of the present invention, then insists on them more specifically.

The prior techniques for co-immobilization use supports such as cellulose, semipermeable membranes of synthetic polymers, liposomes, etc. In  the present invention, the support, the matrix, is one of the microorganisms to be immobilized, therefore the immobilization expense since no support is used external inert. Thus, it is a simple, easy and easy method cheap, without the need for an external inert carrier. For another On the other hand, the unions and interactions that occur between organisms are natural, so their catalytic activities are not they must be affected since it is not an immobilization forced.

A "coinmobilization" is achieved in which  the two active microorganisms can be used for processes simultaneous as a saccharification and fermentation, or only active yeast in processes without aeration (fermentation), where the filamentous fungus would act as support inert.

An additional advantage is the possibility of emptying the inside of the biocapsule and filling it with other types of cells or biocatalysts to perform other processes by confinement of a large density of cells within the biocapsule It is therefore a renewable source of cells, already that the cells grow inside the spheres and part go  freeing the environment by continually regenerating itself functioning as transporters and cell sources. In addition, they are usually easily separable from the environment in which the process is carried out which in addition to speeding it up makes it cheaper.

The biocapsules of the present invention have a wide diversity of industrial applications especially in the field of fermentation processes and in the industry of feeding.

As illustrative of any of these applications, specifically the following can be mentioned:

one)

Fermentative processes for ethanol production from different carbon substrates; molasses, starch, cellulose, inulin, among others.

2)

Use as support for others biocatalysts in processes of obtaining and recovering High added value products.

3)

Obtaining drinks fermented

4)

Liquid clarification

In addition, the biocapsules of the present invention can be used in the aforementioned processes, whether they are carried out continuously or in batches or batches.

Brief description of the figure

Figure 1 and only attached is a representation graph of the evolution of the CO2 production speed of the immobilized and free yeast cells (control) on the molasses medium (9% sugars, 0.3% ammonium sulfate, Ph = 7), corresponding to the embodiment of Example 2.

Embodiments of the invention

The present invention is further illustrated. through the following examples, which should not be considered, at all, as limiting the scope of the invention, defined solely and exclusively by the note attached claim.

Example 1: Obtaining biocapsules of S.cerevisiae (capensis) and A.niger .

To obtain biocapsules of S.cerevisiae (capensis) and A. niger , the following conditions were used:

one)

Media composition

-

YNB without amino acids

-

Acid gluconic

-

Na_2HPO_4; KH_2PO_4 and NaOH

2)

Media Preparation

-

YNB dissolution in distilled water according to the manufacturer's instructions and addition of gluconic acid up to about 5 g / L.

-

Adjustment from pH to a value of 7 with phosphoric acid salts and with sodium hydroxide.

3)

Sterilization of the autoclave medium (120 ° C, 15 min) and inoculation with yeast cells (1x10 ^ 6 cel / mL) and filamentous fungus spores (several handles of sowing).

4)

Thermostat at 28 ° C and stirring at 90 rpm in orbital shaker for 48 h.

Thus spherical biocapsules were obtained with a average sphere diameter of 15 mm in diameter and were collected with a 0.2 mm sieve.

Example 2: Application of biocapsules to obtain ethanol.

Biocapsules obtained from S. cerevisiae (capensis) and a species of Penicillium were used , by a process analogous to that described in Example 1 above, in obtaining ethanol from the fermentation of molasses, reused seven times without that a decrease in its fermentative activity was observed.

The effect of immobilization on fermentation kinetics, whose results are shown in the Figure 1 attached.

This figure graphically represents the evolution of the production rate of CO_2 of the cells of immobilized and free yeasts (control) on a medium of said molasses consisting of 9% sugars, 0.3% ammonium sulfate, pH = 7; the data have been obtained by weight difference with respect to to the initial total weight of molasses.

In said Figure 1 it can be seen that:

(one). The profile of the fermentation curve in the free cell fermented molasses medium is different from those observed when immobilized cells are used, which are more soft and similar to each other. The delay observed, the first time which was fermented with immobilized cells, is due to a period of adaptation to the fermentation medium. (2). The maximum speed of fermentation was achieved with free yeast cells when They are inoculated in full fermentation activity. (3). The duration of process (about 5 days) and ethanol content reached (41 gl -1) are similar in all cases.

The capsules used in this experiment are typical capsules of the present invention in the form of spheres hollow filamentous fungus and yeast cells, various sizes, flexible, elastic and very stable to situations drastic These balls allow the growth of the cells of yeast and its constant regeneration, functioning as capsules of yeast cells, without altering the properties of the carrier, and being able to reuse a large number of times in adequate conditions and without loss of activity or viability of yeast cells. The filamentous fungus serves as a mere carrier.

The size of the spheres used ranged from 2 and 20 mm in diameter, with thicknesses of 0.5 and 1.5 mm respectively.

Claims (18)

1. Procedure for obtaining yeast biocapsules, characterized in that it comprises the operation of causing spontaneous co-immobilization between two microorganisms, namely a filamentous fungus and a yeast, artificially creating the appropriate conditions to induce the corresponding symbiosis between both microorganisms, and all this in the absence of conventional physical-chemical supports. 2. Method according to claim 1, characterized in that a first of said conditions suitable for inducing said symbiosis is the use of a culture medium provided with a carbon source usable by said filamentous fungus and not usable by said yeast. 3. Method according to claim 2, characterized in that said carbon source is selected from gluconic acid, starch, cellulose, inulin and other related colloidal molecules. 4. Method according to claim 1, characterized in that a second of said conditions suitable for inducing said symbiosis, is the use of a buffered culture medium, to control that the pH does not drop to a value at which the cells of the cell die. yeast. 5. Method according to claim 1, characterized in that a third of said conditions suitable for inducing said symbiosis is the use of a permanently agitated culture medium that allows the formation of said biocapsules having a spherical shape with a diameter between millimeters and several centimeters. Method according to claim 1, characterized in that said filamentous fungus is chosen among those of the Aspergillus and Penicillium species. Method according to claim 1, characterized in that said yeast is selected from the races of Saccharomyces cerevisiae . 8. Method according to claim 7, characterized in that said yeast breed is capensis . 9. Method according to claim 7, characterized in that said yeast breed is bayanus . Method according to claim 4, characterized in that said pH is maintained between 3 and 7 during the symbiosis co-immobilization operation of said fungus and said yeast. Method according to any one of the preceding claims, characterized in that other additional conditions for inducing said symbiosis, include carrying out the culture at an incubation temperature of 20-30 ° C at a stirring speed of 90-270 rpm, and over a period of 24 hours to 120 hours. 12. Biocapsules obtained by the method defined by any one of the preceding claims 1 to 11, characterized in the form of hollow spheres, whose wall has a smooth surface composed of mycelium of said fungus and cells of said trapped yeast, limiting said wall a interior space partially occupied by free yeast cells and others associated with hyphae, forming clusters. 13. Biocapsules according to claim 12, characterized in that said spheres have a diameter between 0.2 mm and 5 cm. 14. Use of the biocapsules defined in the preceding claims 12 and 13, as agents of fermentation in industrial fermentation processes. 15. Use according to claim 14, characterized in that said fermentative processes are intended for the production of ethanol from a carbon source selected from molasses, starch, cellulose and inulin. 16. Use of the biocapsules defined in the preceding claims 12 and 13, as support for others biocatalysts in processes of obtaining and recovering industrial products with high added value. 17. Use of the biocapsules defined in the preceding claims 12 and 13, for obtaining fermented beverages 18. Use of the biocapsules defined in the preceding claims 12 and 13, for the clarification of liquids in industrial processes.