Method to produce foaming agent

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce a foaming agent. The method includes cultivation of a microorganism in a fermentative medium, the cells of which produce a foaming agent extracellularly. At the same time the fermentative medium contains a defoaming agent, which has opacity temperature. Then the defoaming agent is removed at the temperature of the fermentative medium of at least 10°C higher than the opacity temperature of the defoaming agent.

EFFECT: method makes it possible to simplify removal of a defoaming agent from a fermentative medium.

15 cl, 2 dwg, 6 tbl, 4 ex

 

The present invention relates to methods of fermentation. In particular, the invention relates to extracellular production of foam by means of fermentation.

The level of technology

Foaming is a common problem in aerobic subsurface fermentation. Foaming is caused by the injection of gas in the enzymatic environment to ensure cultivated aerobic organisms, oxygen for growth (e.g., bacteria, yeast, fungi, algae, cell cultures). If the enzymatic environment contains surface-active components, such as proteins, polysaccharides or fatty acids, the foam can be formed on the surface of the medium when the bubbles injected gas released from the liquid. The foam creates a number of problems, including undesirable displacement of the product, nutrients and cells in the foam, and may complicate the localization process. In a known manner to control the foaming is the use of defoamers, several types of which are widely used: defoamers based on silicon-containing compounds (for example, polydimethylsiloxane), polyalkylene glycols (e.g., polypropylenglycol), fatty acids, polyesters and natural oils (such as linseed oil, soybean oil). Defoamers replace the foam components on the surface of bubbles, which leads to the destruction of the foam when the coalescence of bubbles. Defoamers are added at the beginning and/or during fermentation.

If the fermentation product is intended for use in food, personal care products or pharmaceuticals, it is highly desirable that the product is distinguished by the body-producer in the enzymatic environment (i.e., was extracellular, but not intracellular product). This avoids the need for the destruction of cells by physical or chemical way to release the product to highlight. While maintaining the integrity of cell material can be easily separated from the product so that the product purified from the cell and the genetic material that are generally regarded as undesirable contamination. This can be particularly important if the body is producing has been genetically modified. However, extracellular products can increase the degree of foaming in the fermenter, especially if the product enhances the formation of foam or increases its stability, for example, surface-active substance of biological origin or hydrophobin. The use of defoamers is a particular problem when extracellular products such blowing means for two reasons: first, increases required if estvo of antifoam, so as a self-foaming tool contributes to foaming in the fermenter. Secondly, there is no need to remove the antifoam most fermentation products, as it is present in low concentrations that do not affect the functionality of the product. However, when the fermentation product is a foaming agent, antifoam should be essentially removed because the presence of antifoam in the product will break the functionality of the latter.

Baileyet al, Appl. Environ. Biotechnol. 58 (2002) pp 721-727 reveal the products of hydrophobins HFB I and HFB II through the fermentation of transformantsTrichoderma reesei. To prevent foaming was used antifoam (Struktol J633), and hydrophobin was purified using two-phase aqueous extraction. However, such methods of separation, as a two-phase aqueous extraction or process chromatography are expensive and may require incompatible with food substances.

Daviset al, Enzyme and Microbial Technology28 (2001) pp 346-354 disclose an alternative method that avoids the need for antifoam. According to this method, the foam formed during the fermentation, is collected, and the product separated from her. This method was successfully applied for separation and concentration of surface-active substances of biological origin, lipopeptides bio-surfactants. what, however, this method has several disadvantages: first, continuous removal of foam could jeopardize the aseptic nature of fermentation; secondly, the removal of the foam may affect the number of living cells (because some cells may be removed from the foam), the volume of fluid and nutrient levels in the fermenter, making it difficult to control the fermentation process; and thirdly, the extraction of the product from the foam can be difficult, especially if the product forms a very stable foam. Thus, a need remains for an improved fermentation method for extracellular production of the blowing means.

The invention

We found that when using a certain group of defoamers to suppress foaming in the process of extracellular production of foam by means of fermentation, the antifoam can be easily removed from the product. Accordingly, first of all, the present invention provides a method for the production of foaming means, including: (i) culturing the host cell in an enzymatic environment where: a host cell secretes foaming agent in the environment; and enzymatic environment contains antifoam, which has a temperature (temperature) turbidity; (ii) removing agent at a temperature of enzymatic environment above the cloud point.

The use of antifoam lower the t foaming during the fermentation to a minimum. The choice of antifoam, which has a cloud point, and confirmation that the temperature of the enzymatic environment exceeds this temperature, turbidity, causes the loss of antifoam precipitated in the form of a dispersion. This provides a simple way antifoam may be removed upon completion of the fermentation, for example, filtration, centrifugation or adsorption. For comparison, defoamers, not having a cloud point, require a more complicated and/or expensive separation techniques such as extraction in aqueous two-phase system or chromatography.

Preferably, stage (i) enzymatic environment aeronaut by injection of air or air enriched with oxygen.

Preferably, stage (i) the temperature of the enzymatic environment above the cloud point of the antifoam.

Preferably, stage (ii) the agent is removed by filtration, centrifugation or adsorption. More preferably, the defoamer is removed by means of filtration cross-flow through a semipermeable membrane.

Preferably, stage (ii) remove at least 75% of antifoam, more preferably at least 85%, most preferably at least 90%.

Preferably, stage ii), the temperature of the enzymatic environment is at least 10°C above the cloud point, more is predpochtitelno not less than 20°C above the cloud point, most preferably at least 30°C above the cloud point.

Cell owners are also preferably removed from the enzymatic environment in phase ii).

Foaming tool preferably purify and/or concentrate of the enzyme environment after stage ii), for example, by ultrafiltration.

Preferred food antifoam.

The agent preferably contains at least one non-ionic surfactant/polymer, such as polyester, poly(allenglish), the block copolymer is a propylene oxide, polisport-based block copolymer is a propylene oxide, polyester dispersion based polypropylenglycol or alkoxycarbonyl ester of fatty acid.

Preferably, the blowing agent is a food.

Foaming tool preferably is hydrophobinum, more preferably by hydrophobinum class II, most preferably HFBI or HFBII fromTrichoderma reesei.

A host cell preferably is a genetically modified fungus, more preferably a yeast cell, most preferablySaccharomyces cerevisiae.

Preferably, after stage ii), the weight ratio of agent to the foaming agent is less than 0.2, more preferably less than 0.15, most preferably less than ,1.

Detailed description of the invention

If not stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art (for example, cell culture, molecular genetics, chemistry of nucleic acids, hybridization techniques and biochemistry). The standard techniques used for molecular and biochemical processes, can be found in Sambrooket al., Molecular Cloning: A Laboratory Manual, 3rded. (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubelet al., Short Protocols in Molecular Biology (1999) 4thEd, John Wiley & Sons, Inc. and the full version, entitled "Current Protocols in Molecular Biology".

Foaming funds

In the context of the present invention, the term "foaming agent" refers to surface-active substance of biological origin, which contributes to the formation of foam and/or increases its stability by suppressing the merger of bubbles.

Preferably the blowing means such that an aqueous solution of the foaming agent causes the formation of foam with a volume of the gaseous phase is not less than 20%not less than 50% retained when stored for 1 hour at 5°C, more preferably after 2 hours, most preferably after 4 hours, in accordance with the following test.

Preparing 80 ml volnogorskoe blowing funds (0.5% by weight). The solution aeronaut when shear deformation solution into chilled (2°C) the prescribed vertical cylindrical vessel of stainless steel, of a closed casing, with the inner proportions of 105 mm and a diameter of 72 mm, Cover the vessel fills with 54% of the inner volume, leaving 46% (180 ml) for sample. The rotor is used to create a shear deformation of the sample, consists of a rectangular impeller appropriate proportions to touch the inner surface of the container during the rotation (72 mm×41.5 mm). Also attached to the rotor by two semi-circular (diameter 60 mm) knife blade, creating a large shear force, are at an angle of 45° to a rectangular fixture. 80 ml of the solution was poured into a vessel and covered with a lid. Then the solution was subjected to shear deformation at 1250 rpm for 10 minutes. The solution is saturated with gas, immediately poured into the measuring cylinder. The volume of the foam was calculated in the measuring cylinder immediately and again after storage at 5°C. the Volume of the gaseous phase was determined based on the measured amount of foam and the known volume of the aqueous phase (i.e., 80 ml) as follows:

The volume of the gaseous phase=[(volume of foam - 80 ml)/volume of foam)]×100

The liquid in the foam over time, drains, which leads to the formation of two separate and distinct layers: foam top and an aqueous solution of the bottom. However, in this case the interest is the stability of the foam. To calculate the volume of the gaseous phase, the volume of foam is considered as a total system, that is, the gaseous phase and aqueous phase, regardless divided whether they are two distinct layers. Therefore, the volume of the gaseous phase gives a quantitative indicator of the stability of the foam to the loss of gas. Thus, if the initial volume of the gaseous phase is 50%, after storage volume of the gaseous phase must be at least 25%; if the initial volume of the gaseous phase is 20%, after storage it must be at least 10%.

Foaming tools include hydrophobin and surface-active substances of biological origin, such as glycolipids (e.g., rhamnolipid, regularity, callability, sophorolipids); lipopetides and lipoproteins (e.g., peptidome, serafettin, wiscosin, surfactin, subtilisin, gramicidines, polymyxins); fatty acids, neutral lipids and phospholipids; polymeric surface-active substance of biological origin (e.g., emulsan, biodispersant, mannan-lipid-protein, liposan, carbohydrate-protein-lipid, protein-PA), dispersion of surface-active substances of biological origin (vesicles and cilia, whole cells), glycosides (e.g., saponin) and f is ballernia proteins (for example, fibroin). Milk proteins and soy/protein hydrolysates are also foaming means, although they are usually not produced by means of fermentation. Foaming tool preferably is not a milk protein or soy or hydrolyzed protein. In a particularly preferred embodiment of the invention, the foaming agent is hydrophobinum.

Foaming means can be obtained by culturing organisms owners that produce foaming agent in an enzymatic environment in nature. For example, hydrophobin can be obtained by culturing filamentous fungi such as hyphomycetes (for example,Trichoderma), basidiomycetes and records of Ascomycetes. Particularly preferred hosts are microorganisms used in the food industry, such asCryphonectria parasiticathat produce hydrophobin called riparin (MacCabe and Van Alfen, 1999, App. Environ. Environ 65: 5431-5435). Similarly, surfactin can be obtained fromBacillus subtilisand glycolipids from, for example,Pseudomanas aeruginosa,Rhodococcus erythropolis,Mycobacterium speciesandTorulopsis bombicola(Desai and Banat, Microbiology and Molecular Biology Reviews, Mar. 1997, pp 47-64).

Alternatively, the blowing means can be obtained using recombinant technology. For example, the cells of the host, usually a microorganism, can be modified for expression inoob asousi funds. Methods for the introduction of structures of nucleic acids encoding the blowing means (where the foaming agent is a polypeptide or an enzyme necessary for the production of foam funds (where the foaming agent is not a peptide, for example, biosurfactant), in cells of the hosts, are well known in the art. Recombinant technology can also be used to modify the DNA sequence of a foaming tool or synthesis of new foaming tools required/improved properties.

Typically, suitable a host cell or organism transformed design nucleic acid that encodes the desired polypeptide foaming agent. The nucleotide sequence encoding the polypeptide, can be inserted into a suitable expression vector that encodes the necessary elements for transcription and translation, and thus that they will be expressed under appropriate conditions (e.g., in proper orientation and correct reading frame, and with adequate target and expression sequence). The technology required to create these expression vectors are well known to specialists in this field.

For the expression of the sequence encoding the polypeptide, can be used a number of expression systems is m They include, but are not limited to, bacteria, fungi (including yeast), insect cells and plant cell cultures, which were transformed with the appropriate expression vectors. The preferred hosts are those that are considered as food, safe (GRAS) organisms.

Suitable species of fungi, including (but not limited to) such yeast, as the genusSaccharomyces,Kluyveromyces,Pichia,Hansenula,Candida,Schizosaccharomycesand the like, and these types of filamentous fungi (but not limited to), as the fungi of the genusAspergillus,Trichoderma,Mucor,Neurospora,Fusariumand similar.

Sequence encoding the polypeptide of the blowing means, preferably at least 80% identical at the amino acid level the foaming agent found in nature, more preferably identical, at least 95% or 100%. However, experts in this field can make conservative substitutions or other changes in amino acid sequences which do not reduce the biological activity of the blowing means.

Hydrophobin are the most preferred class of the blowing means. Previously, EP1623631, we found that hydrophobin make it possible to obtain foam water-based with excellent resistance to disproportionation and coalescence. As hydrophobin predstavlyaet a high-foaming tools, their presence in the enzymatic environment is a special problem to control the formation of foam.

Hydrophobin are well-studied class of proteins (Wessels, 1997, Adv. Microb. Physio. 38: 1-45; Wosten, 2001, Annu Rev. Environ. 55: 625-646), capable of self-Assembly at the interface of hydrophobic and hydrophilic phases and has a conservative sequence:

XnC-X5-9-C-C-X11-39C-X8-23C-X5-9-C-C-X6-18C-Xm(SEQ ID No. 1),

where X represents any amino acid and n and m are independent integers. Usually hydrophobin has a length of up to 125 amino acids. Residues of cysteine (C) in the conserved sequence are part of disulfide bridges. In the context of the present invention, the term hydrophobin has a wider meaning, including functionally equivalent proteins, also demonstrating the properties of self-Assembly at the interface of hydrophobic and hydrophilic phases, which leads to the formation of protein films, such as proteins with the sequence:

XnC-X1-50C-X0-5C-X1-100C-X1-100C-X1-50C-X0-5C-X1-50C-Xm(SEQ ID No. 2)

or parts thereof, also showing the properties of self-Assembly at the interface of hydrophobic and hydrophilic phases, which leads to the formation of protein films. In accordance with the definition of the present invention, Samos is an Orc can be detected by protein adsorption on Teflon and use of circular dichroism to establish the presence of secondary structure (usually α-helices (De Vocht et al., 1998, Biophys. J. 74: 2059-68).

The fact of the formation of the film can be installed by incubation sheet of Teflon in the protein solution followed by leaching (at least three) with water or buffer (Wosten et al., 1994, Embo. J. 13: 5848-54). Protein film can be graphically represented by any suitable method, such as tagging fluorescent marker or using fluorescent antibodies, as well established in the art. The values m and n are typically in the range from 0 to 2000, but more usual values of m and n is generally less than 100 or 200. Definition hydrophobin in the context of the present invention includes fused proteins hydrophobin and another polypeptide, as compared hydrophobia and other molecules, such as polysaccharides.

Hydrophobin discovered to date, generally classified as belonging to class I or class II. Both types were found in mushrooms as secreted proteins, which self-organizes on the boundary of the hydrophobic phase in amphipatic film. Collected system hydrophobinum class I, as a rule, relatively insoluble, whereas hydrophobinum class II easily soluble in some solvents. Preferred hydrophobin, soluble in water, which means that it is not less than 0.1% soluble in water, preferably not Mapecem 0.5%. Not less than 0.1% soluble means that does not form any precipitate hydrophobin when 0.1 g hydrophobin 99.9 ml of water are subjected to centrifugation at 30000 g for 30 minutes at 20°C.

Also, in these filamentous bacteria, asActinomyceteandStreptomyces sp.were found hydrophobin-like proteins (for example, "Chaplin" ("chaplins")) (WO01/74864; Talbot, 2003, Curr. Biol, 13: R696-R698). These bacterial proteins, in contrast to the mushroom hydrophobinum, can form only no more than one disulfide bridge, as they may have only two cysteine residue. Such proteins are examples of functional equivalents of hydrophobins with the consensus sequence shown in SEQ ID No. 1 and 2, and are within the scope of the present invention.

More than 16 species of fungi were cloned more than 34 genes encoding hydrophobin (see, for example, WO96/41882, which includes a sequence of hydrophobins detected inAgaricus bisporus; and Wosten, 2001, Annu Rev. Environ. 55: 625-646). For the purposes of the present invention hydrophobin having at least 80% identity at the amino acid level to hydrophobin occurring in nature, are also covered by the term "hydrophobin".

Defoamers

The term "agent" includes those defoamers, which are usually added before will come foaming, and also those that are the usual added, when the foam has formed (sometimes called proclaime additives). A specific group of defoamers that are suitable for the present invention are defoamers, with turbidity. The cloud point is the temperature at which an aqueous solution of antifoam becomes optically turbid when the separation of the phases (i.e., molecules of antifoam form aggregates, light-scattering), as described on page 63 of the text "Surfactant Aggregation and Adsorption at Interfaces", J. Eastoe, in the publication ofColloid Science: Principles, Methods and Applications, ed. T. Cosgrove, Blackwell Publishing, 2005.

Examples of defoamers that demonstrate temperature opacities include compounds based on poly(alkalophiles) (PAG), such as block copolymers, ethylene oxide/propylene oxide, polyols based on block copolymers of ethylene oxide/propylene oxide and polymers of simple ether of ethylene oxide and propylene; and compounds based on esters of fatty acids.

The cloud point depends on the composition and chemical structure of the surfactants. For example, for polyoxyethylene (PEO) non-ionic surfactants, the cloud point increases with increase in the content of ethylene oxide (EO) taken for hydrophobic groups. Preferably, the cloud point of the antifoam is between 0°C and 90°C, b is more preferably between 5°C and 60°C.

The agent preferably contains at least one non-ionic polymeric surfactants, such as polyester, poly(allenglish), a block copolymer of ethylene/propylene oxide, polyhydric alcohol based block copolymer of ethylene/propylene oxide, dispersion of polymer a simple broadcast-based polypropylenglycol, or alkoxycarbonyl ester fatty acids. Defoamers based on the PAG (such as struktol (Struktol) J647, available from Schill & Seilacher), polyhydric alcohols based on block copolymers of ethylene oxide/propylene oxide (such as Struktol (Struktol) J647, available from Schill & Seilacher), and other non-ionic surfactants defoamers are particularly effective for the destruction of the foam, even in the presence of such a powerful foaming means, as hydrophobin.

You can apply a mixture of defoamers, in this case, the cloud point of a mixture is defined as the highest temperature opacities of the individual components.

Some common commercially available defoamers, with turbidity, are shown in table 1.

td align="justify"> Polyalkyleneglycol
Table 1
AntifoamTemperature °C
Struktol J647, Schill &Seilacher24
Struktol SB2121ca.30
UCON LB 65, Dow Chemical Company25
UCON LB 28515
UCON LB 62510
UCON LB 17158
KFO673, Lubrizol25
ST934, Pennwhite Ltdca.20
Block copolymers, ethylene/propylene oxide
Pluronic PE3100, BASF41
Pluronic PE610023
Pluronic PE620033
Pluronic PE810036
Pluronic PE1010035
Application DF204, BASF18-21
Polyhydric alcohols based block copolymer of ethylene/propylene oxide
Struktol J650, Schill &Seilacher13
Polymer dispersion of a simple broadcast-based polypropylenglycol
Antifoam 204, Sigma15
Alkoxycarbonyl ester fatty acids
Struktol J673, Schill &Seilacher30

Fermentation technology and the destruction of antifoam

Fermentation to obtain the blowing means is carried out by culturing the host cell in a liquid enzymatic environment within the bioreactor (e.g., industrial fermenter). The composition of the medium (e.g., nutrients, carbon source, and so on), temperature and pH are chosen to ensure adequate growth conditions of the culture and/or products of the blowing means. Air or air enriched with oxygen, is usually injected into the environment, providing oxygen for breathing culture.

The defoamer may be included in the initial composition of the medium and/or added as needed during fermentation. In practice, the widespread use of such technologies detection (detection) foam, as probes, which are automatic the ski start adding antifoam. In the present invention, the defoamer is preferably present in a concentration of from 0.1 to 20 g/l, more preferably from 1 to 10 g/L.

The temperature of the fermenter at stage i), that is, during fermentation, may be above or below the cloud point of the antifoam. Preferred fermentation temperature above the cloud point of antifoam, as antifoam most effective in bringing about coalescence of bubbles and the destruction of the foam at a temperature above the cloud point. Usually chosen such temperature of the fermenter to achieve optimal conditions for the growth of host cells and/or products.

At the end of fermentation the antifoam must be almost completely removed to ensure undisturbed functionality of the blowing means. Preferably remove at least 75% of antifoam, more preferably at least 85%, most preferably at least 90%. For example, after stage ii) the weight ratio of agent to the foaming agent is preferably less than 0.2, more preferably less than 0.15, most preferably less than 0.1.

Remove antifoam is achieved by ensuring that the temperature of the enzymatic environment above the cloud point of antifoam, so that the antifoam is divided into phases. In phases of penagos the tel can be removed from the enzymatic environment suitable way, such as:

filtering, for example, dead-end filtration or filtration in a filter press

- filtration cross-flow through a semipermeable membrane, for example, microfiltration or ultrafiltration

- centrifugation

- adsorption using, for example, activated carbon, silica or hard-shelled land as adsorbent.

Remove antifoam may be performed using, for example, one of these techniques in a single phase. Alternatively, the technique can be repeated or combined. For example, after the first stage of filtration, the filtrate can be re-heated (if necessary) and again filtered.

We found that the antifoam is removed to a greater extent if the temperature of the enzymatic environment is at least 10°C above the cloud point, preferably not less than 20°C above the cloud point, most preferably at least 30°C above the cloud point.

The temperature of the enzymatic environment should not be so high that the foaming tool denaturised. For this reason, preferably the blowing agent, resistant to heat, for example, hydrophobin. Preferably, the temperature of the enzymatic environment is less than 90°C, more preferably less than 75°C. In a preferred embodiment, done by the means, the antifoam has a cloud point in the range of 20-30°C and the temperature of the enzymatic environment in phase ii) is in the range of 40-60°C. For comparison, if you adhere to the conventional technology, such a temperature increase of the enzymatic environment deliberately avoided to reduce to a minimum the possibility of degradation reactions (which can cause discoloration and odor), inactivation of the enzyme, protein denaturation and loss of functionality (see for example page 7 edition "Separation Processes in the Food and Biotechnology Industries, Eds. Grandison, A. S.; Lewis, M.J.).

The preferred method of separation of antifoam is filtered through a semipermeable membrane. It was considered that the conduct of enzymatic environments containing antifoam, filtration through a semipermeable membrane at temperatures above its cloud point will lead to clogging of the membrane sediment defoamer, will cause a reduction in the leakage flow and the subsequent technological difficulties: see, for example, Yamagiwaet al., J. Chem. Eng. Japan, 26 (1993) pp 13-18, and WO 01/014521. Thus, it was previously thought that filtered through a semipermeable membrane should be carried out at temperatures below the cloud point. However, we have found that acceptable flow settings are achieved with the procedures ultrafiltration and microfiltration when the temperature is roughly 25°C above the cloud point of the antifoam.

To confirm that the product foaming tool is released from intracellular and genetic material (which is usually regarded as undesirable contamination), the cells must be removed from the enzymatic environment. In a preferred embodiment, the cells are separated from the environment at the same time remove precipitated defoamer, for example, at the stage microfiltration carried out at a temperature above the cloud point.

In an alternative embodiment, cells can be removed from the environment for additional stages of separation before removing antifoam - for example, by filtering (e.g., dead-end filtration or filtration in a filter press), filtering, cross-flow through a semipermeable membrane (e.g., microfiltration or ultrafiltration), or centrifugation is at a temperature below the cloud point. In this embodiment of the invention, the stage of purification and/or concentration (for example, by ultrafiltration) may be performed (again, at a temperature below the cloud point) after removal of the cells, but before the separation of the antifoam. Then the medium is heated to a temperature above the cloud point so that the antifoam can be removed, as already described.

As soon as antifoam the cells were removed from the enzyme environment, product foaming agent can be subjected to further purification and concentration, as necessary, for example by ultrafiltration. If the foaming agent is hydrophobin, it can be cleaned from the enzymatic environment using, for example, the techniques described in WO01/57076, which includes adsorption hydrophobin on the surface and subsequent contact surface with a surface-active agent such as Tween 20, for elution hydrophobin from the surface. Cm. also Collen et al., 2002, Biochim Biophys Acta. 1569: 139-50; Calonje et al., 2002, Can. J. Environ. 48: 1030-4; Askolin et al., 2001, Appl Environ Biotechnol. 57: 124-30; and De Vries et al., 1999, Eur J Biochem. 262: 377-85.

Hereinafter the present invention will be described by reference to the following examples, which are illustrative only and not limiting, and figures, where:

Fig. 1 shows the % of transfer as a function of temperature, 0.2% by weight aqueous solutions of defoamers Struktol J647 and J633.

Fig. 2 shows a calibration curve specified in example 2.

Examples

Example 1: the Definition of turbidity defoamers

Turbidity of the antifoam is measured by the following method, shown here for two commercially available defoamers, one of which has a turbidity (Struktol J647), and the second is not (Struktol J633).

0,2% (by weight) solution of each penagos is a dye was prepared in aqueous solution at room temperature. Samples with a volume of 20 ml was poured into a cylindrical glass vessels (Turbiscan). The samples were balanced at the measurement temperature for 1 hour in a water bath. The turbidity of the samples was determined with the use of device Turbiscan Lab Expert (Formulaction, France). This device has a light source with a wavelength of λ = 880 nm and an optical sensor of the incident light with an angle of view of 180°, which measures the percentage of incident light transmitted through a sample at 25 mm above the bottom of the vessel containing the sample solution. When the turbidity of the solution decreases the amount of transmitted light. Vessels containing samples were introduced into the instrument Turbiscan Lab Expert, which was set at the desired temperature measurement. Transmittance (%) was determined as a function of temperature in increments of 5°C from 5°C, the results are shown in Fig. 1. For antifoam J647, the transmittance sharply declined from 75% to 0% between 20°C and 25°C, indicating that this temperature was reached the cloud point. These data are consistent with a value of 24°C, as claimed by the manufacturer. (If you want a more accurate value of cloud temperature, the measurement can be conducted with a smaller stepper temperature interval, e.g. 1 or 2°C.) In contrast, the antifoam J633 has very small changes in turbidity, as it has no temperature pomote the Oia. Therefore, J647 is a suitable antifoam for use in the present invention, while J633 not suitable.

Example 2: remove the agent from the model solution

The experiment was conducted in order to show that the defoamers may be removed from model solutions by increasing the solution temperature to a value above the cloud point, and removing the precipitate by filtration. The solution of antifoam Struktol J647 (0.3% (weight/volume)) was prepared by dissolving an aliquot Struktol J647 weight 3.00 g in 1 liter of water Milli Q. Sample of this solution was heated to a temperature above the cloud point by placing in a water bath set at the desired temperature, for 1 hour. Then the samples were carefully mixed by rotation and immediately filtered.

We performed two different experiments. First, we studied the inuence of pore size of the filter by applying a filter with pore size 0.45 µm (Pall Life Sciences Acrodisc), 0.2 μm, of 1.20 μm and of 5.00 μm (all Sartorius Minisart) with a 2 ml syringe at a constant solution temperature (50°C). Secondly, varying the solution temperature from 30°C to 70°C (i.e., from 6°C to 46°C above the cloud point), while the pore size of the filter remained unchanged (0.2 μm).

The concentration of the antifoam in the filtrates was determined with the use of the kit for the analysis of water Lange LCK 433 topic ionic surface-active compounds. It uses the principle that non-ionic surface-active compounds (such as J647) form complexes with indicator TBPE (complex ethyl ester tetrabromophenolphthalein), which can be selected in dichloromethane and measured photometrically to determine the concentration. First was constructed calibration curve. The solution of antifoam Struktol J647 (0.3% (weight/volume)) was prepared by dissolving an aliquot Struktol J647 weight 3.00 g in 1 liter of water Milli Q at 15°C. an Aliquot of this solution was diluted with water Milli Q to concentrations: 6, 15, 30, 60, 150 and 300 mg/L. Water Milli Q was used as a blank sample. Samples of each concentration by volume of 0.2 ml was added to test tubes analytic set containing TBPE and dichloromethane. The tube gently mixed for 2 minutes, and then were left for 30 minutes. Then measured on the spectrophotometer Lange DR2800 at 605 nm, according to the instructions to the analytical set. The obtained calibration graph shown in Fig. 2.

Then the filtrates were diluted 1/10 with water Milli Q. Sample volume of 0.2 ml was measured on the spectrophotometer, as described above, and a calibration curve was determined by the concentration of the antifoam in each sample of the effluent. Number (%)of antifoam remaining in the filtrate was calculated as

(the measured concentration in the filtrate)/(known in the real concentration)×100%.

The concentration of antifoam up to 0.2 mg/l (2x10-5% weight/volume) can be measured according to a similar method, with the use of the kit for the analysis of water Lange LCK 333, and a calibration curve in the relevant range of concentrations. In this case, the analytical set add an aliquot of the measured sample volume of 2 ml, instead of 0.2 ml.

The results are shown in Table 2. The difference in the number of remaining antifoam between two measurements with a filter of 0.2 μm at 50°C (i.e., 6%) indicates the amount of error associated with using this method.

Table 2
The pore size of the filter (µm)The solution temperature (°C)Temperature above the cloud point (°C)The remaining share of antifoam (%)
0,250266
0,45502617
1,2502628
5,0 502679
0,230626
0,2502612
0,270469

The data show that the smaller the pore size of the filter, the greater the amount of antifoam removed, that is, reduce the amount of antifoam, remaining in solution, as expected. About J647, the pore size of 5.0 microns, small enough to remove most of the antifoam, whereas the use of filters with a pore size of 0.2 microns removes about 90% of the antifoam. The data also show that for the assumed pore size of the filter, increasing the temperature of the solution leads to more effective removal of antifoam.

Example 3. Remove antifoam model of the enzymatic environment

To demonstrate the removal of the antifoam of the typical enzymatic environment was prepared model of the enzymatic environment. First, there were prepared two Rast is ora, the composition of which is given in Table 3. In a typical periodic fermentation with the addition of the substrate Portion 1 will be the initial environment, and the Portion 2 is gradually fed through the loading intervals.

Table 3
ComponentPortion 1 (g/l)Portion 2 (g/l)
Glucose22440
Galactose010
Yeast extract1025
Dihydroorotase potassium2,112
Magnesium sulfate0,62,5
Antifoam - Struktol J6470,40,8
Water Milli QTo 1 literTo 1 liter

Each serving (1 litre) was autoclaveable 20 minutes at 121°C. Then, the portions were mixed (50:50) for receipt the model of the enzymatic environment with a concentration of antifoam 0.6 g/L. The environment has not been planted and are not subjected to fermentation, and were analyzed in raw form. The samples were heated and filtered to remove antifoam, and measured the number of remaining antifoam, also as described in example 2. The share of the remaining antifoam for each case are given in Table 4.

Table 4
The pore size of the filter (µm)The solution temperature (°C)Temperature above the cloud point (°C)The remaining share of antifoam (%)
0,230616
0,2502610
0,270466
0,4550268
1,2502610

The experiment was repeated, but with the addition of mo is part of the enzymatic environment of an additional quantity of antifoam thus, the initial concentration was 3 g/L. the Results are shown in Table 5.

Table 5
The pore size of the filter (µm)The solution temperature (°C)Temperature above the cloud point (°C)The remaining share of antifoam (%)
0,250268
0,4550268
1,2502615

These data show that the choice of antifoam with turbidity, the antifoam can be almost completely removed from the model of the enzymatic environment simple and convenient way.

Example 4: Remove agent from enzymatic environment containing the foaming agent

Periodic fermentation with the addition of the substrate genetically modified strain ofSaccharomyces cerevisiae. The strain was modified by introducing a gene encoding hydrophobin HFBII from fungusTrichoderma reeei (foaming agent), thus, what is achieved extracellular expression hydrophobin during fermentation. Fermentation was carried out essentially as described by van de Laar Tet al.inBiotechnol Bioeng. 96(3):483-94 (1997), using glucose as carbon source and by changing the scale of the procedure until a total volume of 150 litres in 300-liter fermentation tank. To control foaming during fermentation was used antifoam Struktol J647 (instead of Struktol J673, used van de Laar Tet al.).

At the end of fermentation, enzymatic environment was filtered at 15°C (i.e. below the cloud point of antifoam J647) to remove yeast cells.

Microfiltration was carried out at a pilot plant with ceramic membranes Kerasep with a pore size of 0.1 μm, using two volumes of diafiltration deionized water. Then the medium was subjected to ultrafiltration, again at 15°C for the partial purification of HFBII. Ultrafiltration was carried out on 1 kDa spiral-twisted polymer membranes Kansas state University with a transmembrane pressure of 0.9 bar and four volumes of diafiltration.

Measured (as described in example 2), the concentration of the defoamer in an enzymatic environment after stage ultrafiltration was 0,196 g/l Concentration of HFBII was 0,320 g/l, was measured using high-performance liquid chromatograph and (HPLC) as follows. The sample was dissolved in 60% aqueous solution of ethanol to an approximate concentration of 200 μg/ml prior to analysis. Separation by HPLC was performed on a column of Vydac Protein C4 (250×4.6 mm) at 30°C. Hydrophobin was determined by UV detection at 214 nm, and the concentration was calculated by comparison with samples of known concentration HFBII obtained from VTT Biotechnology, Espoo, Finland).

Then Wednesday, purified from cells, was heated to 50°C, maintained at this temperature for 30 minutes and then filtered to remove antifoam (pore size 0.2 μm), as described in example 2. The amount of remaining in the filtrate of antifoam and HFBII was measured as before and are shown in Table 6 (column entitled "stage 1"). Then the filtrate after the first stage was heated again to 50°C, maintained at this temperature for another 30 minutes, and filtered as before. Measured concentrations of HFBII and defoamer in the resulting filtrate, they are also given in Table 6 ("stage 2").

Table 6
Stage 1Stage 2
The number of HFBII in filtrate (g/l)0,320,30
% remaining HFBII 10093,75
The amount of antifoam in filtrate (g/l)0,050,028
% remaining antifoam25,514,3
The mass ratio of the antifoam/HFBII0,1560,093

The results show that the choice of antifoam with turbidity, the antifoam can be almost completely removed from the enzymatic environment containing cells masters and foaming tool, simple and convenient way.

Various features and embodiments of the present invention described in the above specific sections that apply, if necessary, to other sections, with appropriate amendments. Therefore, the characteristics described in one section may be combined with features described in other sections, as appropriate.

All publications mentioned in the above description, the included fully by reference. Specialists in this field will be apparent various modifications and variations of the described methods of the invention, without departing from the scope of the present invention. Hoteisalmeria described in connection with specific preferred variant implementation, it is clear that the claimed invention is not limited to such specific examples. Indeed, various modifications of the described embodiment of the invention that are obvious to experts in the relevant fields, are included in the scope of the attached claims.

1. The method of obtaining the blowing means, including:
i) culturing a microorganism in an enzymatic environment, the cell which produces extracellular foaming agent, and enzymatic environment contains a defoamer, which has a turbidity;
ii) removing agent at a temperature of enzymatic environment of not less than 10°C above the cloud point of the antifoam.

2. The method according to claim 1, in which stage (i) enzymatic environment aeronaut by injecting air or air enriched with oxygen.

3. The method according to claim 1, in which stage (i) the temperature of the enzymatic environment above the cloud point of the antifoam.

4. The method according to any one of claims 1 to 3, wherein in stage (ii) antifoam removed by filtration, centrifugation or adsorption.

5. The method according to claim 4, in which the antifoam is removed by filtration through a semipermeable membrane.

6. The method according to any one of claims 1 to 3, 5 in which at least 75% of antifoam remove stage ii).

7. The method according to any one of claims 1 to 3, 5 in which microorg the mechanisms are removed from the enzymatic environment in phase ii).

8. The method according to any one of claims 1 to 3, 5 in which the blowing means to purify and/or concentrate of the enzyme environment after stage ii).

9. The method according to any one of claims 1 to 3, 5 in which the antifoam is food.

10. The method according to any one of claims 1 to 3, 5 in which the antifoam contains at least one non-ionic surfactant/polymer interface.

11. The method according to claim 10, in which the antifoam is a polyester, poly(allenglish), the block copolymer is a propylene oxide, polisport-based block copolymer is a propylene oxide, polyester dispersion based polypropylenglycol or alkoxycarbonyl ester of fatty acid.

12. The method according to any one of claims 1 to 3, 5, 11, in which the foaming agent is a food.

13. The method according to any one of claims 1 to 3, 5, 11, in which the foaming agent is hydrophobinum.

14. The method according to any one of claims 1 to 3, 5, 11, in which the microorganism is a genetically modified fungus.

15. The method according to any one of claims 1 to 3, 5, 11, in which the weight ratio of agent to the foaming tool after stage ii) is less than 0.2.



 

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