The invention concerns the use, in dry form, of a combination of substantially amorphous microfibrils with a crystallinity index not more than 50%, with at least a polyhydroxylated compound, as additive for food formulations, the content of this combination being more than 0% and less than 20% by weight relative to the total weight of the food formulation.

This application is an application under 35 U.S.C. Section 371 of International Application Number PCT/FR98/02228, filed on Oct. 16, 1998.
The present invention relates to the use, in dry form, of cellulose microfibrils combined with at least one polyhydroxylated compound, as an additive in food formulations.
Microfibrillar cellulose is a product that is well known and used in many fields, such as the food sector, since it gives the media in which it is present specific properties in terms of viscosity and stability, for example.
In general, cellulose microfibrils are used in the form of dispersions, which correspond, in fact, to the form in which they are obtained by carrying out fibrillation operations.
Thus, European patent EP 295 865 proposes the use of cellulose microfibril suspensions containing parenchymal cells as an additive for food formulations. These microfibrils are obtained by carrying out a process which consists in treating, in a first step, the plant pulp at an acidic or basic pH, and then, in a second step, in carrying out a heat treatment at high temperature for a short period. The pulp thus treated is then subjected to a mechanical shear so as to obtain microfibrils, which are then separated and optionally blanched. This process has the particular feature of entirely removing the pectins and hemicelluloses present at the surface of the microfibrils.
European patent application EP 726 356 proposes, inter alia, to use, as an additive in food formulations, essentially amorphous cellulose microfibrils whose surface is charged with carboxylic acids.
Although the advantage of microfibrils is, of course, not brought into doubt here, the fact that they are used in the form of dispersions of relatively low concentration (conventionally less than 10%, by dry weight, of microfibrils) does not represent an advantage as regards the storage (location and stability of the suspension) or even the use (metering).
Now, microfibrils are not compounds which can be dried without specific precautions. If such is the case, microfibrils in dry form are not redispersible, on account of the very strong hydrogen bonding which binds the fibrils together.
It is thus known practice to carry out the drying of the said microfibrils in the presence of additives. However, the amounts generally used are very high. It is not uncommon for these contents to represent at least 50% by weight, or even 100% by weight, relative to the weight of microfibrils.
This may be disadvantageous, especially for cellulose microfibrils which have specific rheological properties that are different from those of the additive added. The reason for this is that the additive introduced in such amounts masks the rheological profile of the microfibrils in a certain manner.
As may be observed, no food formulations obtained from cellulose microfibrils in dry form which are readily redispersible are currently available.
Thus, a subject of the present invention is the use, in dry form, of a combination of essentially amorphous cellulose microfibrils having a degree of crystallinity of less than or equal to 50%, with at least one polyhydroxylated compound, as an additive for food formulations, the content of this combination being greater than 0% and less than 20% by weight relative to the total weight of the food formulation.
It has been observed that the combined cellulose microfibrils, in dry form, used according to the invention are, in the first place, readily redispersible. The term xe2x80x9creadilyxe2x80x9d means that it is not necessary to use high shear means such as those resulting from the use of a machine of Ultra-Turrax type.
In addition, combined microfibrils, in dry form, once redispersed, regain at least 50% of their initial viscosity.
Moreover, it has been found that the dried, combined microfibrils have very good texturizing properties, i.e. they have a twin role as stabilizer and thickener in the formulation into which they are introduced.
The dried combined cellulose microfibrils can also legitimately at least partially replace fats for the preparation of reduced-fat formulations.
It should be noted that the organoleptic properties of the dried, combined cellulose microfibrils are such that they give the formulation similar consistency and unctuousness to those of the products which they replace.
The dried combined cellulose microfibrils similarly have noteworthy overrun properties. In other words, the incorporation of combined microfibrils gives the overrun formulations very large stability and foam-elasticity properties.
The dried combined microfibrils similarly have the advantage of replacing one or more products, which simplifies the use of the formulation.
In addition, the amounts of dried combined microfibrils, relative to the additive(s) which they replace, are lower.
It should be noted that the dried combined microfibrils can be used as substitutes for animal gelatin, usually of bovine origin, which represents an appreciable advantage in terms of health.
Other advantages and characteristics of the present invention will emerge more clearly on reading the description and the examples which follow.
As has been indicated previously, a subject of the present invention is the use, in dry form, of a combination of essentially amorphous cellulose microfibrils with at least one polyhydroxylated compound as an additive for food formulations.
The expression xe2x80x9cessentially amorphousxe2x80x9d refers to microfibrils whose degree of crystallinity is less than or equal to 50%. According to one specific variant of the present invention, the degree of crystallinity is between 15 and 50%. Preferably, the degree of crystallinity is less than 50%.
The cellulose microfibrils forming part of the composition of the formulation according to the present invention are obtained from cells preferably consisting of at least about 80% primary walls. Such characteristics are present with cellulose based on parenchymal cells. For example, citrus fruit, such as lemons and grapefruit in particular, and sugar beet, are plants comprising such cells.
Preferably, the amount of primary walls is at least 85% by weight. More particularly, cellulose derived from sugar beet pulp is used.
According to one preferred embodiment of the invention, the microfibrils are surface-charged with carboxylic acids and acidic polysaccharides, alone or as a mixture.
The term xe2x80x9ccarboxylic acidxe2x80x9d refers to simple carboxylic acids, as well as salts thereof. These acids are preferably chosen from uronic acids, or salts thereof. More particularly, the said uronic acids are galacturonic acid, glucuronic acid or salts thereof.
As acidic polysaccharides, mention may be made of pectins, which are more particularly polygalacturonic acids. These acidic polysaccharides can be present as a mixture with hemicelluloses.
One very advantageous embodiment of the invention consists of microfibrils whose surface is at least charged with galacturonic acid and/or polygalacturonic acid.
It should be noted that it is not a case here of a simple mixture between the said microfibrils and the acids and polysaccharides, but rather a strict combination between these two types of compound. The reason for this is that the process for preparing the microfibrils is such that the acids and polysaccharides are not totally separated from the fibres, but instead still remain at the surface thereof, giving them quite specific properties. Thus, it has been observed that it was not possible to obtain the same properties if these acids and/or polysaccharides were totally separated from the microfibrils during their preparation in order for them to be added thereafter.
The cellulose microfibrils moreover have a cross section of between about 2 and about 10 nm. More particularly, the microfibril cross section is between about 2 and about 4 nm.
The specific microfibrils forming part of the composition of food formulations have such characteristics on account of the use of a quite specific preparation process, which will now be described.
It should be noted that this process has been described inter alia in patent application EP 726,356, to which reference may be made for further details.
Firstly, the said process is more particularly carried out on sugar beet pulp, after it has undergone a preliminary step of extraction of the sucrose, according to the methods known in the art.
The preparation process comprises the following steps:
(a) first acidic or basic extraction, after which a first solid residue is recovered,
(b) optionally, second extraction, carried out under alkaline conditions, of the first solid residue, after which a second solid residue is recovered,
(c) washing of the first or second solid residue,
(d) optionally, bleaching of the washed residue,
(e) dilution of the third solid residue obtained after step (d) so as to obtain a solids content of between 2 and 10% by weight,
(f) homogenization of the dilute suspension.
In step (a), the term xe2x80x9cpulpxe2x80x9d is intended to refer to wet, dehydrated pulp stored by ensilage or partially depectinized.
The extraction step (a) can be carried out in acidic medium or in basic medium.
For an acidic extraction, the pulp is suspended in an aqueous solution for a few minutes so as to homogenize the acidified suspension at a pH of between 1 and 3, preferably between 1.5 and 2.5.
This operation is carried out with a concentrated solution of an acid such as hydrochloric acid or sulphuric acid.
This step may be advantageous for removing the calcium oxalate crystals which may be present in the pulp, and which, on account of their highly abrasive nature, can cause difficulties in the homogenization step.
For a basic extraction, the pulp is added to an alkaline solution of a base, for example sodium hydroxide or potassium hydroxide, with a concentration of less than 9% by weight, more particularly less than 6% by weight. Preferably, the concentration of the base is between 1 and 2% by weight.
A small amount of a water-soluble antioxidant, such as sodium sulphite Na2SO3, may be added in order to limit the oxidation reactions of the cellulose.
Step (a) is generally carried out at a temperature of between about 60xc2x0 C. and 100xc2x0 C., preferably between about 70xc2x0 C. and 95xc2x0 C.
The duration of step (a) is between about 1 hour and about 4 hours.
During step (a), partial hydrolysis takes place with release and solubilization of most of the pectins and hemicelluloses, while at the same time retaining the molecular mass of the cellulose.
The solid residue is recovered from the suspension obtained from step (a) by carrying out known methods. Thus, it is possible to separate the solid residue by centrifugation, by filtration under vacuum or under pressure, with filter gauzes or filter presses, for example, or else by evaporation.
The first solid residue obtained is optionally subjected to a second extraction step carried out under alkaline conditions.
A second extraction step is carried out when the first step has been carried out under acidic conditions. If the first extraction has been carried out under alkaline conditions, the second step is only optional.
According to the process, this second extraction is carried out with a base preferably chosen from sodium hydroxide and potassium hydroxide, whose concentration is less than about 9% by weight, preferably between about 1% and about 6% by weight.
The duration of the alkaline extraction step is between about 1 and about 4 hours. It is preferably equal to about 2 hours.
After this second extraction, if it is carried out, a second solid residue is recovered.
In step (c), the residue derived from step (a) or (b) is washed thoroughly with water in order to recover the residue of cellulosic material.
The cellulosic material from step (c) is then optionally bleached, in step (d), according to the standard methods. For example, a treatment with sodium chlorite, with sodium hypochlorite or with hydrogen peroxide in a proportion of 5-20% relative to the amount of solids treated can be carried out.
Different concentrations of bleaching agent can be used, at temperatures of between about 18xc2x0 C. and 80xc2x0 C., preferably between about 50xc2x0 C. and 70xc2x0 C.
The duration of this step (d) is between about 1 hour and about 4 hours, preferably between about 1 and about 2 hours.
A cellulosic material containing between 85 and 95% by weight of cellulose is thus obtained.
After this bleaching step, it may be preferable to wash the cellulose thoroughly with water.
The resulting suspension, which has optionally been bleached, is then rediluted in water to a proportion of 2 to 10% solids, before undergoing a homogenization step.
The homogenization step corresponds to a mixing or blending operation or any operation of high mechanical shear, followed by one or more passages of the cell suspension through an orifice of small diameter, subjecting the suspension to a pressure drop of at least 20 MPa and to a high-speed shear action, followed by a high-speed deceleration impact.
The mixing or blending is carried out, for example, by passage(s) through the mixer or blender for a period ranging from a few minutes to about an hour, in a machine such as a Waring Blendor fitted with a four-blade impeller or a pan mill mixer or any other type of blender, such as a colloidal mill.
The actual homogenization will advantageously be carried out in a homogenizer such as a Manton Gaulin in which the suspension is subjected to a shear action at high speed and pressure in a narrow passage and against an impact ring. Mention may also be made of the Micro Fluidizer, which is a homogenizer mainly consisting of a compressed-air motor which creates very high pressures, an interaction chamber in which the homogenization operation takes place (elongational shear, impacts and cavitations) and of a low-pressure chamber which allows depressurization of the dispersion.
The suspension is introduced into the homogenizer preferably after preheating to a temperature of between 40 and 120xc2x0 C., preferably between 85 and 95xc2x0 C.
The temperature of the homogenization operation is maintained between 95 and 120xc2x0 C., preferably above 100xc2x0 C.
The suspension in the homogenizer is subjected to pressures of between 20 and 100 MPa and preferably above 50 MPa.
Homogenization of the cellulosic suspension is obtained by a number of passages which can range between 1 and 20, preferably between 2 and 5, until a stable suspension is obtained.
The homogenization operation can advantageously be followed by a high mechanical shear operation, for example in a machine such as the Sylverson Ultra-Turrax machine.
The cellulose microfibrils according to the invention are combined with at least one polyhydroxylated compound.
More particularly, the polyhydroxylated compound is chosen from carbohydrates and derivatives thereof, and polyols.
As regards the carbohydrates, mention may be made most particularly of linear or cyclic C3-C6 and preferably C5 or C6 monosaccharides, oligosaccharides, polysaccharides and fatty derivatives thereof such as sucroesters or fatty acid sucroesters, carbohydrate alcohols and mixtures thereof.
Non-limiting examples of monosaccharides which are suitable are fructose, mannose, galactose, glucose, talose, gulose, allose, altrose, idose, arabinose, xylose, lyxose and ribose.
Oligosaccharides which may be mentioned, inter alia, are sucrose, maltose and lactose.
The polysaccharides may be of animal, plant or bacterial origin.
In addition, they can be used in anionic or nonionic form.
Xanthan gum, succinoglycans, carrageenans and alginates are representative components of anionic polysaccharides.
As regards the nonionic polysaccharides, mention may be made in particular of galactomannans, such as guar gum, carob gum, starch and its nonionic derivatives, and nonionic cellulose derivatives.
As regards the carbohydrates, mention may be made, without any intention of being limited thereto, of fatty acid sucroesters, fatty acid esters, carbohydrate alcohols such as sorbitol or mannitol; carbohydrate acids such as gluconic acid, uronic acids such as galacturonic acid or glucuronic acid, as well as their salts, and carbohydrate ethers such as carboxymethyl cellulose.
As regards the polyols, glycerol, pentaerythritol, propylene glycol, ethylene glycol and/or polyvinyl alcohols can be used in the food formulations.
It should be noted that the compounds described above can be used alone or as a mixture.
According to a first particularly advantageous variant, the polyhydroxylated compound is at least carboxymethyl cellulose.
Cellulose is a polymer consisting of glucose monomer units. The carboxylic group is introduced in a manner which is known per se, by reacting chloroacetic acid with cellulose.
The degree of substitution corresponds to the number of carboxymethyl groups per glucose unit. The maximum theoretical degree is 3.
Depending on whether or not the degree of substitution is greater than 0.95, or less than or equal to this value, it is specified that the carboxymethyl cellulose has a high or low degree of substitution, respectively.
According to a second variant, the polyhydroxylated compound is a combination of carboxymethyl cellulose with at least one compound chosen from nonionic and anionic monosaccharides, oligosaccharides and polysaccharides and derivatives thereof, and carbohydrate derivatives such as carbohydrate alcohols, acids and ethers.
In particular, the carboxymethyl cellulose is used in combination with at least one of the following compounds: xanthan gum, sorbitol, sucrose.
The total content of polyhydroxylated compound is at least 5% by weight relative to the microfibrils and to the said polyhydroxylated compound, and not more than 50% by weight relative to the same reference.
Preferably, this content is between 5 and 30% by weight relative to the same reference. In an entirely advantageous manner, the content is between 10 and 30% and preferably between 15 and 30% by weight.
Optionally, besides the abovementioned polyhydroxylated compound, the cellulose microfibrils can be combined with at least one co-additive chosen from:
compounds of formula (R1R2N)COA, in which formula R1 or R2, which may be identical or different, represent hydrogen or a C1-C10, preferably C1-C5, alkyl radical, A represents hydrogen, a C1-C10, preferably a C1-C15, alkyl radical or the group Rxe2x80x21Rxe2x80x22N with Rxe2x80x21 and Rxe2x80x22, which may be identical or different, representing hydrogen or a C1-C10, preferably C1-C5, alkyl radical.
As regards the compounds of (R1R2N)COA type, it is preferred to use compounds comprising two amide functions. Preferably, urea is used as co-additive.
The cellulose microfibrils forming part of the composition of the food formulations are obtained from the drying of a dispersion of microfibrils in the presence of an additive and optionally of a co-additive.
It may likewise be envisaged to use a dispersion of such a composition.
Such compositions have especially been the subject of the patent applications PCT/FR 97/01290 published under the number WO 98/02486 and PCT/FR 97/01291 published under the number WO 98/02487; reference may be made to these patents as regards the nature of the additives, co-additives and combinations preferred, the respective proportions of the additives and co-additives, and their method of preparation.
The co-additive content is such that the total content of polyhydroxylated compound and of co-additive is at least 5% by weight relative to the microfibrils and to the said polyhydroxylated compound, and not more than 50% by weight relative to the same reference.
Preferably, the said co-additive content is such that the abovementioned total content is between 5 and 30% by weight. In an entirely advantageous manner, the content is between 10 and 30% and preferably between 15 and 30% by weight.
The process for preparing such microfibril compositions consists, firstly, in preparing the cellulose microfibrils from suitable cellulosic pulp by carrying out a hydrolysis, optionally followed by at least one step of bleaching of the pulp thus treated. Everything which has been mentioned previously in this respect remains valid and will not be repeated here.
More particularly, in a first step, at least some of the additive and optionally co-additive(s) are added to the microfibril suspension, which has optionally undergone at least one homogenization cycle. Next, in a second step, a step of drying of the suspension thus supplemented is carried out.
According to a first variant, the addition of at least some of the additive and optionally co-additive is carried out after the homogenization step.
A first particularly suitable method consists in adding at least some of the additive and optionally co-additive to the suspension after the homogenization step, after this suspension has undergone at least one concentration step.
As a guide, the concentration step(s) take(s) place by filtration, centrifugation or evaporation of some of the water from the suspension, by precipitation, for example in an alcohol, by freezing-thawing, by dialysis, etc.
According to this embodiment, the concentration operation can be carried out until a solids content of about 35% by weight is obtained.
The introduction of the additive and optionally co-additive is carried out in a manner which is known per se, i.e. by any means which allows homogeneous introduction of a solution, a suspension or a powder to a suspension which tends to have the consistency of a paste. For example, mention may be made of blenders, extruders and mixers.
This operation can be carried out over a wide temperature range, more particularly between room temperature and 80xc2x0 C.
A second method consists in adding at least some of the additive and optionally co-additive to the suspension after the homogenization step, before this suspension has undergone at least one concentration step.
In this case, the concentration step(s) which take(s) place after the addition of additive and optionally of co-additive is (are) carried out in the same way as indicated above.
According to a second advantageous variant, the introduction of at least some of the additive and optionally co-additive is carried out before or during the homogenization step. When it is indicated that the supplementation takes place during the homogenization step, this means that the additive and optionally the co-additive are introduced when the pulp has undergone at least one cycle of the homogenization step.
The supplementation takes place according to the two methods indicated in the context of the first variant.
Prior to the actual drying step, it may be advantageous to carry out shaping of the suspension which has been concentrated as mentioned previously. This shaping is carried out in a manner which is known to those skilled in the art. Mention may be made in particular, without, however, intending to be limited thereto, of extrusion and granulation.
According to a particularly advantageous variant, the drying step is carried out so as to maintain not less than 3% by weight of water relative to the weight of the solid obtained. More particularly, the weight of water maintained is between 10 and 30% by weight.
The drying advantageously takes place in air, although it may be envisaged to carry it out under an inert gas, such as nitrogen.
It should also be noted that it is preferred to carry out the drying in an atmosphere whose degree of humidity is controlled so as to be able to maintain the desired moisture content in the composition.
The drying temperature should limit any degradation of the carboxylic acids, of the acidic polysaccharides, of the hemicelluloses and/or of the additives and co-additives. It is more particularly between 30 and 80xc2x0 C., preferably between 30 and 60xc2x0 C.
It should be noted that it would not constitute a departure from the context of the present invention to carry out a drying operation in several steps, some of which would use the means indicated above for the concentration step.
After the drying step, the composition obtained can be blended.
The content of cellulose microfibrils, comprising the polyhydroxylated compound, and optionally the co-additive, is greater than 0% and less than or equal to 20% by weight relative to the total weight of the food formulation. More particularly, this content is between 0.01 and 5% by weight relative to the same reference. Preferably, it is between 0.03 and 3% by weight relative to the same reference.
The food formulations in which the combined microfibrils are added are conventionally dispersions. The term xe2x80x9cdispersionxe2x80x9d denotes emulsions of liquids, emulsions of gases and liquids, suspensions of liquids and solids, or any other system combining these possibilities.
The food formulations are obtained very simply, using the conventional methods for preparing food formulations depending on their type. Thus, the combined microfibrils, in dried form, are mixed with other required ingredients of the formulation and the whole is then homogenized.
The temperature at which the formulation is prepared does not present any particular problems. It should advantageously be noted, in this respect, that the formulations comprising the combined microfibrils can be sterilized without any damage to their use properties. Another advantage of the combined cellulose microfibrils according to the invention is that it is possible to prepare the food formulations without needing to preheat the ingredients.
It should moreover be pointed out that the cellulose microfibrils are compatible and substantially retain their properties despite the diversity of the food formulations (pH values, ionic strengths, composition).
The parameters which can be measured to determine the texture of the food formulations are of rheological nature, and consist essentially in measuring the viscosity and the elasticity. The viscosity corresponds to the resistance to flow under an irreversible deformation, and the elasticity is the response of the formulation to a reversible deformation. They also make it possible to give indications regarding the stability of the formulations, which is particularly desired, especially when the formulation comprises at least two components that are not miscible with each other. Thus, a high viscosity slows down certain emulsion-destabilizing phenomena, such as creaming and flocculation. A high value for the elasticity, which represents the structural result of interactions between the various components, allows the formulation tp undergo shear stresses, such as those encountered when the formulation is served with a spoon or spread, for example, without becoming destabilized, without any breaking of the droplets formed, thus avoiding coalescence of the formulation.
The Theological measurements of flow viscosity are measured using a controlled-stress rheometer or a controlled-shear-gradient rheometer, or more simply using a Brookfield viscometer.
The flow rheology makes it possible to evaluate the flow threshold of the formulation, which represents the force to be supplied to destroy the structure of the medium and force it to flow. The flow rheology also makes it possible to quantify the ease of a formulation to flow when the imposed shear increases (shear-thinning behaviour) as well as the viscosity level at a given shear rate.
These flow rheology data are representative of the behaviour of the formulation while it is being chewed, transferred, overrun, etc.
The elasticity measurements are carried out under a dynamic regime using a rheometer which makes it possible to function in oscillations. It is thus possible to determine the values of Gxe2x80x2, the storage modulus (or elastic modulus), and Gxe2x80x3, the loss modulus (or viscous modulus), as well as the deformation range in which the moduli do not vary. It should be noted that, in general, the behaviour is linear at low deformations.
The objective of these rheological characteristics is to demonstrate the viscoelastic behaviour of the formulations and to compare them with each other.
The combined microfibrils, in dry form, used in the context of the present invention give food formulations a specific Theological profile of shear-thinning type. This type of Theological behaviour has the consequence of entailing a reduction in the viscosity of the formulation containing it when this formulation is subjected to shear stresses, whether these are during the preparation of the formulation or during its consumption. It should be noted that this reduction in viscosity in the mouth also makes it possible to promote the release of the flavours present in the formulation.
The combined microfibrils used, in dry form, have very good properties as agents for bringing about an overrun state. Specifically, the introduction of the microfibrils makes it possible to improve the stability of the film separating the gas from the other ingredients of the formulation, and to give it good elasticity.
By way of illustration, food formulations comprising the dried combined microfibrils have high storage modulus Gxe2x80x2 values, for example of between 1500 and 4000 Pa, measured at a frequency of 1 Hz, Which are higher than those measured for formulations comprising xanthan gum or gelatin.
Thus, the combined microfibrils, in dry form, can be used legitimately as additives in formulations intended to be rendered in an overrun state, such as whipped creams, chantilly creams, toppings and ice-creams. The combined microfibrils can similarly be used in compositions such as mayonnaises, vegetable mousses, mousses comprising proteins, such as meat mousses, fish mousses, and mousses comprising albumin, such as meringues.
Besides the overrun properties, the combined microfibrils also have an action of controlling and inhibiting crystal growth. Now, this property is highly desired for foods which undergo temperature cycles (freezing, heating), such as ice-creams, or frozen foods. The reason for this is that it is essential for such formulations to be free, for as long as possible, of ice crystals which would give the said formulations an undesirable texture, such as the loss of a smooth constitution.
Moreover, the combined microfibrils give the formulations in which they are used a texture similar to that of formulations comprising the replaced additives.
Another advantage of the combined microfibrils used in the invention is that they can advantageously replace several compounds ordinarily used in food formulations intended to obtain overrun products. The formulations are thus all the more simplified.
Furthermore, combined cellulose microfibrils represent a means for effectively replacing animal gelatin, which is an additional advantage in terms of hygiene.
The combined cellulose microfibrils used according to the invention can similarly be used as additives in the formulations of vinaigrettes, fruit juices, vegetable juices, milk-based drinks and more particularly chocolate-flavoured milk-based drinks.
Specifically, combined microfibrils are good stabilizers and thickeners of emulsions and/or dispersions of this type.
In the specific case of chocolate-flavoured milk-based drinks, the dried combined cellulose microfibrils used according to the invention are not only compatible with milk-based media, but also make it possible to reduce the phenomenon of depletion (sedimentation of the chocolate) which appears in conventional formulations based on xanthan gum as stabilizer.
The combined cellulose microfibrils can also form a part of the composition of yoghurts, for example as viscosity modifiers. In an entirely advantageous manner, the combined microfibrils according to the invention do not prevent fermentation from taking place.
The viscosity-modifying properties and their xe2x80x9ccreamyxe2x80x9d consistency make combined cellulose microfibrils particularly suitable for incorporation into formulations for pastes for spreading and margarines.
In addition, and this represents an additional advantage, since they represent only a small supply of calories, they can legitimately, totally or partially replace fats, in order to prepare reduced-fat compositions.
It should be noted that the combined cellulose microfibrils do not impair the tastes of the foods into which they are introduced.
It has even been found that they have flavour-enhancing properties.
Concrete but non-limiting examples of the invention will now be given.
In the examples which follow, the flow viscosities were measured using a Brookfield RVT 20-2 viscometer, at room temperature.
The viscosities are expressed in mPa.s.
The storage modulus values were determined using a Carrimed CSL100 controlled-stress rheometer.
They were measured under a dynamic regime: oscillating regimexe2x80x94frequency from 0.01 to 10 Hz.
The deformation range (or storage plateau) is determined by carrying out coupled scanning at a given frequency and the range in which the moduli Gxe2x80x2 do not vary is determined.
The values are expressed in Pa.
The measurements of the degrees of overrun were carried out in the following manner:
Mousse is introduced into a beaker of known volume (v) and known mass, it is struck three times and is then levelled out.
The beaker is weighed to determine the mass of mousse it contains (m).
The degree of overrun corresponds to the following ratio: [m(g)/v(ml)xc3x97100.
The degree of overrun is expressed in %.

Use of cellulose microfibrils in dry form in food formulations


A can body loader assembly includes a turret starwheel having asymmetrical pockets and a vacuum belt that provides a precise pitch dimension between adjacent cans.

Beverage can bodies are produced in vast quantities. And the can bodies must be decorated in the same vast quantities in automated processes that are fast and reliable. A common type of can body decorator includes a large wheel that rotates the cans over printing blankets. Rotating decorators are commercially available from Stolle Machinery Company and from CMB Engineering Company, which is a sister company to the assignee of the present invention.
Digital printing of cans is under development. For example, a process disclosed in U.S. Pat. No. 9,770,922, entitled "Printing System and Method," uses inkjet printers at a printing station. A carriage moves an array of cans on a track, such as a Rockwell iTRAK™, and into and out of the printing station. The carriage includes mandrels that are inserted into the cans.


A shied formed by a pair of substantially C-shaped metal shield plates 10,10 that have substantially the same shape and dimensions. Contact parts 13 that contact the other shield plate are defined at both ends of each shield plate. The contact parts 13,13 are edge portions of both shield plates that are accommodated together and caused to contact each other inside grooves 7 that are formed in the end wall surfaces of the housing 2. Housing-engaging projections 13b and 13c are elastically engaged with the inside wall surfaces 7b,7b of grooves 7 at points above and below the contact point embossments 13a with the mating shield plate, so that the shield plates are clamped inside grooves 7.

Conventionally, certain shielded connectors have a long, slender rectangular insulating housing made of an 1.0 insulating synthetic resin, and are attached to a circuit board so that the connectors can receive a mating connector from a direction that is substantially perpendicular to the surface of the circuit board. The shield member is installed on the outside of the housing and has plate surfaces that are substantially parallel to the direction from which the mating connector is received. For example, see Japanese Patent Application Kokai No. 5-74522, Japanese Utility Model Application Kokai No. 6-54259 and Japanese Patent Application Kokai No. 5-21111.
However, conventional shielded connectors of this type have proved to be unsatisfactory in the following respects: i. e., the shielding characteristics are inadequate, a high degree of dimensional precision is required for assembly, and the manufacturing process is complicated.
For example, in the connector described in Japanese Patent Application Kokai No. 5-74522 mentioned above, two metal plates are installed as shield members on both sides of the housing; however, since there are no shield members on the ends of the housing, the shielding characteristics are inadequate. The connector described in Japanese Utility Model Application Kokai No. 6-54259 is equipped with a rectangular frame-form metal shield as a shield member, and therefore has good shielding characteristics; however, since the shield member is formed as a frame, a high degree of dimensional precision is required in order to attach this frame to the housing. In the connector described in Japanese Patent Application Kokai No. 5-21111, the housing is shielded by applying a conductive plating to the outside of the housing; accordingly, although this connector similarly has good shielding characteristics, the manufacturing process is complicated so that the cost of the connector is increased.
Thus, the object of the present invention is to provide a shield type connector which solves all of the problems described above.


A double-row angular-contact antifriction bearing including an outer bearing ring, and an inner bearing ring and rolling elements between the bearing rings in two rows next to one another on raceways of the bearing rings. A bearing cage for the rolling elements. The inner bearing ring is axially split and is of two individual rings which are connected and between which a distance ring defines axial prestress between the rolling elements of the both rows. The distance ring between the individual rings of the axially split inner ring is a connecting element of the individual rings.

DE 90 17 861 U1 discloses such an angular-contact antifriction bearing and establishes the generic type. This angular-contact antifriction bearing is designed as a double-row tapered roller bearing in O arrangement. It essentially comprises an outer bearing ring and an inner bearing ring and a number of tapered rollers which roll between the bearing rings in two rows next to one another on the raceways of the bearing rings and are kept apart at uniform distances by a respective bearing cage. In this case, the outer bearing ring is of one-piece design, whereas the inner bearing ring of the tapered roller bearing is axially split and comprised of two individual rings. With the fitting of the bearing in machines or the like, the individual rings are usually positioned relative to one another by an interference fit on a shaft in such a way that production-related tolerances during the production of the individual parts of the antifriction bearing are compensated for, and defined axial prestress is produced between the tapered rollers of both rows. This axial prestress enables the bearing to roll free of play to the greatest possible extent under load.
The measures for defining the degree of the axial prestressing of the individual rings of an axially split bearing ring relative to one another, which measures were known up to the time of the application of DE 90 17 861 U1 and are still customary today, were in this case either to grind down the end faces of the touching end faces of the individual rings in an appropriately dimensionally accurate manner or to grind down in a dimensionally accurate manner the end faces of a separate distance ring arranged between the individual rings and then to join together all the individual parts in an accurately fitting manner. However, since these measures only made it possible to compensate for the tolerances occurring during the production of the individual parts of the antifriction bearing and did not take into account the tolerance variations of the shaft to be equipped with the antifriction bearing, DE 90 17 861 U1 additionally proposed to arrange a distance ring, consisting of two ring segments, between the individual rings of the axially split bearing ring. In this case, one ring segment of this distance ring is determined in its width in such a way that it takes into account the tolerances during the production of the antifriction bearing, whereas the other ring segment is adapted in its width to the tolerance variations of the shaft.
In practice, however, it has been found that such compensation of tolerances or the achievement of defined axial prestress between the rolling elements by joining the individual rings of the axially split bearing ring to one another, both with and without an inserted distance ring and with a multi-piece distance ring by an interference fit on the shaft, involves considerable difficulties, since the high setting forces required for the interference fit not infrequently result in an elastic deformation of the individual rings of the axially split bearing ring up to their raceways, and the desired play between the rolling elements of the inner bearing ring is usually not reached due to this elastic deformation. As a result, the rolling elements are not prestressed in a defined manner but are adversely restrained against one another, so that jamming of the rolling elements on the ring flange of their raceway and subsequent increased wear of the bearing right up to failure of the bearing may occur. In this case, a basic cause of the usually imperfect prestressing of the bearing fitted on the shaft is the fact that, although the bearing manufacture can dimensionally produce all the individual parts of the bearing in such a way that the individual rings or the axially split bearing ring or the rolling elements of both rows in the fitted state of the bearing ought to theoretically have ideal axial prestress relative to one another, the rolling elements actually only rarely have such defined prestress, since the bearing is assembled by the user himself.
A conceivable improvement of this state would therefore be to already connect together the individual rings of the axially split bearing at the bearing manufacture by means of clamping rings of U-shaped cross section, as disclosed, for example, by DE 84 29 899 U1 or by DE 36 21 381 A1. However, such clamping rings, which are usually made of thin metal sheets or of plastic, are not suitable for producing or for maintaining defined axial prestress between the individual rings of the axially split bearing ring or between the rolling elements of both rows as they roll on their raceways.

Double-row angular-contact antifriction bearing


A data imprinting optical system that records a date, data, etc. on a predetermined surface, e.g. a film in a camera, uses a diffractive optical element to achieve savings in space and a reduction in cost. The data imprinting optical system has a display device (1a) and imprints an image displayed on the display device (1a) onto a predetermined surface (1c). The optical system has a diffractive optical element (1b). The diffractive optical element (1b) may have a plurality of different image-formation points to enable a plurality of data imprinting modes. The diffractive optical element (1b) may be integral with a part of another optical system, e.g. a photographic optical system.

The present invention relates to a data imprinting optical system and, more particularly, to a data imprinting optical system capable of imprinting date and other information on the surface of a film used in a camera, for example.
A conventional data imprinting optical system is disposed behind a film. Because the conventional arrangement causes the camera body to become undesirably thick, there have recently been proposed data imprinting optical systems designed to imprint data from the front side of the film.
For example, Japanese Patent Application Unexamined Publication Number hereinafter referred to as "JP(A)"! 4-256918 proposes a retrofocus type data imprinting optical system comprising two lenses. JP(A) 7-104370 proposes a data imprinting optical system wherein an optical axis is bent by a prism. Recently, data imprinting optical systems adapted for panoramic photography have also been proposed in which the image frame size is changed by cutting upper and lower end portions of the film. For example, in JP(A) 6-35061, two lenses are arranged in parallel to each other so as to be selectively used for ordinary photography and panoramic photography. In JP(A) 7-295066, two prisms are arranged in parallel to each other so as to be selectively used for ordinary photography and panoramic photography. In JP(A) 7-287295, a lens is inserted to cause a magnification to change between ordinary photography and panoramic photography.
To provide a data imprinting optical system in front of a film in a camera, it must be disposed in a photographic lens, a finder optical system, an autofocus system, etc. This hinders achievement of further reductions in the size and cost.
In JP(A) 4-256918, the back focus of an objective lens is lengthened to increase the degree of freedom with which a data imprinting optical system can be disposed in a camera or the like. However, the number of constituent members increases, which is disadvantageous from the viewpoint of cost.
In JP(A) 7-104370, the optical axis is bent by a prism to increase the degree of freedom with which a data imprinting optical system can be disposed in a camera or the like. However, the constituent member is larger in size than a lens, which is disadvantageous from the viewpoint of space.
Recently, there has also been proposed a data imprinting optical system that imprints data on a plurality of positions as in panoramic photographs, for example. In this case, extra members and space are needed to effect imprinting on a plurality of positions. JP(A) 6-35061 uses two lenses and hence causes the number of constituent members to increase and also causes the required space to increase, undesirably. JP(A) 7-295066 uses two prisms; therefore, the number of constituent members increases, and the required space also undesirably increases. JP(A) 7-287295 proposes a data imprinting optical system improved in terms of space in comparison to the above two conventional techniques. However, the problem of the increased number of constituent members still remains unsolved.


A method and apparatus for hardware input devices provides context sensitive labels which can change according to an application running on a computer and/or the function assigned to an input element. An illustrative implementation of the method and apparatus includes employing ink having two or more electroluminescent pattern layers with an input device. Based on the context of a computer associated with the input device, an illuminated pattern identifies the current functionality of one or more of the inputs of the input device.

In today's computer systems, multiple applications with varying functionality are available to a user. Some of the functionality and functions found in one application are not found in every other application. In the early days of the personal computer, control and function keys were added to the standard QWERTY keyboard to provide additional functionality. The function keys, typically arranged in a line across the top of the keyboard, could be assigned specific commands by the current application or the operating system. Control keys provided cursor and screen control. The control keys allowed the user to make large jumps in most applications. Common control keys include: Home, End, Insert, Delete, Page Up, Page Down, Control (Ctrl), Alternate (Alt), and Escape (Esc).
As more and more functionality and applications have come on line, further keys have been added to keyboards. Some keyboards adapted to operate with the Microsoft Windows® operating system include some extra control keys: two Windows® or Start Keys and an Application Key. As more keys have been added to a keyboard, it has become increasingly difficult for a user to remember the specific functionality assigned to each key or combination of keys, much less find the keys providing a particular functionality. This can be particularly confusing when the functionality of a key changes from one application to another.
The functionality of a key and a key combination has been placed on the keys themselves in the form of text labels or graphic symbols. In some applications, such as Microsoft Word®, functionality associated with certain key combinations is not identified on the keys themselves. With those key combinations, to determine their functionality a user can press the key combination or through the Tools menu access a customize tool, followed by selecting keyboard. By navigating through a list of functions, a user can see which function is assigned to what key or key combination. Also, a user may assign a shortcut for a function to a key or key combination, so-called "hot keys", resulting in a user-customizable keyboard. The user may set the function assigned to a hot key such that subsequent pressing of the hot key causes the desired function to be performed. Not surprisingly, many users do not take advantage of conventional hot keys because it is inconvenient to assign the functions to the keys, or because they may not even appreciate that such a capability exists. Even when hot keys are programmed, the user may have difficulty in recalling which function is assigned to each hot key. This is especially true when there are multiple hot keys on a keyboard.
Another prior art solution involves providing flip chart above keys as labels to identify the functions assigned to keys or key combinations for different applications.
Generally speaking, a keyboard has a limited number of keys for receiving user input commands. To account for the limited number of keys, certain keystroke commands can perform different functions from one application to another. However, it can be difficult for the user to remember which keys have what functionality at any given time and requiring a user to search through menus in the application to determine the functionality of various key and key combinations can be cumbersome. Consequently, there is a need to make it simpler for a user to understand the functionality for key input commands in a respective application.
Another consideration in improving a user's understanding of various key input commands is to do so without a substantial increase in cost of the keyboard. With input devices such as a keyboard, there is a need to provide an input device that is flexible, inexpensive and can be utilized with relative ease with applications supported by a computer.

This patent is subject to a terminal disclaimer.

Context sensitive labels for a hardware input device


A balance material is applied to the inner surface of a tire at one or more selected locations to offset an imbalance of the tire. The balance material is fluently applied to the inner surface at a first viscosity, whereafter the balance material transforms to exhibit a second viscosity that is higher than the first viscosity and remains affixed to the inner surface of the tire at the point of application. A method of balancing a tire comprises determining the location and amount of balance material needed to correct an imbalance of the tire, applying the balance material at a first viscosity, and transforming the balance material to have a second viscosity sufficient to maintain the balance material at the desired location.

Pneumatic tires have found many applications in a variety of vehicles, such as passenger automobiles, aircraft and industrial machinery. It is well known that a small amount of imbalance in a pneumatic tire mounted to a vehicle can cause undesirable vibration and noise when the tire is rotating at the operating speeds of the vehicle. This imbalance can be attributed to non-uniformities or imperfections in the tire wheel rim, or to imperfections or non-uniformities in the molded tire itself. Such imbalance is particularly noticeable on tires used on aircraft landing gear, due to the high wheel speeds attained during take-off and landing of the aircraft.
One approach to eliminating or reducing the effects of imbalance in formed tires has been to secure discrete patches of rubber material to the tire inner liner in an effort to offset the imbalance. This approach requires determining the amount and location of imbalance of the formed tire, typically in a dynamic testing machine. Based upon the results of the imbalance testing, a determination is made as to the location and mass of the patch material needed to counteract the imbalance of the tire. The inner surface of the tire is thereafter cleaned at the appropriate location, typically using an organic solvent, and the patch is adhered at the desired location. Rubber patches commonly used for balancing tires are generally provided in fixed weight increments, thereby limiting the precision to which the imbalance may be corrected, and requiring users to maintain an inventory of patches in various sizes.
The present invention seeks to provide a balance material that can be added to the inner liner of pneumatic tires to more easily and more precisely correct tire imbalance and to eliminate the need for maintaining an extensive inventory of balance patches.


Conventionally gas turbine engine compressors have fixed inlet guide vanes to give a predetermined swirl to incoming air at an engine design speed, so that air enters the compressor at an optimum angle. At speeds much lower, for example, than the design speed the guide vane is less efficient. The guide vanes are thereby made variable in camber. Each vane comprises a number of spanwise hinged members which can be moved relative one to another to vary the vane camber.

It is common for current axial flow gas turbine engines to employ variable stator vanes to vary the flow characteristics of, in particular, the compressor.
For example, the inlet guide vanes are used to widen the design performance of the compressor by changing the air swirl velocity component of inlet air to match the speed of the compressor. This variation of swirl can be achieved by mounting the inlet guide vanes on simple pivots and regulating the angular setting of the vanes in accordance with the compressor speed.
However, if the whole vane is turned the leading edge incidence ceases to be optimal causing significant losses and some of the potential benefits are lost. It is often the case that the swirl angle of air entering the front face of the stator ring is substantially constant. In the case of the first stage fan inlet guide vanes the angle may even be zero, i.e. air flow is virtually wholly axial. The leading edge portion of the guide vane is, therefore, preferably fixed.
Variable camber guide vanes with fixed leading edge incidence are known from GB Patent No 736,796 Rolls-Royce Ltd in which the stator blades are divided longitudinally into a fixed leading edge portion and a relatively pivotable trailing edge for imparting the adjustable swirl characteristics. The trailing part of the vanes are pivotally mounted in the outer engine casing structure, and may be mounted also in the inner stator structure.
The angularly adjustable parts of the whole stator ring are preferably interconnected for simultaneous angular adjustment by means of a coupling ring attached to radius arms carried by spindles which extend from each vane.
A similar type of arrangement is also know from GB Patent No 774,501 in the name of PowerJets (Research and Development) Limited. In this the stator ring is located in the turbine entry and the fixed leading edge part receives substantially axially flowing hot gas from a combustor outlet.
A problem inherent in this type of arrangement arises from an abrupt transition in the airfoil surfaces of the vane at the junction between the fixed and pivoted parts of the structure which precipitate breakaway of the airflow. The present invention attempts to solve this problem by providing a variable camber vane in which the full turning effect is achieved more progressively.


The Gutter Cleaner is a plastic and steel combination, in which the handle is a hard plastic and the U shaped rod is made of steel. The dipper is made of a hard plastic. The plastic is molded around the steel rod. The handle has threaded female grooves inside the handle for the extension to be screwed into. The dipper has three slotted holes for the release of the water when removing out the leaves or debris.

The present invention relates to a Gutter Cleaner for removing of leaves and debris from gutters without using a ladder. Gutter Cleaner is a plastic and steel combination, in which the handle is a hard plastic and the U shaped rod is made of steel. The dipper is made of a hard plastic, in which the plastic is molded around the steel rod. The handle has threaded female grooves inside the handle for the extension to be screwed into. The dipper has three slotted holes for the release of the water dipping out the leaves from the gutter. This Gutter Cleaner is used for the cleaning of one story and two story homes by standing from the ground, by which inserting a extendible handle into the Gutter Cleaner.


A method and composition for treating liquefied petroleum gas containing acid gases such as H.sub.2 S, CO.sub.2, and COS to sweeten such liquefied petroleum gas by removal of a substantial portion of such acid gases while minimizing losses of amines due to solubility in LPG and enhancing CO.sub.2 slip, said method comprising contacting said liquefied petroleum gas with an absorbent mixture comprising an aqueous solution of TEA and at least another amine preferably selected from the group consisting of MEA, DEA, MDEA, DIPA, and mixtures thereof.

Petroleum gas often contains a variety of acidic, gaseous contaminants, of which the principal ones are hydrogen sulfide, mercaptans and other diverse sulfur compounds, carbon dioxide, and carbonyl sulfide (COS). It is well known in the gas treating industry that such contaminants can be successfully removed by contacting the gas with aqueous solutions of one or more amines, which may be either selective or non-selective in their ability to absorb various of the acid gases. After such absorption, the acidic compounds are stripped from the amines and the amines are returned to the system, except to the extent they may have been lost in the process. It has been theorized that many different amines would provide some level of utility for removal of acid gases, but as a practical matter, the amines actually in commercial use are monoethanolamine (MEA), diethanolamine (DEA), methyldiethanolamine (MDEA), and diisopropanolamine (DIPA). Triethanolamine (TEA) is also frequently disclosed in the art as useful in gas treatment, but its actual commercial use appears to be very limited to non-existent. Use of MDEA/DIPA mixtures has also been reported (U.S. Pat. No. 4,808,765) for the purpose of removing H.sub.2 S and COS from liquefied petroleum gas (LPG). More specifically, U.S. Pat. No. 4,808,765 teaches that MDEA, a selective H.sub.2 S absorbant, may be formulated with DIPA, a COS absorbent, to reduce amine losses due to solubility in LPG. This patent also teaches that MDEA is less soluble than MEA or DEA in liquid hydrocarbons.
Treatment of LPG presents particular problems in that amines tend to be significantly soluble in the LPG, leading to a corresponding economic penalty due to the need to make up the lost amine(s). Many refineries use aqueous DIPA or MDEA to remove the acidic impurities from LPG; however, the concentration of these amines is typically limited to the range of about 20-35 weight percent of the aqueous stream in which they are supplied to the process. Operation at higher concentrations, which is desirable for capacity reasons, generally results in undesirably high levels of LPG contamination with amine(s). The problem is particularly acute at refineries treating cracked (i.e., highly unsaturated) LPG. Often, the loss rate of MDEA is sufficient to negate the economic justification for substituting MDEA for DEA. In addition to the high amine replacement costs, specialized remediation equipment is required, which increases the financial burden. Moreover, failure to remove dissolved MDEA can negatively affect downstream processes, e.g., poisoning of alkylation catalyst beds, and the like.
It would be highly desirable to have an amine composition which maximizes the effective amine concentration circulating in the LPG system, while yet minimizes the amount of amine(s) lost due to solubility in the LPG and increases desirable CO.sub.2 slip.

Method for sweetening of liquid petroleum gas by contacting with tea and another amine

Union Carbide Chemicals & Plastics Technology Corporation


The present invention provides compositions and methods for the culture and maintenance of cancer stem cells. More particularly, the present invention provides the identification of cancer stem cell specific markers and methods of recognizing the same for the detection of tumors, for facilitating the prognosis of a patient with a tumor, and for the treatment of various cancers. The invention also provides antibodies that specifically recognize the disulfide linked Erbb2Δ16 homodimer, an Erbb2Δ16/Erbb3 heterodimer, or post-translational modifications of Erbb2 that are specific to Erbb2 of variant hESCs. In addition, the invention provides a modified defined media useful in the absence of a feeder layer and in the absence of serum or serum replacement, that comprises a basal salt nutrient solution, bFGF, IGF-I, and Activin A, and wherein the composition does not comprise heregulin. The invention further relates to the use of an Erbb2 variant isoform to generate robust cell cultures that are independent of heregulin.

1. Field of the Invention
The present invention generally relates to compositions and methods for culturing pluripotent stem cells and/or primary tumor cells comprising an Erbb2 variant, the cells created by these methods, and the uses thereof. Particularly, the invention relates to the identification of cancer stem cell specific markers. In addition, the invention relates to the use of a modified defined culture medium for producing cultures of cancer stem cells in the absence of a feeder cell layer, in the absence of serum or serum replacement, and independent of heregulin.
2. Background Art
The process of embryonic development establishes the differentiated lineages of the body and sets aside tissue specific progenitor cells, which are also called stem cells. These progenitor cells are capable of regenerating all the relevant lineages of individual tissues during normal cellular turnover, or after injury. Examples of this regeneration include the constant regeneration of the skin, and reconstitution of the hematopoetic system following transplantation of hematopoetic stem cells. These progenitor cells typically reside in a "stem cell niche" and are relatively long lived as compared to their differentiated progeny. Embryonic stem (ES) cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation of mammals and resultant commercial, medical, and agricultural applications. Furthermore, appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells suited to study cell differentiation and/or suited for transplantation for treatment of diseases that result from cell damage or dysfunction.
Accumulated genetic change leading to unregulated cell growth is a hallmark of cancer progression. Because of their longevity, tissue specific stem cells may have a greater chance of accumulating mutations than differentiated cells, which exhibit comparatively rapid turnover in many tissues. Because tissue specific stem cells are the progenitor cells of other cells within that tissue, deregulation of the balance between quiescence, self-renewal, differentiation, or apoptosis of the tissue specific stem cells can have severe consequences. Any increase in the proliferation of these cells could lead to a magnified over-proliferation of downstream cell populations, leading to tumorigenesis. Furthermore, should mutations enable maintenance or expansion of these progenitor cells, self-perpetuating "cancer stem cells" (CSC) could be generated within tumors. It has long been known that a relatively small proportion of cells within differentiated tumors have the capacity to regenerate tumors at high frequency after transplantation (See, e.g., U.S. Pat. No. 6,984,522). This is indicative of rare populations of transformed cells that have the capacity to expand and differentiate to all lineages of the tumor. There is a need in the art for a natural model system in which to study the development of and signaling within a cancer stem cell population.
Human embryonic stem cells (hESCs) are pluripotent cells that can be isolated from the human blastocyst and maintained in culture as self-renewing cells, or differentiated to representatives of all mature tissues in the body. hESCs may therefore be a valuable model of human development in vitro, and are the focus of substantial research aimed at generating differentiated populations for cellular therapies. hESCs are "master" stem cells and theoretically capable of differentiating to all lineages, including tissue specific progenitor cells. Because hESCs are a self-renewing progenitor population, they are likely to use growth factors and molecular signaling pathways that are overlapping with some tissue specific stem cells and their related CSCs.
The successful isolation, long-term clonal maintenance, genetic manipulation, and germ-line transmission of pluripotent cells has generally been difficult, and the biochemical mechanisms regulating ES cell pluripotency and differentiation are very poorly understood. However, the limited empirical data available and much anecdotal evidence suggest that the continued maintenance of pluripotent ES cells under in vitro culture conditions is dependent upon the presence of cytokines and growth factors present in the extracellular milieu. Until recently, the standard growth conditions for hESCs were relatively undefined. The inventors recently developed a simple media for HESC growth that has several advantages over other reported defined media. The media relies on growth factor signaling through the EGF receptor family of cell surface proteins to maintain hESC pluripotency. This signaling is primarily transmitted through the Erbb2 receptor, a strong activator of the PI3 kinase pathway when present as heterodimers with other Erbb receptor family members. Four growth factors (bFGF, TGF-I, Activin A, and Heregulin) were required to inhibit spontaneous differentiation and promote self renewal of hESCs in this defined media. This system enabled the robust expansion of hFSCs and facilitated the examination of such cells in a standardized and simple background.
Neuregulin-1 (NRG1; heregulin) is a large gene that exhibits multiple splicing and protein processing variants. This generates a large number of protein isoforms, which are referred to herein collectively as neuregulin. Neuregulin is predominantly expressed as a cell surface transmembrane protein. The extracellular region contains an immunoglobulin-like domain, a carbohydrate modified region and the EGF domain. NRG1 expression isoforms have been reviewed previously (Falls, 2003, Exp. Cell Res., 284:14-30). The cell membrane metalloproteases ADAM17 and ADAM19 have been shown to process the transmembrane form(s) of neuregulin-1 to soluble neuregulin/heregulin. HRG-α and -β are the cleaved ectodomains of neuregulin, containing the EGF and other domains. As the EGF domain is responsible for binding and activation of the Erbb receptors, a recombinant molecule containing only this domain can exhibit essentially all of the soluble growth factor effects of this protein (Jones et al., 1999, FEBS Lett., 447:227-231). Also, there are processed transmembrane isoforms of neuregulin that are thought to trigger juxtacrine signaling in adjacent cells via interaction of the EGF domain with Erbb receptors.
The EGF growth factor family has at least 14 members, including, but not limited to, EGF, TGFα, heparin binding-EGF (hb-EGF), neuregulin-β (also named heregulin-β (HRG-β), glial growth factor and others), HRG-α, amphiregulin, betacellulin, and epiregulin. All these growth factors contain an EGF domain and are typically first expressed as transmembrane proteins that are processed by metalloproteinase (specifically, ADAMs) proteins to generate soluble ectodomain growth factors. EGF family members interact with both homo- and hetero-dimers of the Erbb1, 2, 3, and 4 cell surface receptors with different affinities (Jones et at, 1999, FEBS Lett, 447:227-231). EGF, TGFα, and hbEGF bind Erbb1/1 (EGFR) homodimers and Erbb1/2 heterodimers at high affinity (1-100 nM range), whereas HRG-β binds Erbb2/3 and Erbb2/4 heterodimers at very high affinity (<1 nM range). Activated Erbb receptors signal through the PI3 Kinase/AKT pathway, the MAPK pathway, and several other pathways (Oda et al., 2005, Mol. Sys. Biol., 1:2005.0010. Epub May 25).
Erbb2 and Erbb3 are amongst the most highly expressed growth factor receptors in hESCs (Sperger et al., 2003, PNAS, 100(23):13350-13355), and HRG-β has been shown previously to support the expansion of mouse primordial germ cells (Toyoda-Ohno et al., 1999, Dev. Biol., 215(2):399-406). Furthermore, over-expression and subsequent inappropriate activation of Erbb2 is associated with tumorigenesis (Neve et al., 2001, Ann. Oncol., 12 Suppl 1:S9-13; Zhou & Hung, 2003, Semin. Oncol., 30(5 Suppl 16):38-48; Yarden, 2001, Oncology, 61 Suppl 2:1-13). Human Erbb2 (Chromosome 17q between positions 11.2 and 12), and Erbb3 (Chromosome 12q13) are present on chromosomes that have been observed to accumulate as trisomies in some hESCs (Draper et al., 2004, Nat. Biotechnol., 22(1):53-4; Cowan et al., 2004, N Engl. J. Med., 350(13):1353-6; Brimble et al., 2004, Stem Cells Dev., 13(6):585-97; Maitra et al., 2005, Nat. Genet., 37(10):1099-103; Mitalipova et al., 2005, Nat. Biotechnol., 23(1):19-20; Draper et al., 2004, Stem Cells Dev., 13(4):325-36; Ludwig et al., 2006, Nature Biotechnol., 24(2):185-87), possibly suggesting that over-expression and/or activation of these receptors could be associated with the purported growth/survival advantage conferred by trisomies of these chromosomes.
The proto-oncogene Erbb2 is known in the art by several additional names, including, among others, human epidermal growth factor receptor 2 (HER2), C-erbB-2, ERB2_HUMAN, Her-2/neu, MLN 19, NEU, NEU proto-oncogene, NGL, Oncogene NGL, neuroblastoma- or glioblastoma-derived, p185erbB2, TKR1, Tyrosine kinase-type cell surface receptor HER2, V-Erb-B2, and Oncogene Erbb2. Various groups identified the genes and/or mapped the location to the long arm q of Chromosome 17 (Coussens et al., 1985, Science, 230:1132-1139; Semba et al., 1985, Proc. Nat. Acad. Sci., 82:6497-6501; Yang-Feng et al., 1985, Cytogenet. Cell Genet., 40:784; Di Fiore et al., 1987, Science, 237:178-182; Fukushige et al., 1986, Mol. Cell. Biol., 6:955-958; Kaneko et al., 1987, Jpn. J. Cancer Res., 78:16-19; Popescu et alt, 1989, Genomics, 4:362-366; Anderson et al., 1993, Genomics, 17:618-623; Muleris et al., 1997, Cytogenet. Cell Genet., 76:34-35). The Erbb2 gene consists of 27 exons, and the mRNA is approximately 3,768 nucleotides. Akiyama et al. (1986, Science 232:1644-46) raised antibodies against a synthetic peptide corresponding to 14 amino acid residues at the COOH terminus of the predicted Erbb2 protein, and they immunoprecipitated an 185-kD Erbb2 glycoprotein with tyrosine kinase activity from adenocarcinoma cells. The Erbb2 protein consists of 1255 amino acids. Erbb2 is a tyrosine kinase with a single transmembrane domain that separates an intracellular kinase domain from an extracellular domain. Erbb2 protein is expressed in several human organs and tissues, including normal epithelium, endometrium, ovarian epithelium, prostate, pancreas, lung, kidney, liver, heart, and hematopoietic cells. Erbb2 plays a role in normal development and differentiation.
Several researchers have identified the over-expression of Erbb2 as being involved in various cancers and have analyzed Erbb2's role as an oncogene. For example, increased expression of Erbb2 was noted in a human adenocarcinoma of the salivary gland (Semba et al., 1985, Proc. Nat. Acad. Sci., 82:6497-6501), a gastric cancer cell line (Fukushige et al., 1986, Mol. Cell. Biol., 6:955-958), a large-cell, comedo growth type of ductal carcinoma (Van de Vijver et al., 1988, New Eng. J. Med., 319:1239-1245), breast and ovarian cancer (Slamon et al., 1989, Science, 244:707-71 2; Yu et al., 1998, Mol. Cell, 2:581-591; Kun et al., 2003, Hum. Mol. Genet. 12:3245-3258; Menendez et al., 2004, Proc. Nat. Acad. Sci., 101:10715-10720), prostate cancer (Qiu et al., 1998, Nature, 393:83-85), acute lymphoblastic leukemia, bladder cancer, cervical cancer, childhood medulloblastoma, colorectal cancer, oral squamous cell carcinoma, germ-cell testicular cancer, cholangiocarcinoma, lung cancer, osteosarcoma, pancreatic adenocarcinoma, primary fallopian tube carcinoma, and synovial sarcoma. Di Fiore et al. (1987, Science, 237:178-182) demonstrated that over-expression alone can convert the Erbb2 gene into an oncogene. Other researchers have suggested that levels of Erbb2 expression could be used in determining the prognosis and/or chemosensitivity of human cancers, especially breast and ovarian cancer (Pegram et al., 1997, Oncogene, 15:537-547; Mehta et al., 1998, Oncol., 16:2409-2416; De Placido et al., 1998, Breast Cancer Res. Treat., 52:55-64). Erbb2 over-expression has been reported in 30-50% of ovarian carcinomas and is associated with advanced disease stage, worse prognosis, and decreased response to therapy in ovarian carcinoma patients; however, the molecular mechanisms underlying Erbb2 oncogenic activities in human cancer are unclear. Erbb2 over-expression is thought to be the mechanism of Erbb2 activation in certain cancers.
Other researchers have attempted to elucidate the function and activity of Erbb2. Qiu et al. (1998, Nature, 393:83-85) showed that Erbb2 forms a complex with the gp130 subunit of the IL6 receptor (IL6R) in an IL6-dependent manner and that Erbb2 is a critical component of IL6 signaling through the MAP kinase pathway. Yu et al. (1998, Mol. Cell, 2:581-591) found that over-expression of Erbb2 inhibits Taxol-induced apoptosis in breast cancers. The resistance to taxol-induced apoptosis is thought to be through the inhibition of p34 (CDC2) activation, via Erbb2-mediated upregulation of p21(CIP1), or CDKN1A, which inhibits CDC2. Tan et al. (2002, Mol. Cell, 9:993-1004) reported that the inhibitory phosphorylation on tyr15 (Y15) of CDC2 was elevated in Erbb2-overexpressing breast cancer cells and primary tumors, and concluded that Erbb2 can confer resistance to taxol-induced apoptosis by directly phosphorylating CDC2. In addition, Menendez et al. (2004, Proc. Nat. Acad. Sci., 101:10715-10720) identified a molecular link between the biosynthetic enzyme fatty acid synthase (FASN), which is associated with more aggressive breast and ovarian cancers, and the Erbb2 oncogene. Pharmacologic and RNAi FASN inhibitors were found to suppress Erbb2 expression and tyrosine kinase activity in breast and ovarian cancers over-expressing Erbb2.
Certain mutations and polymorphisms in the Erbb2 protein have been identified as having an association with breast cancer, adenocarcinoma, glioblastoma, gastric cancer, and ovarian carcinoma (die et al., 2000, J. Nat. Cancer Inst., 92:412-417; The Cancer Genome Project and Collaborative Group, 2004). Alternative splicing results in several additional transcript variants, some encoding different isoforms and others that have not been fully characterized. Doherty et al. (1999, Proc. Nat. Acad. Sci., 96:10869-10874) described a secreted protein of approximately 68 kDa, designated herstatin, as the product of an alternative Erbb2 transcript that retains intron 8. Herstatin appears to be an inhibitor of p185Erbb2, because it disrupts dimers, reduces tyrosine phosphorylation of p185, and inhibits the anchorage-independent growth of transformed cells that over-express Erbb2.
Another Erbb2 splice variant (referred to here as Erbb2Δ16) has been identified which harbors a deletion of exon 16, resulting in a 16 amino acid in-frame deletion in a small extracellular region of wild type Erbb2 (Siegel et al., 1999, EMBO J., 18(8):2149-64; Castiglioni et al., 2006, Endocr. Relat. Cancer, 13(1):221-32). Erbb2Δ16 has been implicated as an oncogeneic isoform of Erbb2. Exon 16 also contains a cleavage site for ectodomain shedding of wild type Erbb2 (Yuan et al., 2003, Prot. Exp. Pur., 29:217:222), and shedding may, therefore, be substantially altered in Erbb2Δ16. Exon 16 also contains two cysteine residues that are usually involved in disulfide bonds with other regions of the molecule, and Erbb2Δ16 may therefore have a different structure than wild type Erbb2. Disulfide linked homodimers and/or heterodimers of Erbb2Δ16 may form in cells transfected with an Erbb2Δ16 expression construct. The region of the deletion in Erbb2Δ16 is also a hotspot for mutations in Erbb2 that lead to breast cancer in mouse and rat models. Some researchers have reported that this oncogenic Erbb2 splice variant has been detected in several breast cancer cell lines, but was not over-expressed in breast tumors.
Brumlik et al. (Poster, 2003, Faseb Summer Research Conference: Growth Factor Receptor Tyrosine Kinases in Mitogenesis. Omni-Tucson, Tucson, Ariz.) developed an RT-PCR assay to quantitate Erbb2Δ16 expression and have generated preliminary data that identified over-expression of Erbb2Δ16 in ovarian and breast cancer cell lines and primary ovarian tumors from patients with advanced disease, suggesting that Erbb2Δ16 may in fact contribute to disease progression and would therefore represent a viable target for therapeutic intervention of ovarian cancer. In addition, this group targeted a unique sequence at the Erbb2Δ16 exon 15 and exon 17 junction for suppression by RNA interference (RNAi) and demonstrated the efficacy and specificity of RNAi to suppress Erbb2Δ16 expression in ovarian cancer cells.
Similarly, Castiglioni et al. (2006, Endocrine Related Cancer, 13:221-232) have reported that the Erbb2Δ16 splice variant represents about 9% of the Erbb2 mRNA obtained from most of the 46 breast carcinoma samples in that study. They found that human cells transfected with wild type Erbb2 cDNA revealed no growth of wild type cells in nude mice, whereas clones expressing 10-fold less Erbb2Δ16 were tumorigenic. In addition, they noted that unlike wild type Erbb2-transfectants, Erbb2Δ16-expressing cells showed low sensitivity to two therapeutic drugs targeting receptors of the HER family (ZD1839 and Trastuzumab (Herceptin®), whereas an inhibitor of the HER2 tyrosine kinase domain (Emodin) blocked activation of both Erbb2Δ16 and wild type Erbb2 transfectants. They suggested that their data indicate that the Erbb2Δ16 transcript encodes the transforming form of the oncoprotein and that malignant transformation arises when a critical threshold of Erbb2Δ16 is reached in Erbb2 over-expressing tumors.
Therapies directed toward Erbb2 are currently being used effectively in breast cancer patients; however, these treatments have significant drawbacks. In particular, a humanized anti-Erbb2 monoclonal antibody, Herceptin® (Genentech), has been shown to be effective in slowing the progression of approximately 30% of breast cancers, demonstrating the role of this receptor in tumor growth. Slamon et al. (2001, Science, 244:707-712) found in a large-scale clinical trial that treatment with Herceptin® increased the clinical benefit of first-line chemotherapy in metastatic breast cancer that over-expresses Erbb2. Although Hercepti® demonstrated clinical efficacy, a significant number of women suffered from the severe side effect of treatment-induced cardiotoxicity.
In addition, small molecule inhibitors of the Erbb2 tyrosine kinase (TK) domain have been shown to effectively inhibit proliferation of breast cancer cells in culture and in animal models of tumor growth. The inventors also have shown that three different Erbb2 TK inhibitors, AG879, AG825, and emodin, inhibit the growth of BG01v cells in defined media, in the absence of an exogenous Erbb2 activating growth factor. This confirms that endogenous Erbb2 signaling is critical for the self-renewal of BG01v cells and represents a major difference between variant and normal hESCs.
What is needed in the art are model systems for studying the transformation of tissue progenitor cells to cancer stem cells and the elucidation of media and cell culture conditions that are capable of effecting such transformation. Also needed are new methods for the specific targeted treatment of breast cancer and other tumors. There is a need, therefore, to identify methods and compositions for the culture of a population of cancer stem cells that are able to be used for research purposes to study tumorigenesis. There is also a need to identify markers that are specific to the cancer stem cells that may be used as targets for therapeutic treatments of cancer and that may be used to facilitate the prognosis of patients with various tumors.

Human cancer stem cell culture compositions comprising Erbb2 variants and methods of use thereof


A light curing structure and related methods are disclosed wherein a framework defining an interior space for containing an object having applied thereon a light-cured material is provided, and means associated with the framework for exposing the object to UV radiation from natural sunlight is used to cure the light-cured material. Preferred embodiments include a booth for applying and curing light-cured materials comprising a housing having a ceiling and walls that define an enclosed interior space for holding an object, means for applying a light-cured material to the object; means for preventing the exposure of the light-cured material to UV radiation during application of the light-cured material to the object, and means for exposing the light-cured material to UV radiation from natural sunlight after application of the light-cured material to the object. Apparatus and methods according to the invention are particularly suited for use with UV-cured paints in the automotive industry.

Most metallic and many plastic surfaces of useful articles are painted to prevent weathering and corrosion and for decorative reasons. One common way to paint a large object, e.g. an automobile or automobile part, is by using a housing or booth in which the paint is sprayed on the object. The object then usually remains in the booth while the paint is cured by heating or other means. Such paint spray booths are commonly prefabricated building structures having a ceiling, floor and walls that define an interior space large enough to hold the object to be painted, with sufficient room on all sides for workmen to effectively operate paint spray and other equipment. These booths provide advantages such as reducing particulates, confining paint overspray and evaporated solvents and reducing drying times.
One common type of paint spray booth is the downdraft and semi-vertical spray booth that uses a housing positioned over an open floor grate or an exhaust outlet near the bottom of the walls. Air from the ceiling and any entrained paint overspray and solvents are drawn downward over the vehicle during spraying and drying and are then exhausted through the floor grate or exhaust opening. One example of such a spray booth is described in U.S. Pat. No. 6,533,654, incorporated herein by reference. Curing of the paint applied to the object is usually accomplished by using heaters to increase the temperature within the booth. The paint curing time in conventional paint spray booths may take about one hour.
Materials that are cured (hardened) by exposure to light have recently shown promise in a variety of industries because such materials have very short cure times without the application of heat, and have several advantages in speed and ease of use. Some of the general benefits of light-cured materials include rapid curing times (in some cases almost instantaneous, allowing for immediate further processing), on demand curing (requiring only exposure to UV light), no solvents (100% solids—no environmental pollution due to solvent evaporation), no heat (low thermal stressing of substrate materials), single component systems (ready-to-use with no mixing, no waste, no cleaning of mixing containers, no problems with pot life or mixed materials, no dangerous isocyanate catalysts), and more efficient use of raw materials and energy. Examples of light-cured materials include structural adhesives for bonding glass, ceramics, ferrites, plastics and metals; UV hot melt pressure sensitive adhesives; UV glob tops, chip coats and conformal coatings; UV potting compounds and encapsulants; UV paints, inks and coatings; and UV hardening polyester resin/glass fiber composite materials.
The use of light-cured materials can be found in an extremely wide variety of industries such as, for example, the electronics, printing, furniture, floor covering, medical device, dental, packaging, marine and paper industries. The use of light-cured paints and coatings in the automotive refinish and repair markets has grown considerably in recent years, but to date has typically been limited to primer applications. However, it is likely that light-cured products will be expanded to possibly include basecoats, topcoats, fillers and glazes, adhesives and sealers.
Light-cured materials contain chemicals known as photo initiators that comprise certain reactive groups, e.g. acrylate or methacrylate groups, which are sensitive to UV radiation. When photo initiators are illuminated with intense UV light, they initiate a polymerization reaction that hardens the material. If a light-cured material is used as a paint or other coating to cover an object, the hardened material provides a protective finished surface, or one that is ready for sanding and application of other coats in a short period of time, e.g. in about 2-5 minutes.
Curing of light-cured materials currently requires the use of a UV lamp. These UV lamps typically use a relatively small high-intensity discharge (HID) quartz bulb that is filled with mercury and traces of other elements. A high voltage applied to electrodes in the lamp creates an arc that heats the gaseous atoms to the point at which they emit UV light, visible light and infrared light. Full intensity is usually reached within about 2-5 minutes. The bulb is positioned inside a reflector that collects and focuses the light toward the lamp opening, which is typically designed to filter out more-harmful UV-B and UV-C radiation and allow only UV-A radiation to be emitted by the lamp.
UV-curing lamps have certain disadvantages. In addition to being relatively expensive, the HID bulbs slowly decrease in light output over time (thereby causing progressively longer cure times) and should be replaced after about 500 hours of use. It is recommended to turn off the bulb immediately after each use to maximize bulb life, minimize stray UV radiation, heat buildup in the workplace, and make the lamp easier to handle and move. UV-curing lamps are intended to be used only in a restricted area, accessed only by qualified professional operators, due to the dangers of being exposed to the emitted UV radiation and the generation of ozone by the lamp. In addition, UV lamps generally only have a very limited exposure area or footprint. Currently the coverage area of UV-curing lamps may be limited to an area of about 6 square feet, which results in frequent repositioning of the lamp. Although this disadvantage may be limited somewhat by mounting the lamp on a computer guided robot which automatically repositions the lamp to a different exposure area, this solution is very expensive.