Oil-in-water emulsion stabilized with recombinant...

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Reexamination Certificate

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C430S546000, C430S627000, C430S628000, C430S631000

Reexamination Certificate

active

06645712

ABSTRACT:

SUMMARY OF THE INVENTION
The subject invention is directed at an oil-in-water emulsion in which recombinant collagen-like polymer is applied as a stabiliser. The stabilising effect occurs already at the stage of formation i.e. on the initial size of the droplets in the emulsion. Also the stabilising effect is visible when assessing the ageing of the oil-in-water emulsion. In both cases the droplet size is significantly reduced vis-à-vis the prior art oil-in-water emulsions comprising gelatin. The stabilising effect occurs at a range of temperatures and a range of pH values. It now in fact has become possible to operate processes requiring oil-in-water emulsions at lower temperatures than was possible to date and also at lower pH values than normally are applied to date. The same holds true for the storage temperature and pH at which the oil-in-water emulsions according to the invention can currently be maintained. The oil-in-water emulsions can now be stored longer than was previously the case. Also the oil-in-water emulsions according to the invention can be as stable as the prior art oil-in-water emulsions comprising gelatin at lower concentrations of surfactant than used in the prior art oil-in-water emulsions.
In addition the subject invention provides the possibility to use recombinant collagen-like polymer which are composed of polar and apolar end tails for oil-in-water emulsions and also provides for the first time the bipolar, or more specifically amphiphilic, compounds as such and a description of a method to achieve the production of such compounds.
FIELD OF THE INVENTION
Oil-in-water emulsions consist of hydrophobic droplets in a hydrophilic continuous phase. The interfacial area between these hydrophobic droplets and the hydrophilic continuous phase is stabilised with surfactants and/or polymers.
In the manufacturing process, the size of the droplets in the oil-in-water emulsion is a factor needing careful control. The average size of the droplets in the oil-in-water emulsion should be small i.e. the initial size of the droplets should be as low as possible. Also the size stability of the droplets after making the oil-in-water emulsion should be high i.e. the ageing stability should be as high as possible thus ensuring the increase in droplet size in time is kept as low as possible. To realise this small initial size and this limited ageing, the oil droplets can be stabilised by gelatin.
Present stabilisation methods however have several disadvantages:
1. The initial size of the oil-in-water emulsion is rather large.
2. The stabilisation capability of the present gelatin is limited, meaning that in the manufacturing process the oil-in-water emulsions have a limited life time in which they can be applied for specific functions.
The presently used polymer-like materials (like gelatin) originate from natural sources and the structure and the related rheological and surface chemical characteristics can be modified only in a limited manner,
J. Colloid Polym. Sci
. 272: 433-439 for example reveals experimental data about the relation between the molecular mass distribution of non-recombinant natural gelatin and it's effectiveness in the stabilisation of oil-in-water emulsions. In the case of gelatin samples with a content of more than 30% of the low molecular weight fraction as described in the article an improved stabilisation was obtained in comparison to the native non recombinant non hydrolysed gelatin. The problem still remained with this modified gelatin that the reproducibility of such processes using natural gelatin sources is not extremely good. In particular this is a preferred requirement for photographic applications.
In addition when considering use of oil-in-water emulsions for consumption purposes e.g. in foodstuffs the risk associated with mad cows disease for example can have a prohibitive effect on the use of gelatin derived from natural sources as a stabiliser.
DETAILED DESCRIPTION OF THE INVENTION
The invention is directed at an oil-in-water emulsion comprising recombinant collagen-like (or gelatin-like) polymer in an amount sufficient to act as a stabiliser of the emulsion. The advantages thereof are described in detail elsewhere in the description. An oil-in-water emulsion according to the invention suitably is one wherein the recombinant collagen-like polymer is free of triple helix structure. The recombinant collagen-like polymer is suitably free of any helix structure. It is a preferred embodiment of the invention that the recombinant collagen-like polymer of the oil-in-water emulsion is free of hydroxyproline as this ensures the absence of (triple) helix formation. The triple helical structure is present in natural gelatin. The absence of the (triple) helical structure is advantageous, because the emulsification can be operated at lower temperatures (15-40° C.) than the traditional temperature during the emulsification process (T higher than 40° C.).
The method of arriving at recombinant collagen-like polymer has been described in detail in commonly owned U.S. Pat. No. 6,150,081, inventors van Heerde et al., for example at column 14, line 48 to column 15, line 17, at column 22, line 51 to column 25 line 18 and elsewhere throughout the specification, the entire disclosure of which patent is hereby incorporated herein by reference thereto. The methodology is described in the publication ‘High yield secretion of recombinant gelatins by
Pichia pastoris
’, M. W. T. Werten et al.,
Yeast
15, 1087-1096 (1999), in press.
To be defined as collagen-like at least one GXY domain should be present of at least a length of 5 consecutive GXY triplets and at least 20% of the amino acids of the recombinant collagen-like polymer should be present in the form of consecutive GXY triplets, wherein a GXY triplet consists of G representing glycine and X and Y representing any amino acid. Suitably at least 5% of X and/or Y can represent proline and in particular at least 5%, more in particular between 10 and 33% of the amino acids of the GXY part of the recombinant collagen-like polymer is proline. For the purposes of this patent application the recombinant collagen-like polymer consists of at least 4 different amino acids, preferably more than 10 different amino acids, more preferably more than 15 different amino acids. It can comprise any of the amino acids known. A preferred oil-in-water emulsion according to the invention is one, wherein the recombinant collagen-like polymer comprises at least one lysine residue.
Any of the embodiments disclosed in the van Heerde et al. U.S. Pat. No. 6,150,081 can be applied for the oil-in-water emulsions according to the invention. A preferred embodiment of an oil-in-water emulsion according to the invention is one wherein the recombinant collagen-like polymer has an isoelectric point at least 0,5 pH units removed from the pH of the oil-in-water emulsion itself. Suitably one pH unit removed or even more. The advantage hereof is that the pH at which the emulsion needs to be maintained or used or prepared can vary depending on the isoelectric point (pI) of the applied recombinant collagen-like polymer. The recombinant technology enables variation previously unavailable for tailoring the polymer and thus tailoring the pI. It will be appreciated that not all processes requiring an oil-in-water emulsion are best carried out at pH 6 which is the pH value at which prior art gelatin comprising oil-in-water emulsions were optimally used. Naturally the pH=6 can also be used in those cases where it is still useful or in fact optimal. However the oil-in-water emulsions according to the invention no longer need the strict control of the pH during any of the processes e.g. preparation, storage or application as was previously the case. Now it has in addition become possible to use the oil-in-water emulsions according to the invention at pH=5. It has now become possible to develop oil-in-water emulsions with recombinant collagen-like polymers of extremely divergent pI values. Suitable embodiments involve pI anywhere from 4

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