WO2003079805A1

WO2003079805A1 - Cross-linked gelatin

Info

Publication number: WO2003079805A1
Application number: PCT/NL2003/000076
Authority: WO
Inventors: Govardus Adrianus Hubertus De Jong
Original assignee: Nederlandse Organisatie Voor Toegepast- Natuurwetenschappelijk Onderzoek Tno
Priority date: 2002-02-08
Filing date: 2003-02-04
Publication date: 2003-10-02
Also published as: NL1019932C2; AU2003211315A1

Abstract

The present invention relates to a method for preparation of reversibly gelling with increased bloom number. A method according to the invention comprises the use of an enzymatic cross-linking process wherein a gelatin with special properties is formed. Further, the present invention relates to a cross-linked gelatin which can be used great advantage in a large variety of industrial processes wherein high grade gelatin is required or desired.

Description

Title: Cross-linked gelatin

The invention relates to a cross-linked gelatin as well as to a method for the preparation thereof.

Gelatin is an important food ingredient which is generally produced from collagen from the skin and bones of animals. The most important uses of gelatin are in jelly, candied products, meat products and cooled dairy products, while use is made of the good gelling properties of gelatin.

Thus far, the special gelling properties of gelatins have not been equalled with other materials. In contrast with gels of other proteins, which are only formed by heating (egg-protein) or by heating followed by cooling, gelatins can, inter alia, form gels by cooling alone. These gelatin gels can be easily liquefied again through heating at relatively low temperatures (approximately 40°C).

This reversible nature of a gel on the basis of gelatin renders gelatin particularly suitable for, for instance, desserts, because the gelled material melts in the mouth. The special properties of gelatin are also used in photography. The glossy top layer of photographs consists, for instance, of gelatin. On this type of gelatin, high requirements are imposed. The gelatin must have a high bloom number, i.e. it must yield a gel with a high gel strength, and upon cooling, the gelling behavior should be such that the gel forms rapidly.

In general, a higher bloom number means that the price of the associated gelatin is higher. This holds both for the food industry and, for instance, for the photo industry. In the production of gelatin, as a result of the production process, gelatin types with good and with poor gelling properties are obtained. In addition to physical aspects, the gelling properties of gelatin also comprise organoleptic aspects. From an economic point of view, the poorly gellable gelatin types are of less significance. Nowadays, the commercial market for gelatin is still expanding, notwithstanding the decreased image of animal products in general. In particular the demand for gelatin types with good gelling properties increases. In the past, it has been attempted to cross-link gelatin with the aid of the enzyme trans glutaminase, in the hope of thus improving the gellability. This would, in particular, be of interest for the types of gelatin of lesser quality because, thus, the price can be considerably increased. The French patent specification 2 659 352 describes a method wherein gelatin is treated with the enzyme transglutaminase. With this treatment, an increased gelling point and an increased viscosity can be obtained. Unfortunately, it has appeared impossible to improve the gelling properties, and in particular the gel strength, of gelatin with the aid of transglutaminase. Cross-linking gelatin with transglutaminase even results in deteriorated gellability and a reduction of the gel strength. Now, it has been found that the gellability of gelatin can be strongly enhanced by cross-linking the gelatin with the aid of a cross-linking enzyme. The gelatin cross-linked according to a method of the present invention has a very much higher gel strength. The corresponding bloom numbers have not been realized earlier for gelatin. As a result, completely new applications become possible. This also means that lower concentrations of gelatin can be used for obtaining the same gel strength.

Without wishing to be limited by theory, it is useful here to discuss some facts about proteins in general and gelatin in particular.

Proteins are built up from individual a ino acids, in a sequence specific to a particular protein, linked together by peptide bonds. These amino acids comprise inter alia reactive groups, such as sulfhydryl groups of cysteine residues through which parts of polypeptide chains or different polypeptide chains can be mutually cross-linked. Further, particular groups can be ionized, thereby creating charge. Due to interaction between different amino acids in a protein, further, a characteristic tertiary structure is formed. The hydrogen atom bonded to a nitrogen atom in the chain can form a hydrogen bridge with a carboxyl group of the chain.

Collagen, the most important protein in the connective tissue from which, inter alia, skin, bones, tendons, cartilage and blood vessels are built up, has a very unique protein structure, about which relatively much is known. Gelatin can be obtained from collagen by means of denaturation and partial hydrolysis. Still, however, many fundamental questions about the gelling mechanism of gelatin remain unanswered.

Gels of proteins can be formed in different manners. When heating a protein solution to a temperature of about 80 to 90 °C, in particular the disulfide bridges break so that the proteins unfold. Upon cooling, disulfide bridges are formed again between different protein molecules and a network of proteins is formed. Depending on the protein concentration, the network causes gel formation. This form of gelling is called heat gelling and is used, inter alia, in the preparation of sausages.

A second form of gelling is acid gelling: a gel is formed in that interactions between protein molecules change as a result of change of charge. Still another manner in which a gel of proteins can be obtained is by cross-linking the proteins substantially completely enzymatically. Enzymes such as transglutaminase and laccase can cross-link protein molecules, so that a network is formed. Here, it also holds that with a sufficiently high protein concentration, a gel can be formed. Such a gelling is described, inter alia, in the European patent specification 0 947 142. The drawback of such gels is that they can no longer gel reversibly, i.e., the gel has a permanent character through the formation of covalent bonds between the protein chains.

The form of gelling connected with the present invention is a fourth form of gel-formation of proteins, viz. gelatin gelling. Gelatin gelling is unique because this gelling takes place as a result of the formation of hydrogen bridges between the polypeptide chains. With gelatin gelling, as a rule, no covalent bridges are formed. This means that, in most cases, a solution with a sufficient amount of gelatin is liquid at a temperature over approximately 35°C, and below that, forms a gel. The process of dissolving and gelling of gelatin is completely reversible. This provides gelatin with several special possibilities for use. Surprisingly, it has been found that a cross-linked gelatin prepared by a method according to the present invention reaches gel formation more rapidly and that, also, a higher gel strength is achieved. The accelerated gelling process is highly advantageous, inter alia, for the photo industry because through faster gelling of the gelatin on the photo, the processing rate of photos can be considerably increased.

It is an object of the invention to improve the quality of gelatin and, more specifically, the gel strength and the gelling properties. By the use of a method according to the invention, gelatins of relative low quality and having a low bloom number can gain in quality considerably. Also, gelatins can be prepared with a bloom number that was not reached previously.

The present invention provides a method for the preparation of a reversible gelling gelatin with increased bloom number, wherein a gelatin is partially cross-linked with the aid of a cross-linking enzyme for obtaining a reversibly gelling gelatin with increased bloom number. The present invention also provides a reversibly gelling gelatin with increased bloom number obtained by means of a method according to the invention.

Fig. 1 shows the effect on the gel strength after cross-linking of gelatin with laccase as worked out in Example 1. Fig. 2 represents in a graph the gelling of a gelatin which has been cross-linked with laccase for a shorter and a longer period of time. The maximum value of the gel strength of a gel which has been prepared on the basis of a reversibly gelling gelatin with increased bloom number according to the invention is higher than the corresponding value of a non-cross-linked product. Fig. 3 is a detail of Fig. 2 and represents the rate at which gels form, while it can be seen that a gel on the basis of a reversibly gelling gelatin with increased bloom number according to the invention forms quicker than a comparable gel on the basis of a non-cross-linked gelatin. Fig. 4 represents in a graph the gelling of a gelatin which has been cross-linked with transglutaminase as worked out in Comparative example 1.

Gelatin of any random origin can be used in embodiments of the present invention. Gelatin derived from gelatin from the skin or bones of pigs or bovines as well as gelatins derived from, for example, fish, can be used. In an alternative embodiment according to the invention, also a recombinant gelatin from, for example, bacteria or yeasts can be used. Such gelatins produced through recombination can offer a safe alternative to material of animal origin, since possible contamination of the material with pathogens is thereby precluded. Preferably, in the present invention, a bovine, pig or fish gelatin is used, or a combination thereof.

For obtaining a reversibly gelling gelatin according to the invention, an enzymatic cross-linking reaction is carried out. A reaction mixture for carrying out an enzymatic cross-linking reaction according to the invention can be prepared by suspending, preferably dissolving the gelatin, preferably in the form of a dried gelatin powder, it in a solvent. Preferably, the solvent is water.

A suitable amount of gelatin which is used in a reaction mixture according to the invention is between 1 and 99 % by weight of gelatin, based on the weight of the reaction mixture. Preferably, between 1 and 20% by weight of gelatin is used. More preferably, an amount of gelatin of approximately 10% by weight is used.

To a reaction mixture for carrying out a cross-linking reaction, optionally, buffering substances can be added for maintaining the acidity. Preferably, the pH of the solution is in a range between approximately 4 and approximately 10. More preferably, the pH is between approximately 5 and approximately 8, still more preferably, the pH is approximately 6 to approximately 7.

The presence of emulsifiers and surface active agents such as stabilizers, and optionally different additives can promote the cross-linking reaction in that they keep the enzymes in an active form, and forms a part of the present invention.

To a reaction mixture comprising a gelatin, subsequently, a cross- linking enzyme is added. However, the order in which the different components are added to the reaction mixture is not limitative. The cross-linking reaction can be carried out at a temperature between 5°C and 80°C, preferably between 35°C and 40°C.

The cross-linking reaction can be carried out for a period varying from a few minutes to a few days. Preferably, a cross-linking reaction is carried out for a period of between 10 minutes and 24 hours. Cross-linking enzymes which can be used in the present invention are, inter alia, but not exclusively, polyphenol oxidases, alone as well as in combination. Preferably, as cross-linking enzyme, laccase (EC 1.10.3.2) is used.

As different cross-linking enzymes cross-link different amino acids, in view of this, modification of the reaction conditions is possible. In principle, the reaction conditions during the cross-linking reaction are selected such that an optimal cross-linking can take place. These reaction conditions comprise conditions such as concentration of the cross-linking enzyme, temperature, duration of time of the reaction, pH, salt concentration, gelatin concentration and the presence of optional auxiliary substances. The optimal reaction conditions during a cross-linking reaction of gelatin can differ for different cross-linking enzymes. For instance, in a cross-linking reaction with laccase, oxygen needs to be present, and when using peroxidase, if necessary, hydrogen peroxide can be added to the reaction mixture. When setting the reaction temperature, the temperature optimum and the temperature stability of the enzyme to be used can be taken into account. The amount of enzyme required to cross-link an amount of 1 gram of gelatin is usually some milligrams, but can be much lower for certain enzymes. For example, in the case a laccase of high purity from Trametes υersicolor is used, as represented in Example 1, an amount of enzyme of approximately 0.01 to approximately 100 U/ml reaction mixture suffices. Preferably, in that case, an amount of enzyme of approximately 1 to approximately 10 U/ml reaction mixture is used, which corresponds to an amount of preferably 2 to 20 μg enzyme, while as definition it is taken that at an optimal acidity and temperature, 1 U of enzyme catalyses the formation of 1.0 μmol of product per minute. In the case of laccase, this can be determined by following the oxidation of 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) at 420 nm in the presence of sodium acetate, a pH of 4.0 and 37°C.

An enzymatic cross-linking reaction of gelatin according to the invention can be carried out in a paste, a slurry, a dispersion or in a solution of the gelatin. Depending on the enzyme used for cross-linking, and the desired extent of cross-linking of the gelatin, the skilled person can adjust the reaction conditions, optimize them and deploy particular auxiliary substances to that end. For instance, the reaction period can be prolonged with the object of increasing the extent of cross-linking and thereby the bloom number to be achieved.

The amount of enzyme used, the duration and the temperature during the reaction and the pH of the reaction mixture are not limitative. Depending on the desired end result, for instance the extent of cross-linking, the desired bloom number, and the period of time within which it is desired that this be achieved, the temperature of the reaction mixture and the time of cross-linking can be adjusted.

According to the present invention, it is essential that the gelatin preserves the capacity of reversible gel formation. For that reason, it is not desired that the gelatin is completely cross-linked. In fact, only a partial cross- linking is desired. When the gelatin, as a result of the cross-linking reaction, is cross-linked such that the solution starts to gel, in many cases, there is no longer any question of the capacity of reversibly gelling by the gelatin.

In a reaction mixture according to the invention, the gelatin is preferably cross-linked to the extent where the solution remains substantially liquid. Very low concentrations of gelatin in the reaction mixture can mask the occurrence of complete cross-linking in that with such concentrations, gel formation cannot occur at all. Yet, it is of importance that a cross-linked gelatin according to the present invention preserves its capacity to gel reversibly as described hereinabove. The extent of cross-linking can be determined, inter alia, by determination of the molecular weight. When this is increased, cross-linking is involved. Suitable manners for determining the molecular weight, such as gel filtration or SDS/PAGE, are known to the skilled person.

A partial cross-linking of gelatin according to the invention can be achieved by timely terminating the cross-linking reaction. This can be done by inactivating the cross-linking enzyme through, for instance, denaturation or by modifying the reaction conditions such that the activity of the cross-linking enzyme is substantially arrested. A suitable method for inactivating the cross- linking enzyme is heat inactivation. Preferably, heat inactivation is carried out at a temperature of between 60 - 150 °C. Highly usable is a heat inactivation between 70 - 100 °C for a duration of approximately 10 minutes.

After the inactivation of the cross-linking enzyme, a substantially liquid solution or dispersion is obtained which comprises a partially cross- linked gelatin according to the invention. This cross-linked gelatin is a reversibly gelling gelatin with increased bloom number. Optionally, the solution or dispersion can be dried and ground for obtaining a reversibly gelling gelatin with increased bloom number in powder form.

The reversibly gelling gelatin with increased bloom number according to a method of the invention is characterized in that the bloom number, measured by means of the generally adopted method according to the British Standards, is higher relative to the non-cross-linked starting material. Preferably, by means of a method of the invention, a reversibly gelling gelatin is obtained with a bloom number which is 1.1 - 20 times greater, more preferably 1.5 - 10 times greater than the bloom number of the gelatin in non- cross-linked form.

A gel which is prepared on the basis of a reversibly gelling gelatin with increased bloom number according to the invention forms faster than a comparable gel on the basis of a non-cross-linked gelatin. Also, the maximum value of the gel strength of a gel prepared on the basis of a reversibly gelling gelatin with increased bloom number according to the invention is generally much higher than the corresponding value of a non-cross-linked product (see Figs. 2 and 3).

Reversibly gelling gelatin with increased bloom number prepared according to a method of the present invention finds its particular application in those fields of the industry where high demands are imposed on the gelling properties of the gelatin used, such as in the photo industry, in particular as protective layer on photos. Gelatin which would normally be used as cattle feed because of the lesser quality can be worked up by means of a method according to the invention to a high-grade gelatin. Further, the cross-linked gelatin prepared according to a method of the present invention can be used in the food industry and in pharmaceutical preparations and tablets, in particular in capsules. Also (bio)medical materials can be manufactured on the basis of gelatins according to the invention.

A method according to the present invention finds highly suitable use in the gelatin producing industry. For instance, the quality of gelatin with poor gelling properties as a result of the production process can be considerably improved by means of a method according to the present invention. The cross-linked gelatin is of a higher quality and can be sold at a higher price. Further, a method according to the invention enables the gelatin producing industry to provide a completely new product with a bloom number not previously achieved for gelatins.

The invention will now be illustrated on the basis of the following examples which should not be taken as being limitative.

Example 1. Method

To a 100 ml solution (pH 6) of 10% by weight of bovine gelatin (150 bloom), 1 ml of laccase solution (EC 1.10.3.2) was added. The enzyme preparation used was pure for > 95% by weight, based on the total protein content of the preparation and was obtained from Trametes υersicolor after purification over an anion exchanger followed by a gel filtration step, according to methods known to the skilled person. The specific activity of the enzyme preparation was 238 U/ml (0.5 mg/ml). During the incubation with laccase at 37°C, the solution was stirred well and oxygen was fed in the Erlenmeyer. At different moments, samples were taken (t=0, 20, 40, 120, 180 and 240 min). These samples were heated for 10 min at 70°C to inactivate the enzyme. After the inactivation, of all samples, the gel strength was measured by cooling the solution to 5°C in a Bohlin rheometer and by measuring the gel strength over time. In Fig. 1, the gel strengths are indicated of the different gelatin solutions after 800 min at 5°C in the Bohlin rheometer. The values are indicated in Pascal units of the elastic modulus G'. The formation of the gels is represented in Figs. 2 and 3.

Result

During the incubation of gelatin with laccase, the viscosity increased. Eventually, a far advanced cross-linking led to gelling of the gelatin. After 300 min, the gelatin solution had gelled and no more samples could be taken. The gel formed could therefore no longer be processed into a good ingredient. The samples that were taken up to a period of 240 min remained liquid and still possessed the property of forming a gel upon cooling. These solutions could be dried, and thereupon could be processed well in several applications. In this example, the cross-linking led to a four times stronger gel and a corresponding increase of the bloom number of the gelatin.

Comparative example 1. Method

The method of cross-linking gelatin with transglutaminase was as follows: To 100 ml solution (pH 6) of 2% by weight of bovine gelatin (150 bloom), 500 μl of transglutaminase solution (EC 2.3.2.13) was added. The enzyme preparation used was pure for > 95 % by weight and had been obtained from Streptoυerticillium mobaraense after purification over an anion exchanger according to methods known to the skilled person. The specific activity was 12 U/ml (0.5 mg/ml). During the experiment, the solution was maintained at 37°C in a water bath. At different times during the cross-linking reaction with transglutaminase, samples were taken (t-0, 2 and 24 hours). These samples were heated at 90 °C for 30 min to inactivate the enzyme. After the inactivation, the sample was freeze-dried. The freeze-dried samples were dissolved to a protein concentration of 10 % by weight. The gel strength of these solutions was measured by cooling the solution to 5 °C in a Bohlin rheometer and measuring the gel strength over time.

Fig. 4 shows the formation of gels through cooling of the different gelatin solutions to 5°C in the Bohlin rheometer. The values are represented in Pascal units of the elastic modulus G'. Clearly visible is the reduction of the gel strength after cross-linking with transglutaminase.

Claims

1. A method for the preparation of a reversibly gelling gelatin with increased bloom number, wherein the gelatin is partially cross-linked with the aid of a cross-linking enzyme for obtaining the reversibly gelling gelatin with increased bloom number.

2. A method according to claim 1, wherein a polyphenol oxidase is used as cross-linking enzyme.

3. A method according to claim 1, wherein laccase is used as cross- linking enzyme.

4. A method according to any one of claims 1 — 3, wherein the gelatin is preferably present in a reaction mixture for a cross-linking reaction in an amount of 1 — 20 % by weight based on the weight of the reaction mixture.

5. A method according to any one of the preceding claims, wherein a reaction mixture for the cross-linking reaction remains substantially liquid.

6. A method according to any one of the preceding claims, wherein the gelatin is a bovine, porcine and/or fish gelatin.

7. A method according to any one of the preceding claims, wherein the cross-linking of the gelatin is terminated by inactivation of the cross-linking enzyme.

8. A method according to any one of the preceding claims, wherein the cross-linking is carried out for a period of between 10 minutes and 24 hours.

9. A method according to any one of the preceding claims, wherein the cross-linking enzyme is inactivated by heat inactivation.

10. A reversibly gelling gelatin with increased bloom number obtained by means of a method according to any one of claims 1 — 9.

11. A reversibly gelling gelatin according to claim 10, with a bloom number which is 1.5 — 10 times higher than the bloom number of the gelatin in non-cross-linked form.

12. A composition comprising a reversibly gelling gelatin with increased bloom number according to claim 10 or 11.