WO2016012375A1 - Gels de gélatine sensibles au calcium - Google Patents

Gels de gélatine sensibles au calcium Download PDF

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Publication number
WO2016012375A1
WO2016012375A1 PCT/EP2015/066467 EP2015066467W WO2016012375A1 WO 2016012375 A1 WO2016012375 A1 WO 2016012375A1 EP 2015066467 W EP2015066467 W EP 2015066467W WO 2016012375 A1 WO2016012375 A1 WO 2016012375A1
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Prior art keywords
gelatin
gel
calcium
liquid composition
collagen
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PCT/EP2015/066467
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English (en)
Inventor
Harmen Henri Jacobus De Jongh
Diana Karina BAIGTS ALLENDE
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Stichting Top Institute Food And Nutrition
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Publication of WO2016012375A1 publication Critical patent/WO2016012375A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • A23L29/284—Gelatin; Collagen

Definitions

  • the invention relates to a method for the production of a gel-based product.
  • the invention further relates to a product obtainable with such method, such as a food or non-food product.
  • the invention also relates to the use of functionalized gelatin.
  • compositions comprising a cross-linkable protein or polypeptide, and a non-toxic material which induces cross-linking of the cross-linkable protein.
  • the compositions are prepared in a non-phosphate buffer solvent.
  • the cross-linkable protein includes gelatin and any gelatin variant or variant protein as described herein.
  • the non-toxic material comprises transglutaminase (TG), which may optionally comprise any type of calcium dependent or independent transglutaminase, which may for example optionally be a microbial transglutaminase (mTG).
  • US5232727 describes a frozen food produced by adding a gelling agent comprising gelatin to a dough mainly consisting of wheat flour, fat and egg.
  • the process for producing the frozen food comprises adding a gelling agent comprising gelatin to dough materials mainly consisting of wheat flour, fat and egg to thereby give a dough and then molding and freezing said dough.
  • US3266906 describes a composition suitable for producing a high bloom strength alginate gel by incorporation with an aqueous medium comprising a water soluble alginate.
  • GB 1474891 describes synthetic caviar comprises granules of an aqueous gel of gelatin containing edible proteins and enclosed in two pellicles, namely an inner pellicle consisting of the products of tanning the gel with a vegetable tannin and an outer pellicle containing a calcium and/or aluminum salt of an edible polysaccharide. Both pellicles and the gel may also contain ferric salts, and an edible eno or annatto dye may also be present in the gel.
  • EP1367066 describes a process for preparing a gelatin composition suitable for use in the preparation of a blood plasma extender, which process comprises: (a) pre-conditioning, under alkaline conditions, a gelatin-producing raw material; (b) preparing, from the pre-conditioned raw material product of step (a), a gelatin starting material having a pi (iso-ionic point) in the range of from 4.5 to 6; and (c) derivatising the gelatin starting material product of step (b), whereby there is produced derivatised gelatin having a pi in the range of from 4 to 5.
  • the derivatised gelatin thereby produced preferably has a pi in the range of from 4 to 4.8 and a degree of derivatisation in the range of 50-60%.
  • a succinylated gelatin thereby produced is particularly suitable for use in the preparation of a blood plasma expansion products.
  • Collagen is an abundant protein organized in tissues with a highly ordered molecular axis in fibrous networks contributing to the spatial cellular structure.
  • Gelatin is the product of structural and chemical degradation of collagen that still resembles most of the functional properties of the original molecule. As indicated above, gelatin is widely used in a variety of applications in different sectors of the food industry as bakery (to promote emulsification, or gelling and stabilization properties), dairy (stabilization and texture), meat products (water-binding), and confectionery as well as in specific products like jam, jelly and low-fat spreads (promoting creaminess, fat reduction and mouth feel).
  • the versatility of gelatin in functional properties in production of foods makes it a valuable tool for developing new and more attractive or tailored products for consumers.
  • One of the most important quality parameters in processed foods is the textural attribute.
  • gelatin may also be used in non-food applications, such as coatings or encapsulates of pharmaceuticals.
  • a primordial attribute of gelatin-gels is the ability to store energy in the network (elasticity) that can be recovered when an applied deformation relaxes.
  • Profound stiffness of gelatin gels has been related to the presence of long triple helices connected by flexible strands.
  • a number of structural motifs seem to dictate the macroscopic functionality. These are: (i) the mesh size of the network as set by the average distance between junction zones, which is protein concentration dependent, (ii) the length of the dominant structural motif, like the triple helix in gelatins, and (iii) the thickness, and thereby inherent flexibility of the structural motifs.
  • a disadvantage of functionalizing gelatin is that the gelling properties may change, or some properties may improve while other properties get worse.
  • gel strength may be reduced when gelatin is functionalized. In some instances, this may be desired, but in other instances, a desired functionality may be accompanied by an undesired gel strength change and/or an undesired change of the gelling temperature.
  • the gelling time is not always easily controllable, whereas in processing food or non-food products, it is desirable to control the process and gelling time.
  • collagens are cross-linked. In such instances, gel formation, if any, is not reversible. However, reversibility of the gelling may be desired.
  • the invention provides a method ("method") for the production of a gel-based product (“product”), the method comprising (i) providing a liquid composition comprising a collagen, especially gelatin, especially in an amount of 0.1-30 wt.%, such as especially 0.1-10 wt.%, and (ii) adding a multivalent cation salt, especially a divalent cation salt, especially a calcium salt, to the liquid composition, especially to provide a multivalent cation concentration (especially calcium concentration) in the liquid composition up to 150 mM, such as up to 100 mM, especially up to 50 mM, wherein when combining the collagen, such as gelatin, and (calcium) salt in the liquid composition, in a specific embodiment substantially does not comprise a cross- linker, and wherein the collagen, especially gelatin, comprises amino acids with especially at least 1% of the total amount of amino acids being succinylated, phtalated or deamidated (i.e.
  • the invention also provides a gel-based product obtainable by the method as described herein.
  • the invention provides a gel-based product comprising 0.1-30 wt.%, such as especially 0.1-10 wt.% gelatin, comprising a multivalent cation, especially calcium, in an amount up to 2 wt.%, the gel-based product comprising a gel phase which substantially does not comprises a cross-linker.
  • the gel phase especially comprises said multivalent cation, such as calcium, especially in an amount up to 2 wt.% relative to the gel phase.
  • succinylation, phtalation or deamidation may be applied to gelatin type A. Further, especially succinylation or phtalation may be applied to gelatin type B.
  • succinylation, phtalation or deamidation or “succinylation or phtalation” or similar phrases may especially indicate that one or more amino acids may be succinylated, or one or more amino acids may be phtalated, or - where applicable - one or more amino acids may be deamidated.
  • these phrases or similar phrases may also indicate that one or more amino acids may be succinylated, and one or more other amino acids may be phtalated, and - where applicable - yet one or more other amino acids may be deamidated.
  • combinations of two (or more) of these functionalizations may also be applied. Therefore, especially the invention is direct to the succinylation or phtalation of gelatin A or B, or combinations of these functionalizations and/or combinations of these gelatins.
  • Non-functionalized collagen such as gelatin
  • gel formation by enzymatic cross- linking as mentioned in the prior art also takes substantially more time, even hours.
  • specific functionalized collagen such as gelatin
  • a gel is formed with good gelling properties and with properties that may be comparable to the non- functionalized collagen (non-functionalized gelatin).
  • the gel properties are restored, especially when the functionalization is chosen under the conditions as described herein. It further appears that gel formation is reversible.
  • gelatin may be that it is a molecule wherein the negative and positive charges are relative remote from each other, unlike e.g. in fibrinogen or globular proteins.
  • the invention provides amongst others calcium sensitive gelatin gels or other multivalent cation gelatin gels, and more in general multivalent cation collagen gels.
  • a calcium salt (or other multivalent cation salt) can be used to initiate gelling of gelatin in a liquid composition comprising the gelatin (which gelatin is functionalized with carboxylate groups by one or more of succinylation, phtalation and deamidation, especially succinylation), while not substantially changing one or more of the temperature and pH (though optionally also one or more these parameters may be used to induce and/or control gelling).
  • Gelling can now be controlled much better.
  • the gel can be formed within seconds (upon addition of the salt) at the moment desired by the process controller. Even at low temperatures, gel formation can occur within seconds.
  • a calcium salt and gelatin in a liquid composition comprising the gelatin, wherein the gelatin is functionalized with carboxylate groups by one or more of succinylation or deamidation, to provide a reversible gel.
  • succinylation also other routes to introduce the carboxylate groups may be used.
  • other dicarboxylic acids may be applied, like phtalic acid.
  • the gelatin is functionalized with carboxylate groups by one or more of succinylation, phtalation, etc..
  • one or more of succinic anhydride and phthalic anhydride, or another anhydride may be applied to introduce the carboxylate group(s).
  • multivalent cations may be relevant, such as magnesium, iron, aluminum, copper, cobalt, etc., respectively.
  • combinations of two or more different types of collagens, such as different types of gelatins may be applied.
  • the terms “collagen” or “gelatin”, and similar terms may also refer to a combination of different types of collagen or different types of gelatin, respectively.
  • two or more different types of multivalent cations may be applied.
  • the term “multivalent cation” or “calcium salt”, and similar terms may also refer to a combination of different types of multivalent cations or different types of calcium salts, respectively.
  • the gelatin is especially functionalized with carboxylate groups. Good results are obtained when at least 10% of all lysines (of the collagen, especially gelatin) are succinylated (and/or phtalated). Succinylation is known in the art. It is a posttranslational modification where a succinyl group is added to a lysine residue in e.g. gelatin or another protein molecule. The addition of the succinyl group changes lysine's charge from +1 to -1.
  • the invention also provides a method and/or product embodiment, wherein at least 10% of all lysines are succinylated (and/or phtalated), especially wherein in the range of 40-90 % of all lysines are succinylated (and/or phtalated).
  • succinylation or another process wherein the carboxylates are added, such as phthalation
  • succinylated and/or phtalated indicates that from a subset of lysines all may be phtalated, all may be succinylated, or part of the subset are phtalated and part of the subset are succinylated.
  • deamidation may be applied, to change amide groups to carboxylic acid groups.
  • at least 2% of all asparagines and glutamines are functionalized with a carboxylic acid group, especially by deamidation.
  • the invention also provides a method and/or product wherein one or more of (i) at least 50% of all asparagines and (ii) at least 15% of all glutamines are functionalized with carboxylic acid groups, apply.
  • the liquid composition will in general be an aqueous liquid composition, such as water.
  • the liquid composition may comprise other components (in addition to gelatin) than the pure liquid (or liquid phase).
  • the liquid composition may comprise further ingredients to form a food product or the liquid composition may comprise further ingredients to form an encapsulate.
  • the liquid composition may be a viscous liquid composition.
  • the liquid composition may thus comprise in addition to a liquid phase, such as water, a plurality of components, including the collagen, especially gelatin.
  • One or more components, such especially the collagen, even more especially gelatin may at least partly be solved in the liquid composition. However, one or more components may also not be solved.
  • the liquid composition may comprise a collagen, especially gelatin, especially in an amount of 0.1-30 wt.%, such as especially 0.1-10 wt.%, even more especially at least 0.5 wt.% of collagen, especially gelatin. Lower amounts may not lead to efficient formation of a gel, and at higher concentrations very viscous solutions may be obtained at high temperatures, which are difficult to handle.
  • the liquid composition may comprise the collagen, especially gelatin, in an amount of not more than 6 wt.%.
  • the thus obtained gel- based product may thus also comprise in the range of 0.1-30 wt.%, such as especially 0.1- 10 wt.%, of the (gelled) collagen, such as (gelled) gelatin.
  • the weight percentage may be above 10 wt.%).
  • the gel-based product may also an intermediate product, with the final product thus in general comprising a lower amount of (gelled) collagen than the intermediate product.
  • multi-valent cations are cations having a charge of 2+ or higher, such as especially Ca 2+ , which is a divalent cation.
  • Especially calcium salts may be applied, such as calcium nitrate or calcium chloride.
  • other salts may be applied like e.g. salts of one or more of magnesium, iron, aluminum, copper, and cobalt.
  • the liquid composition comprises the collagen, especially gelatin, in an amount of 1-2 wt.%, wherein the multivalent cation salt, especially calcium salt, is added to the liquid composition to provide a multivalent cation, such as calcium, concentration in the liquid composition up to 50 mM, especially up to 20 mM, and wherein the multivalent cation salt, such as calcium salt, comprises especially one or more of calcium chloride and calcium nitrate.
  • the lower concentration of the multivalent cation salt, especially calcium will especially be at least 0.5 mM, especially at least 5 mM.
  • cross-linking indicates a chemical bonding, especially via an organic molecule.
  • collagen molecules are connected to each other via the multivalent cation(s), i.e. via ionic bonding.
  • no cross-linker is necessary.
  • the presence of cross- linkers is undesired, as it leads per definition to a gel that is not reversibly formed.
  • the liquid composition in a specific embodiment thus substantially does not comprise a cross-linker.
  • Such cross-linker may be an enzymatic cross-linker or a chemical cross-linker, as known in the art.
  • Examples of some common cross-linkers are the imidoester cross-linker dimethyl suberimidate, the N- hydroxysuccinimide-ester cross-linker BS3, glutaraldehyde and formaldehyde.
  • the amount of cross-linker (used in the method as described herein) is smaller than 1000 ppm (mg/kg), especially smaller than 500 ppm, such as smaller than 200 ppm, even more especially smaller than 100 ppm, such as smaller than 50 ppm, especially smaller than 10 ppm, relative to the total amount of the liquid composition (or the gel-based product).
  • the amount of cross-linker (used in the method as described herein) is smaller than 1000 ppm (mg/kg), especially smaller than 500 ppm, such as smaller than 200 ppm, even more especially smaller than 100 ppm, such as smaller than 50 ppm, especially smaller than 10 ppm, relative to the total amount of the gelatin (or the gel- based product (see also below)).
  • the enzymatic cross-linker thus especially comprises an enzyme that can induce cross-linking of proteins, like transglutaminase.
  • the concentration of the enzymatic cross-linker is especially smaller than 1 enzyme units (U/ml) (at room temperature and at the pH used), such as smaller than 0.1 enzyme units, even more especially smaller than 0.01 enzyme units.
  • U/ml enzyme units
  • the amount of cross-linker can be even below 100 ppm, such as especially below 50 ppm, such as below 10 ppm, like equal to or below 1 ppm.
  • the gel-based product as described herein may be a product wherein the amount of cross-linker is smaller than 1000 ppm (mg/kg), especially smaller than 500 ppm, especially smaller than 200 ppm, even more especially smaller than 100 ppm, such as smaller than 50 ppm, especially smaller than 10 ppm.
  • ppm ppm (mg/kg) (in total), especially smaller than 500 ppm, of one or more of the following components is available in the liquid composition or the gel phase in the gel-based product, respectively: glucose, a sugar, monosaccharide, a disaccharide, an aldehyde (such as glutaraldehyde, formaldehyde, glyceraldehyde), a peroxide (such as hydrogenperoxide), an epoxide (such as 1,3-butadiene diepoxide), benzene, sulfonic acid, guanidine hydrochloride, a block-aldehyde, n-methylol, a ketone, a carboxylic derivative, a carbonic acid derivate, a sulfonic ester, a sulfonyl halide, an active halogen compound, an s-triazine, an aziridine, an active oleofin,
  • the amount of cross-linking microcomponents such as one or more of a sugar, an aldehyde, etc.
  • the amount of cross-linking microcomponents may be lower than 500 ppm (mg/kg), relative to the liquid composition (or the gel phase in the gel-based product), especially in the case of gelatin type A.
  • the composition and/or the gel-phase of the gel-based product may include a cross-linking inhibitor.
  • the present invention is especially related to the succinylation and/or phtalation and the deliberate addition of a divalent cation containing salt. The presence of cross-linkers may lead to less desired hardening of the gel.
  • the process conditions may be chosen by the person skilled in the art.
  • the liquid composition when combining the gelatin and calcium salt the liquid composition has a pH in the range 2.5-9.
  • a temperature is applied which is especially above the melting temperature of the gelatin, but in general not above temperatures of about 70 °C.
  • characteristic temperatures may be in the range of 30-70 °C, such as 40-70 °C, especially such as 45-50 °C.
  • the collagen may already be functionalized.
  • the functionalization is also included in the method as described herein.
  • the invention also provides a method comprising (ia) providing a collagen, especially gelatin, (ib) functionalizing the collagen, especially gelatin, by one or more of succinylation (or other carboxylation) and deamidation (to provide the herein described (functionalized) collagen, especially gelatin), (ic) providing said liquid composition comprising collagen, especially gelatin, in an amount of especially 0.1-30 wt.%, such as especially 0.1-10 wt.%, and (ii) adding said multivalent cation salt, especially calcium salt, to the liquid composition to provide said multivalent cation, especially said calcium, concentration in the liquid composition especially up to 150 mM, such as up to 100 mM, especially up to 50 mM.
  • the gel obtained by adding the multivalent cation may have a good strength and other desirable properties (such as especially elasticity and brittleness). Especially, the thus obtained gel has a dissociation constant (or binding constant or binding affinity) of 30 mM or lower.
  • the dissociation constant would be much higher, which is indicative of a week gel.
  • the calcium sensitive enzyme be used for cross-linking, then the calcium content would have to be much higher than indicated herein values, such as at least 5 to 10 times higher than the preferred calcium contents as described herein. For non-calcium multi-valent cations the same may apply.
  • gelatin is often defined as a product obtained by the partial hydrolysis of collagen derived from the skin, white connective tissue and bones of animals.
  • Gelatin derived from an acid-treated precursor is known as Type A
  • gelatin derived from an alkali-treated process is known as Type B.
  • Type A Gelatin derived from an acid-treated precursor
  • Type B gelatin derived from an alkali-treated process
  • Gelatin is defined as the product obtained from the acid, alkaline, or enzymatic hydrolysis of collagen, the chief protein component of the of the skin, bones, and connective tissue of animals, including fish and poultry.
  • Gelatin is thus derived from collagen which is the principal constituent of connective tissues and bones of vertebrate animals.
  • Collagen is distinctive in that it contains an unusually high level of the cyclic amino acids proline and hydroxyproline. Collagen consists of three helical polypeptide chains wound around each other and connected by intermolecular crosslinks.
  • Gelatin is recovered from collagen by hydrolysis. There are several varieties of gelatin, the composition of which depends on the source of collagen and the hydrolytic treatment used (see also below). The principal raw materials used in gelatin production are cattle bones, cattle hides, and pork skins. Several alternative sources include poultry and fish. Extraneous substances, such as minerals (in the case of bone), fats and albuminoids (found in skin), are removed by chemical and physical treatment to give purified collagen. These pretreated materials are then hydrolyzed to gelatin which is soluble in hot water.
  • Gelatin may for instance include about 1-7 wt.% lysine.
  • the gelatin comprises gelatin type A (or gelatin type B) having a Bloom in the range of 50-300.
  • the term "gelatin” may also refer to recombinant gelatin. Especially, such gelatin may now relatively easily gel due to the functionalization.
  • the method as described herein may be a method further including the presence of one or more other components, to provide a food product.
  • Specific food examples wherein the thus obtained gel may be applied may e.g. be in confections such as such as gummy bears, fruit snacks, and jelly babies as well as other products such as marshmallows, gelatin desserts, ice creams, trifles, aspic, dips and yogurts.
  • Gelatin may be used as a stabilizer, thickener, or texturizer in foods such as yogurt, cream cheese, and margarine.
  • the method as described herein may be method further including the presence of one or more other components, to provide a coating or encapsulate for a medicament.
  • gelatin is a vital ingredient in the most popular drug delivery systems in the world such as two piece hard capsules, soft capsules, tablets, coated tablets, mini, micro capsules etc.
  • gelatin may be applied in two-piece hard capsules.
  • the manufacture of hard gelatin capsules involves the dipping of stainless steel mold pins into the gelatin solution, drying, stripping from the pin into a collate, trimming of the caps and bodies, and joining them together.
  • the strength, flexibility, clarity and viscous nature of gelatin provide characteristics that make it unique in the manufacture of capsules.
  • a typical hard capsules grade gelatin specification will have a gel strength (bloom) 200 - 270, though lower may also be possible, and may have a viscosity (mps) of 40 - 50.
  • gelatin may also be applied in soft gelatin capsules.
  • Soft gels as they are commonly known today, use a gelatin solution that is plasticized with propylene glycol, sorbitol, glycerin or other approved mixtures.
  • Soft gelatin capsules are one-piece and hermetically sealed to enclose a liquid or semi-liquid fill. Soft gelatin capsules are manufacture-formed, filled and sealed in one continuous operation.
  • a typical soft capsule grade gelatin specification will have a gel Strength (bloom) 150 - 180 and may have a viscosity (mps) of 30 - 40.
  • Gelatin may also be used in the preparation of the active ingredient and as binder in the tablet. For instance, gelatin may be used as a film former in tablet coating. Tablets are generally coated to reduce dusting, mask unpleasant taste and enable printing and color coating for product identification.
  • Other pharmaceutical applications of gelatin include suppositories, micro-encapsulation, surgical sponges, bacterial growth media etc.
  • the method may include combining the liquid comprising gelatin and one or more other components to provide the food product or non-food product and adding the salt to induce gelling.
  • the salt may be added, and the thereby obtained gel may be combined with the one or more other components to provide the food product.
  • the method may include transporting the liquid comprising the gelatin to a vessel wherein or conduit wherein the liquid and the one or more other components to provide the (non-food) food product are combined, and then (also) combining the liquid comprising the gelatin with the salt, to induce gel formation.
  • gelling can be started at the desired time, such as within 5 minutes before combining the liquid comprising the gelatin and the one or more other components to provide the (non-food) food product, during the combining stage or after combining the liquid comprising gelatin and one or more other components to provide the food product or non-food product.
  • This provides a better controlled processing.
  • a food product or non-food product comprising a gel phase may be obtained.
  • the gel-phase may be substantially continuous or may discontinuous (e.g. gel particles).
  • the invention also provides a gel-based product obtainable by the method as described herein.
  • the invention provides (such) gel-based product, wherein the gel-based product is a food product.
  • the invention provides (such) gel-based product, wherein the gel-based product is a coating or an encapsulate for a medicament.
  • gel- based product indicates that the product includes a gel, even as minor component, or has been made in a process wherein a gel has been formed. Examples of such products are the above mentioned food products and non-food products. Other non-food applications of (gelled) collagens, especially (gelled) gelatin are photography, and cosmetic manufacturing. In the overview below, some possible applications are indicated.
  • the gel-based product may be a gel per se or may be a product including other components.
  • the gel-based product may be a food product or a nonfood product.
  • the gel may be prepared in the presence of one or more other components, to provide such food product or non-food product (or an intermediate product for a final food product or non-food product).
  • the gel may be combined with the one or more other components to provide such food product or non-food product (or an intermediate product for a final food product or nonfood product).
  • a preferred degree of functionalization is max 10-80%, more preferred 20- 60%, most preferred 30-50%>. This may apply to both the type A and B gelatin, especially when functionalized with succinate or phatalate.
  • a preferred degree of calcium concentration (for gelling) is max 2.5-30 mM, more preferred 5-20 mM, most preferred 7.5-15 mM. This may apply to both the type A and B gelatin, especially when functionalized with succinate or phatalate.
  • Gelatin type A (especially succinylated) application can e.g. be a pharmaceutical soft/hard capsule production.
  • the capsule production method may e.g. be based on (fast) gelation of the gelatin. It appears that with the gelatin functionalized as described herein, capsule production can be better controlled. Undesired cross-linking can be prevented.
  • Gelatin type B (especially succinylated) application can e.g. be used in microencapsulation, in particular those applications were cross-linking is not desired and gelling is essential in relation to production. However, in principle both gelatin types may be used for both applications. As shown with gelatin type A or B application, instead of (or in addition to) succinylation, also phtalation may be applied.
  • Characteristic molecular weights of gelatins that can be used in the invention may especially be in the range of about 50-250 kDa. Further, characteristic Bloom values are in the range of 50-300, especially 80-300. Further applications of the gelatin as described herein may be in low fat spreads (e.g. mouth feel and/or stabilization of emulsion), in meat and fish (e.g. for texture and/or gelling), desserts/dairy (e.g. for texture, thickening, gelling), confectionary (e.g. for gelling, texture, chewability, stabilization, binding, etc.). Other applications include applications in juice or wine fining.
  • low fat spreads e.g. mouth feel and/or stabilization of emulsion
  • meat and fish e.g. for texture and/or gelling
  • desserts/dairy e.g. for texture, thickening, gelling
  • confectionary e.g. for gelling, texture, chewability, stabilization, binding, etc.
  • Other applications include applications
  • the invention provides a collagen-comprising product comprising a gelled collagen compound, wherein the collagen compound comprises collagen having calcium binding functionality with at least 8 per 1000 amino acids of the collagen compound, and wherein the gelled collagen compound comprises calcium bindings.
  • the collagen compound comprises gelatin.
  • the collagen compound comprises functionalized collagen, especially acylated collagen, such as succinylated collagen.
  • the acylated collagen has a degree of acylation of at least 3 per 1000 amino acids.
  • the collagen compound comprises deamidated collagen.
  • the gelled collagen compound is free of enzymes having cross-linking functionality, especially wherein the gelled collagen compound is free from transglutaminase.
  • the gelled collagen compound is available in an amount of 0.3-1 wt.% relative to the total weight of the collagen- comprising product.
  • the invention provides the use of a calcium salt and a collagen having calcium binding functionality to provide a gel.
  • the invention provides the use of collagen having calcium binding functionality and a calcium salt to provide a gel with increased gelling temperature and improved gel strength.
  • the invention provides a method of making a gel, the method comprising combining collagen having calcium binding functionality with at least 8 per 1000 amino acids of the collagen compound having calcium binding functionality and a calcium source and gelling said collagen.
  • the term “substantially” herein will be understood by the person skilled in the art.
  • the term “substantially” may also include embodiments with “entirely”, “completely”, “all”, etc. Hence, in embodiments the adjective substantially may also be removed.
  • the term “substantially” may also relate to 90% or higher, such as 95% or higher, especially 99% or higher, even more especially 99.5% or higher, including 100%.
  • the term “comprise” includes also embodiments wherein the term “comprises” means “consists of.
  • the term “and/or” especially relates to one or more of the items mentioned before and after "and/or”. For instance, a phrase “item 1 and/or item 2" and similar phrases may relate to one or more of item 1 and item 2.
  • Figure 1 shows the helicity (H) established from optical rotation (OR) measurements as described in the text for aqueous solutions of reference and succinylated gelatin with different degree of substitution (DS) as function of the temperature (T); fraction is indicated with "f ).
  • the large squares, triangles, crosses and asterisks represent a DS of 0% (squares, upper curve between at least about 10-25 °C), 12% (diamonds; are substantially overlapped by the reference 0%), 26% (triangles) and 42% (asterisks) respectively and all collide; the materials with a DS of 65% (circles; indicated with an arrow) and 82% (small squares (between at least about 5-35°C the lowest curve) are indicated in the figure;
  • Figure 2 shows the storage modulus (G') as a function of cooling/heating temperatures at 0.5°C/min scan rate for reference and gelatin solutions with different DS (3 wt %).
  • the DS's of the different materials are indicated for the traces.
  • the small arrows in the figure illustrate the applied temperature ramp of cooling and subsequent heating of the sample;
  • An ensemble averaged molecular weight of 124 kDa is used to calculate B (mol bound calcium / mol protein);
  • Figure 4A and 4B show a qualitative assessment of 3 wt.% gelatin solutions (reference (0%>) (NC refers to no Ca 2+ ) and materials with various DS) indicated on one axis at different protein concentrations (on the other axis) in terms of liquid (Aq), viscous (V), or gel (G) appearance in the absence (4 A) or presence (4B) of 5 mM calcium. Protein solutions are pre-heated for 30 min at 50 °C, subsequently cooled to room temperature and assessed after 1 hour equilibration at room temperature;
  • Figure 6 shows a calorimetric analysis of the cooling ramp from 50 to 5 °C of a 3 wt% reference (left panels; R) and 82% succinylated (right panels; DS 82%) gelatin in the presence of different concentrations of calcium (Ca+ shortly indicates the Ca2+ cation.
  • Upper panels present the onset of the gelling temperature and the lower panels show the concomitant enthalpy (E) change;
  • Figure 7 shows a helicity established from optical rotation (OR) measurements for reference (top panel) and 82% succinylated (lower panel) gelatin (0.5 wt %; 20 mM acetate buffer pH 6.5) in the presence of various calcium concentrations (0- 20 mM) as indicated in the graphs; "f indicates the fraction.
  • the solid diamonds represent samples in the absence, solid squares 4mM, open triangle 10 mM and light square 20 mM of calcium; and
  • Figure 8 shows a uniaxial compression measurements on cylindrical self- supporting specimens (diameter of 21.7 mm and 25 mm in height) of reference gelatin (top panel) and gelatin with a DS of 82% (lower panel) until fracture occurs in the presence of 0,4, 10 or 20 mM calcium, as indicated in the panels, with on the y-axis the true stress (TS); "R” indicates reference.
  • the aim of the experimentation is to try to stabilize strand- strand interactions in the junction zones and thereby 'locking' the junctions. It is hypothesized that in this way the ability to take up en dissipate energy will be reduced.
  • gelatins are chemically modified on the available lysine-residues using succinic anhydride with varying degrees of substitution, basically converting positive charges into negative ones.
  • succinic anhydride with varying degrees of substitution, basically converting positive charges into negative ones.
  • Using calcium the strands, bearing now a higher negatively charge density, could become stabilized at their inter-strand junction zones.
  • the materials are chemically and physically characterized, tested for helix propensity and thermal behavior and the impact of the modification on the mechanical behavior is studied using small and large deformation rheology.
  • Gelatin type A (PGS from pork skin, 150 Bloom and approximately 124,000 Mw), kindly provided by Rousselot® (Gent, Belgium), was used for samples succinylation according to the protocol described by Kosters, H.A., Broersen, K. de Groot, J., Simons, J.W.F.A., Wierenga, P.A. and de Jongh, H.H.J. (2003), Chemical modification as a tool to generate ovalbumin variants with controlled stability, Biotechnol. Bioengineer. 84, 61-70. Samples solutions were prepared (1 wt%) by dissolving in 0.02M phosphate buffer (pH 8) and stirring at 40 °C. Preparation in water without buffer is also possible.
  • Succinic anhydride (239690-250G, Sigma- Aldrich) were gradually and in small aliquots added (10 mg increments) to the gelatin solutions at continuous stirring up to reach the fixed amounts (5, 10, 35 mg per g of protein), in order to obtain different degrees of modification.
  • the pH was continuously adjusted to 8.0 using a pH-stat by titration with 1M NaOH.
  • the solutions were stirred for another 30 min, followed by extensive dialysis against deionized water (at RT) and subsequently lyophilized.
  • a reference sample was subjected to the same procedure without addition of succinic anhydride.
  • the degree of succinylation was determined by OPA (o-Phthaldialdehyde) assay adapted from Church et al.
  • the OPA reagent reacts with primary amino groups (N-terminus and lysine ⁇ -amino groups) in the presence of DMA (2-(dimethyl amino) ethanethiol hydrochloride) and results in the formation of alkyl-iso-indole fluorescent moieties.
  • the OPA reagent was prepared in a 50 ml volumetric flask by mixing 40 mg OPA dissolved in 1 ml methanol, 25 ml 0.1 M Borax solution, 200 mg DMA, 5 ml 10 wt.% SDS solution and demi water.
  • the reagent was freshly prepared before use.
  • the absorbance at 340 nm was measured in the spectrophotometer (UV-1800, Shimadzu) of 1 ml OPA reagent, and 1 ml OPA reagent mixed with 100 ⁇ 0.05% succinylated SP, equilibrated for 1 h at RT in the dark.
  • a calibration curve was obtained by measuring absorbance at 340 nm of 0.08-0.6 mM L- leucine as described above. Using this calibration line, the amount of N3 ⁇ 4 (mM) of protein before and after modification was obtained. Degree of modification was expressed as % of modified groups.
  • the measured absorbance of a sample was corrected with that of a sample containing the non-reacted reagent.
  • a calibration curve was obtained by diluting the OPA reagent with a series of L-leucine (1 mM stock) and solvent yielding concentrations of 0.66, 0.333, 0.166 and 0.083 mM. All assays were done in duplicate. DS is expressed as the percentage relative to the reference material.
  • a 0.1 M stock CaCl 2 was prepared by dissolving calcium chloride dihydrate (Sigma- Aldrich, Germany) in the appropriate acetate buffer. The concentration of free Ca 2+ ions was measured using a calcium ion selective electrode device (Ca ISE, adapted to Orion Star A214 ISE meter). Before to sample measurements, the potentiometric readings (mV) of the electrode were calibrated using standard CaCl 2 solutions (0.5 to 30 mM) with addition of ISA solution to match the ionic strength in all solutions. Gelatin samples (0.9 % w/v) were titrated with the CaCl 2 stock-solution and the corresponding amount of mol calcium bound per mol protein (assuming an average molecular weight of 124 kDa) was established.
  • Ca ISE calcium ion selective electrode device
  • the determination of the recoverable energy (RE) was done by compressing the samples up to a strain of 25% at deformation speeds of 1 mm/s.
  • Table I present the different materials obtained when gelatin is subjected to different degrees of succinylation (DS).
  • the DS shows a rather linear relation with the amount of succinic anhydride added, indicating that the number of lysine groups available for chemical modification is not limiting. It is also clear that, where the reference material has an IEP (iso electric point) of around 7.5, already the lowest DS yields an apparent IEP just below 5, gradually further decreasing with increasing DS. From the amino acid composition this gelatin contains about 27 lysine per 1000 residues and with an average molecular weight of 124 kDa it implies that for the highest DS about 25-27 succinate groups have been introduced per molecule. All experiments described in this work have been carried out at a pH of 6.7, this is above the isoelectric point of all succinylated variants, but below that of the reference material.
  • Figure 1 shows the triple helix propensity as obtained from optical rotation measurements during a cooling ramp from 60 °C to 5 °C for aqueous solutions of reference and succinylated gelatins with different DS. It can be clearly seen that at even high DS the onset temperature for helix formation is not affected. Also, it can be observed that up to a DS of about 60% no significant impact is observed on the helix induction, while the material with a DS of 65% is marginally affected; only for a DS of 82% a reduction of the helix propensity of about 20-25%) is observed. Prolonged incubation at low temperature did not increase the helicity of this latter sample (not shown), illustrating that the observed reduction is not the result of a slower kinetic process.
  • Figure 3 shows the calcium-binding curves for reference and succinylated gelatin at concentrations below the critical gel concentration (0.5% w/v). It can be observed that already the reference gelatin shows some specific calcium-binding and increasing the succinate content steadily increases the number of calcium-ions that are bound with detectable affinity to gelatin. The fact that no plateau value is reached for any of the samples indicates that the binding affinity of molecular gelatin is relatively weak and there is a continuous equilibrium between bound and non-bound calcium that is shifted towards the bound state by increasing the bulk calcium concentration.
  • gelatin with a DS of 26% behaves in the presence of 5 mM calcium physically like reference gelatin.
  • 1% (w/v) gelatin with a DS of 82 behaves like a liquid in the absence of calcium, but shows profound viscosity in the presence of 5 mM calcium.
  • the impact of calcium on gel properties are studied for all materials in more detail. Below the gel properties of reference gelatin and that with a DS of 82% are presented only to illustrate the differences.
  • Figure 5 shows the small deformation rheology of 3% (w/v) gelatin solutions (reference and DS 82%) when applying a cooling ramp to pre-equilibrated samples at 50 °C to 4 °C and subsequently a heating ramp back to 50 °C in the presence of different calcium-concentrations.
  • Figure 5 A For reference gelatin ( Figure 5 A) there is no detectable impact of the presence of added calcium up to a concentration of 20 mM, nor in gelling temperature, or in the maximal gel strength developed. This holds both for the cooling and heating ramps.
  • the succinylated gelatin it was already shown in Figure 2 that in the absence of calcium both the gelling temperature and the gel strength developed were significantly reduced.
  • Figure 5B shows, however, that addition of calcium 'restores' both the gelling temperature and gel strength to those observed for reference gelatin.
  • gelatin molecules to assemble into triple helices (strands) as microstructural building block for network formation also arises from the net charge on molecules, affecting the number of junctions along a strand and their interaction efficiency.
  • gelatin molecules inter-connected at random points produce shorter strands that are less flexible, produce more cross-linked or branched structures and provide consequently weaker networks. It appears that with the calcium ion, the gelling abilites of the functionalized gelatin are restored. For instance, a non-functionalized gelatin with calcium gave a brittle film, whereas gelatin functionalized as descrbed herein provides reversible gelling properties.

Abstract

L'invention concerne un procédé pour la production d'un produit à base de gel, le procédé comprenant les étapes consistant à (i) fournir une composition liquide comprenant de la gélatine en une quantité allant de 0,1 à 30 % en poids, et (ii) ajouter un sel de calcium à la composition liquide en vue d'obtenir une concentration de calcium maximale dans la composition liquide de 50 m M, dans lequel, lorsque la gélatine et le sel de calcium sont combinés, la composition liquide ne comprend essentiellement pas d'agent de réticulation, et dans lequel la gélatine comprend des acides aminés, au moins 1 % de la quantité totale d'acides aminés étant succinylée ou phtalatée.
PCT/EP2015/066467 2014-07-24 2015-07-17 Gels de gélatine sensibles au calcium WO2016012375A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113818095A (zh) * 2021-08-27 2021-12-21 东华大学 一种基于霍夫迈斯特效应的可大规模制备可降解明胶水凝胶纤维的湿法纺丝方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3266906A (en) * 1962-12-13 1966-08-16 Kelco Co Algin gel and gelatin composition having high bloom strength and process
GB1474891A (en) * 1975-03-27 1977-05-25 Inst Elementoorganiche Soedine Synthetic caviar and method of preparing same
EP1367066A2 (fr) * 2002-05-21 2003-12-03 CRODA INTERNATIONAL plc Gélatine succinylée de poisson

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3266906A (en) * 1962-12-13 1966-08-16 Kelco Co Algin gel and gelatin composition having high bloom strength and process
GB1474891A (en) * 1975-03-27 1977-05-25 Inst Elementoorganiche Soedine Synthetic caviar and method of preparing same
EP1367066A2 (fr) * 2002-05-21 2003-12-03 CRODA INTERNATIONAL plc Gélatine succinylée de poisson

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113818095A (zh) * 2021-08-27 2021-12-21 东华大学 一种基于霍夫迈斯特效应的可大规模制备可降解明胶水凝胶纤维的湿法纺丝方法

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