The process of manufacturing gelatin can be traced back over many years.

Perhaps the earliest record is that found in the cave paintings of Rekhmara at Thebes, which date back to 1400BC and show the use of animal glue, a low grade of gelatin, in the application of veneers to wooden panels. A useful reference text for the preparation, properties, and applications of gelatin is to be found in the book"The Science and Technology of Gelatin"edited by A. G.

Ward and A. Courts, published by Academic Press in 1977. The production of gelatin has grown to be a significant industry and its uses are found to be many and varied. It can be used as a food, although it is deficient in some essential amino acids. Gelatin also has excellent emulsifying and stabilising properties; it is a protective colloid and has useful gel-forming and film- forming properties. Thus, gelatin finds widespread application in many industries, particularly in the food and pharmaceutical industries.

Gelatin is a protein derived from the natural protein, collagen, which is found in animal species. The collagen of bone and skin is the primary structural protein in the animal kingdom, and this is the material source used for gelatin manufacture. A major raw material source for gelatin is pigskin. Other major sources are the skins and bones from cattle. Whereas gelatins produced from these raw materials have satisfied the needs of a great many applications over many years, there are certain needs that these raw materials can not satisfy. Thus, people who have strict preferences for Kosher or Halal ingredients (such as for foodstuffs) will have had some difficulty, since pigskins and cattle hides and bones are not desirable sources

for producing such Kosher and Halal gelatins. Others may have concerns surrounding the onset of Bovine Spongiform Encephalopathy and the perceived risk to health that some associate with products derived from such sources of collagen.

An alternative raw material source for producing gelatin is fish collagen, such as is found in fish skins. Careful selection of skins from approved species of fish could therefore allow the production of gelatins that can satisfy Kosher and Halal requirements. Thus, there is an increasing demand for gelatins produced from fish collagen.

One of the more important properties of gelatin is its ability to form thermo- reversible gels. This quality can be assessed using a standard test procedure such as the measurement of Bloom Gel Strength (BS 747 1975), and values such as 50-320g can be achieved. Bloom is the force (in grams) required to depress a standard plunger into a set gelatin of 62/3% concentration that has been kept at 10°C for 16 hours. This gelling ability is related to both the average molecular weight of the gelatin and to the content of the hydroxyproline and proline amino acids in the collagen used. Whereas the content of these amino acids varies little between species of cattle, pigs and many other warm-blooded animals, there is a significant variation among aquatic species. Thus, fish that have evolved in cold waters are found to be somewhat deficient in proline and hydroxyproline content and have a much-reduced tendency to form gels. By contrast, fish evolved in warm waters have higher proline and hydroxyproline content and consequently the gelatin from such species is much more able to form a gel.

Table 1 shows the amino acid composition in residues per 1000 residues for bovine gelatin, non-gelling fish gelatin and gelling fish gelatin, respectively: Another important property of gelatin is its iso-ionic pH in solution. The iso- ionic pH of a gelatin is the pH of a gelatin solution in which there are no other ionic species present (such as when a gelatin solution has been treated with ion-exchange resin). For de-ionised gelatin solutions, iso-ionic

points and iso-electric points are essentially the same. The iso-electric point is the pH at which the gelatin has a net zero charge and thereby shows no net migration on application of an electric field.

Table 1: Amino acid content per 1000 residues Bovine Non-gelling Gelling Amino acid gelatin fish gelatin fish gelatin Aspartic acid 46 52 46 Threonine 16.9 25 26 Serine 36.5 69 37 Glutamic acid 70.7 75 66 Proline 129 102 119 Glycine 333 345 343 Alanine 112 107 121 Valine 20.1 19 17 Methionine 5.5 13 9.5 Isoleucine 12 11 8 Leucine 23.1 23 23 Tyrosine 1.5 3.5 3 Phenylalanine 12.3 13 12 Histidine 4.5 7.5 9.5 Lysine 27.8 25 25 Arginin 46.2 51 54 Hydroxyproline 97.6 53 76 Hydroxylysine 5.5 6 7.5 Fish gelatin and the collagen from which the gelatin is extracted, are, as shown in Table 1, proteins containing about 18 different amino acids. Two of these amino acids, glutamin and asparagine have amide side chains, which can be de-amidated, such as through prolonged exposure to alkaline conditions, to form aspartic and glutamic acids. Exposure to relatively mild acidic conditions does not lead to any significant de-amidation. The de- amidation alters the balance between acidic and basic side chains on the protein, and this accounts for the differences in iso-ionic point of gelatins produced by different processes. Gelatins produced by an alkaline process (hereinafter referred to as Type B gelatins) generally have an iso-ionic pH (pl) in the range 4.8-5.2, whereas gelatins produced by an acidification process (hereinafter referred to as Type A gelatins) generally have an iso- ionic pH in the range of from 6.3-9.2. Type B gelatins tend to have higher

viscosities than Type A gelatins, and consequently pi can give some indication of the Bloom/viscosity ratio that might be expected.

The production of gelatin from fish collagen can prove to be troublesome, and some products may have the undesirable characteristics of fish-like taste or odour. The raw materials also need careful handling and processing to avoid degradation and loss of yield, and to avoid producing a product deemed to be of low quality.

British patent specification no. GB 235 635 describes the use of fish offal for the manufacture of gelatin. The disclosed process includes washing, treating with dilute alkali for 18 to 24 hours, washing and, finally, treating with dilute sulphurous acid, a weak acid. Such a process would produce a Type A gelatin.

European patent specification no. EP 436 266 (also US 5,194,282) discloses a process for preparing fish gelatin comprising (a) treating fish raw material with dilute aqueous alkali, followed by washing with water, (b) treating with dilute aqueous mineral acid, preferably sulphuric acid, followed by washing with water, (c) treating with dilute aqueous organic acid, followed by washing with water and (d) extracting with water at elevated temperatures below 55°C to yield the gelatin product. This process would also produce a Type A gelatin.

Another Type A gelatin-producing process is described in European patent specification no. EP 1016 347. This description indicates the importance of washing the raw fish skins with water containing oxidising agents, such as sodium hypochlorite or hydrogen peroxide, before extracting the washed and acid-treated skins at an acidic pH. However, no alkaline conditioning step is used in this process.

Type B gelatin would be produced by the process described in US patent specification no. 5 484 888 in which fish skins are soaked in an alkaline

solution for 60 days and then the excess alkali is removed before extracting the gelatin from an alkaline solution.

The fish gelatin produced using the processes described herein can be used in a wide variety of applications. A particularly useful application is the making of hard capsules, whereby the fish gelatin may be used in forming the walls of the capsules. The use of gelatin in capsule-making has been known for many years since the original disclosures in the early nineteenth century.

Current production methods are highly sophisticated operations with high machine outputs and this places key requirements on the quality and consistency of the gelatins used. For hard capsules, a typical gelatin specification may include Bloom 235-260 with viscosity 4.3-4.7 mPas (6/3% 60°C) ; for the production of soft capsules, a typical gelatin specification may be Bloom 155-180 with viscosity 3.6-4.2 for a Type B gelatin, or 175-210 Bloom, 2.7-4.2 mPas for a Type A gelatin. Some specifications and much other useful information on capsule production may be found in the text"Hard Capsules"edited by K Ridgeway, published by The Pharmaceutical Press in 1987.

Fish gelatins can also be used for micro-encapsulation, as described in PCT patent specification no. W096/20612 Both gelling and non-gelling fish gelatins may be hydrolyse to produce gelatins with relatively low viscosity and low molecular weights. These hydrolyse fish gelatins may be used to advantage in micro-encapsulation, as well as in tabletting and many other applications. The use of hydrolyse gelatin for encapsulation of vitamin E is described in US patent specification no. US 4 395 422.

It has now been found that fish gelatin with excellent properties can be achieved using processing procedures and sequences that are different from those previously disclosed. We have found that gelatin of excellent quality can be produced from fish sources according to the method of the present

invention. In this process, Type B fish gelatin having a low iso-ionic pH can be obtained.

Accordingly, the present invention provides a process for producing, from fish collagen, a type B gelatin having a pi in the range of from 4.5 to 6.0, which process comprises: (a) treating fish skins with an alkaline medium to provide a mixture thereof having a pH of at least 10; (b) acidifying, using a mineral acid, the mixture produced in step (a) to a pH of less than 7; and (c) acid-extracting the gelatin thereby produced; and, optionally, (d) further processing the extracted gelatin produced in step (c) Preferably, the mixture produced by step (a) has a pH of at least 12, such as about 12.4, and may be as high as 13 or more.

Thus, in step (a), in the case where the alkaline medium is lime, a lime slurry may be prepared to a concentration range such as from 0.6 to 8.0% by weight, such as 2-8% by weight, preferably 4-6% and more preferably 1-2%, especially during the early stages of liming, although these quantities could be reduced, if preferred, for the later stages of liming. Step (a) is undertaken to condition the fish skins in the alkaline (eg saturated lime) solution, such that the amide residues are essentially de-amidated. Preferably, the saturated lime solution and the fish skins are kept in admixture in step (a) for a period in excess of 5 days, and preferably at least 12 days, such as in the range of from 5 to 150 days, such as 15 to 100 days, eg 20 to 70 days.

The lime liquors are preferably replaced at intervals throughout the liming process and the fish skins should be agitated and preferably aerated at intervals. During the early stages of liming, the amide residues in the collagen become de-amidated, thereby releasing ammonia; various non- collagenous proteins and other impurities may be solubilised and removed.

The lime treatment allows the collagen to become conditioned, such that

extraction of gelatin of high quality can be achieved in subsequent processing.

The alkaline conditioning of the fish skins in step (a) is not restricted to the use of excess calcium hydroxide (saturated lime slurry) and may be carried out using various other sources of alkaline materials that allow the production of Type B gelatin. Alkaline materials that could be used include, for example, the oxides and hydroxides of alkali or alkaline earth metals, eg sodium, potassium, lithium, calcium, magnesium and the various mixed salts that incorporate such components. Thus, solutions of sodium hydroxide of suitable concentration may be used over appropriate time periods. When, for example, caustic soda is used in step (a) instead of lime, 0.6-2% sodium hydroxide solution is preferably used and the mixture with the skins allowed to stand for 1 to 20 days, eg about 4 days.

We have therefore found that the use of stronger, more concentrated alkaline conditions over an extended period of time results in the production of a Type B gelatin, in contrast to the Type A gelatins produced by the dilute and mildly basic conditions sometimes used in the prior art.

In step (a), other additives, such as a swelling suppressant, eg sodium sulphate, may be used, if preferred, to suppress excessive swelling and possible subsequent reduction in gelatin yield.

More preferably, step (a) is preceded and/or immediately followed by washing steps. Alternatively or additionally, a preparatory alkalination step, to pre-condition the skins, without causing substantial de-amidation, may precede liming or other strong alkali treatment. Thus, solutions of sodium hydroxide of suitable concentration, such as 0.1-1%, eg 0.2%, may be used over appropriate time periods, such as 0 hours to 5 days, eg about 24 hours; other additives, such as a swelling suppressant, eg sodium sulphate, may be used, if desired, as mentioned above, to suppress excessive swelling and possible subsequent loss of gelatin yield.

It is particularly preferred that frozen or dried skins are used, especially frozen skins, whereby they have been well-preserved. More preferably, the skins are substantially free of other fish offal, such as bones, flesh, heads, and innards. Still more preferred is when the fish skins are substantially free from the skins or other offal of aquatic mammals, including whales and dolphins. The skins may be in de-scaled form.

Preferably, the fish skins are derived from fish that have evolved in warm water, such as certain tuna species, tilapia and Nile perch. Such warm water species tend to exhibit collagen having the preferred amount of proline and hydroxy-proline residues. Accordingly, the invention further provides a process as described above, wherein the fish skins have a collagen content comprising, per 1000 amino acid residues, more than 110 proline residues and/or more than 60 hydroxyproline residues. In cases where the collagen has an amino acid composition outside these criteria, then steps (a) and (b) are preferably undertaken at a reduced temperature in the range of from about 15 to about 5°C.

Especially preferred is when the acidification step (b) is carried out using a dilute mineral acid, such as dilute sulphuric acid, to enable a pH in the range of from 2 to 6, preferably from 3 to 5, such as 2 to 4, eg about 3. Acidification is most preferably undertaken in the absence of added organic acid (s) and/or in the absence of added sulphurous (SO2) moieties. The latter results in a product gelatin having a sulphite content of less than 200 ppm and, preferably, less than 50 ppm. Especially preferred is when the raw material is soaked at the given pH for up to about 24 hours.

In order to prevent deterioration of physical properties, such as Bloom and viscosity, of the extracted gelatin, the acid extraction step (c) is carried out at the lowest temperature possible, typically 30-50°C, although elevation to about 60 to 70°C or higher is practicable, particularly towards the end of the extraction step to help extract all the available gelatin. The acid extraction is

preferably carried out at a pH in the range of from 3 to 5, whereby it may not be necessary to add further mineral acid after acidification step (b).

The further processing step (d) may comprise one or more of the standard separation and/or purification techniques known in the art, including filtration, ion exchange, concentration, sterilisation, drying and the like. Preferably, step (d) includes an ion-exchange reaction using, for example, both anionic and cationic exchange resin to reduce the salts content of extracted liquors.

The use of ion-exchange resins in gelatin manufacture is described by P Caimi in Imaging Science Journal 45 136-138 (1997). Resins can be chosen from any suitable for the purpose, including those manufactured by Rescindion or Rohm & Haas.

In the process of this invention, the fish skins are therefore treated sequentially, preferably in the following stages: defrost the skins, wash, add lime, transfer to process vats (lime pits) and lime for 10-100 days, preferably at least 12 days, transfer to washers, wash, add sulphuric acid to pH about 3, transfer to extractors; extract at 40-50°C, filter, de-ionise, evaporate, dry, grind and store. In this alkali-based process, the gelatin thereby produced is of low iso-ionic pH of around 5 because amide-group (- CONH2)-containing amino acids are converted to acid groups (-COOH).

More preferably, the raw material and process are chosen so as to result in a gelling gelatin.

It is well known by those skilled in the art of gelatin making that gelatin is an excellent medium for the growth of bacteria. Thus, it is desirable that various steps are taken during the manufacturing process to ensure cleanliness of the operation and to reduce risk of bacterial action, which could lead to degradation of product quality and yield. It is not uncommon for process waters to be treated with oxidising agents, such as hypochlorites ; peroxides, such as hydrogen peroxide (H202); free chlorine; or other such agents in order to remove the effects of harmful bacteria. Process waters for washing the raw material or otherwise treating the same may also advantageously

contain such reagents amongst other possible biocidal or biostatic additives.

These procedures are well known in the art, such as from European patent specification no. EP 1 016 347, mentioned hereinabove.

The present invention further provides a gelatin produced by or producible by the alkaline process according to this invention. In particular, it provides a gelatin comprising a thermo-reversible gel at 5% concentration at 20°C.

Gelling fish gelatin (Type B) produced by the alkaline process of this invention is particularly suitable for the making of hard and soft capsules.

Accordingly, this invention further provides the use of a gelatin, produced by or producible by the alkaline process described herein, in the manufacture of capsules for food and/or pharmaceutical applications, especially a soft capsule comprising such a gelatin.

The gelatin of this invention is characterised in having an iso-ionic pH of 4.5 to 6.0, preferably 4.8 to 5.4; the Bloom value is 100-320; preferably 230- 270 for hard capsules and 150-200 for soft capsules ; the viscosity is 3.0-6.5, preferably 4.0-5.0 mPas (62l3%, 60°C) for hard capsules and 3.6-4.2 for soft capsules ; and having no or very little odour or taste. These properties may be obtained through blending various extracts of gelatin to give the required characteristics. Thus, individual batches to be included in such blends may cover a wider range of properties such as Bloom 100-320 and viscosity 3.0- 6.5.

The gelatin prepared according to the process of this invention is also suitable for application to the photographic industry, in view of the particularly high purity of the product. Also, this gelatin has a higher methionine content than gelatin of non-fish origin, which provides other advantages in photographic applications. In particular, this gelatin may act as a carrier for the silver nitrate/potassium bromide reaction and protect the resulting crystals as they form. Accordingly, the present invention further provides a

photographic composition comprising a gelatin produced by or producible by the alkaline process of this invention.

The present invention is illustrated in the following examples.

Example 1-Production of Type B Gelatin A sample of frozen tilapia skins was allowed to thaw out in water containing sodium hypochlorite.

The skins were given a wash in water (1 hour) to remove some of the fat present. A 2191g sample of the drained tilapia skins (42 pieces) was placed in a 10-litre container. Water was added to 8 litres and then 100g lime was added.

The lime treatment was carried out in the laboratory at ambient temperature (19-25°C). The skins were stirred occasionally. Lime changes were carried out after 5 days and after 34 days by draining off the lime liquor and replacing with water and 100g lime. The skins remained tough throughout the liming period.

After 42 days, the skins were divided into two aliquots of 2100g (This is almost double the weight before liming). One portion (B) was returned to the lime liquor to continue liming. The other portion (A) was extracted.

Extraction A: Extraction of Tilapia skins after 42 days in lime The skins were washed in running water over 30 minutes before transferring to a 5 litre polypropylene beaker and covering with water. Sulphuric acid was added over 6 hours to lower the pH to a liquor pH of around pH 3. After 3 hours the liquor pH was 5.0, and the liquor was drained off and replaced with de-ionised water. After a further 2 hours the water was again drained off and replaced with de-ionised water before leaving overnight at pH 3.9. About 18ml concentrated sulphuric acid was used. At this stage, about 20% of the

area of the skins was transparent; some skins were swollen and very soft, whereas others were white opaque and quite tough.

The gelatin was extracted on heating to 40°C in a water bath over 2-4 hours.

The liquor was filtered, de-ionised and the pH adjusted to pH ca 5.5 with sodium hydroxide before allowing to dry on trays at room temperature.

Some of the skins were not extracted within 4 hours and were dried as unextracted residue.

Gelatin yield A1 filtered liquor 2800ml at 3. 3% (92.4g) 76.3g dry A2 filtered liquor 900ml at 7. 0% (63g) 49.6g A3 unextracted residue 71.8g Weight of wet tilapia skins used = 1095. 5g Yield of dry gelatin = 125. 9g 11.5% Total weight recovered = 197. 7g 18.0% Extraction B: Extraction of Tilapia skins after 105 days in lime The skins were washed in running water over 30 minutes to remove excess lime. The weight after draining was 2733g. The skins were transferred to a 5 litre polypropylene beaker and covered with water. Sulphuric acid (13mut) was added over 3 hours to lower the pH to a liquor pH of around pH 5.5. The liquor was drained off and the skins re-weighed (2075g). De-ionised water was added and a further 5ml sulphuric acid added over 2 hours to give a liquor pH of around 3.0. On leaving overnight, the skins had swollen and the liquor pH was 4.1. Most of the skins were soft with some transparency, but about 10-20% were white opaque and less soft.

The gelatin was extracted on heating to 30-40°C in a water bath over 2-4 hours. The liquor was filtered, de-ionised and allowed to dry on trays at room temperature.

Some of the skins were not extracted within 4 hours and were dried as unextracted residue.

Gelatinyield B1 filtered liquor 3820g at 3. 7% (141.3g) 133.4g dry B2 unextracted residue 42.6g Weight of wet tilapia skins used = 1095.5g Yield of dry gelatin = 133.4g 12.2% Total weight recovered = 176. Og 16.1 % Dried gelatin test results Test/Sample A1 B1 Bloomg 288 246 Viscosity mPas (6 2/3% 60°C) 3.67 5.69 pH 5. 24 4. 95 Conductivity uS (6 2/3% @25°C) 220 90 Colour (absorbance of 6 2/3% solution 0.252 0.302 at 420nm) Clarity (absorbance of 6 2/3% solution 0.098 0.098 at 650nm) Ash % <0. 1 <0. 1 Iso-ionic pH 5. 13 5.11 Moisture % 15.2 15.6 Tilapia skins can therefore be used to produce limed gelatin in good yield, and with good chemical and physical properties.

Example 2-Starting Material Preparation of Low pl Fish Gelatin using the Liming Process Tilapia skins (2156g) were added to 0.2% sodium hydroxide solution (8064g), mixed and allowed to stand for 24 hours. The sodium hydroxide solution was drained off and the skins rinsed with water (8000g). The skins (3991 g) were then added to a solution of lime (1.2%, 8100g and left for 12 days. After 12 days the lime solution was removed and the skins washed with water (8000g).

The skins (3764g) were then added to water (5000g), and the pH adjusted and maintained between 2.5-4.0 with the addition of sulphuric acid (77%) over a period of 6 hours. The skins were then allowed to soak at this pH for a further 17 hours. The acid conditioned skins were then heated to 60°C for 2 hours, whilst maintaining the pH at 4.0 with sulphuric acid (77%). After 2 hours the slurry was coarsely filtered to remove the waste skin residue and the resultant extraction filtrate was filtered through a Dicalite 4258 coated W2 filter pad. The filtrate (8812g, 6.4%, pH=4.06) was heated to 55°C and treated with MB6113 mixed ion-exchange resin (950.0g) until the pH was stable (pH=5.65, conductivity=34. S). The filtrate was then filtered through a BECO-KD5G filter pad to remove the ion-exchange resin, followed by filtration through an XE200H filter pad to clarify. The filtrate (7913g, 5.5%) was evaporated to 8.8% (5369g) and poured into a tray and allowed to gel at 4°C, noodle, dried and ground to give Tilapia fish gelatin (446.2g).

Analysis Test Result Moisture (%) 15. 6 Ash (%) <0.1 Nitrogen (%) 15.6 Hydroxyproline (%) 10.2 Bloom @ 6 % (g) 240 pH(1 %, 25°C) 5. 8 Iso-ionic pH 5.7 Viscosity @ 62/3 %, 60°C 4.2 (mPas)

Example 3-Starting Material Preparation of Low pl Gelatin using the Caustic Process Tilapia skins (4341 g) were added to sodium hydroxide solution (1.2%; 19000g), mixed and allowed to stand for 24 hours. The sodium hydroxide solution was then drained off and the skins rinsed with water (19000g). The skins (11297g) were then added to fresh sodium hydroxide (1.2%; 12600g), mixed and allowed to stand for 4 days. The sodium hydroxide solution was then drained off and the skins rinsed with water (12600g).

The skins (15224g) were added to water (10000g), and the pH adjusted and maintained between 2.5-4.0 using sulphuric acid (77%) over a period of 6 hours then allowed to soak at this pH for a further 18 hours. The acid conditioned skins were heated to 55°C for 11/2 hours, whilst maintaining the pH at 4.0 with sodium hydroxide solution (25%). After 11/2 hours the liquor was coarsely filtered to remove the waste skin residue and the resultant extraction filtrate was filtered through a Dicalite 4258 coated W2 filter pad.

The filtrate (pH=4.0) was heated to 55°C and treated with MB6113 mixed ion- exchange resin (3678g) until the pH was stable (pH=5.6, conductivity 7.0, uS).

The filtrate was then filtered through a BECO-KD5G filter pad to remove the ion-exchange resin, followed by filtration through an XE200H filter pad to clarify. The filtrate was evaporated to 10. 2% (9322g) and poured into a tray and allowed to gel at 4°C, noodle, air dried and ground to give low pl Tilapia fish gelatin (906.8g).

Analysis Test Result Moisture (%) 16.5 Ash (%) <0.1 Nitrogen (%) 15.3 Hydroxyproline (% 9.8 Bloom @ 62/3 %(g) 180 pH (1%, 25°C) 5.7 Iso-ionic pH 5. 7 Viscosity @ 62/3 %, 60°C 4.08 (mPas)