WO2002094958A1

WO2002094958A1 - Improved acid process for the preparation of type a fish gelatin

Info

Publication number: WO2002094958A1
Application number: PCT/GB2002/002348
Authority: WO
Inventors: Roger Trevor Jones; David Stanley Field
Original assignee: Croda International PLC
Current assignee: Croda International PLC
Priority date: 2001-05-21
Filing date: 2002-05-20
Publication date: 2002-11-28
Anticipated expiration: 2003-11-21
Also published as: GB2377941B; GB0211557D0; GB2377941A; GB0112327D0

Abstract

A process is described for producing gelatin from fish collagen, which process comprises: (a) treating the fish skins with a 0.1-1 % w/w solution of a base for a period long enough to allow swelling of the skins but less than 5 days; (b) acidifying with a mineral acid the mixture produced in step (a) to a pH in the range of from 1 to 6; and (c) acid-extracting the gelatin from the prepared skins; and, optionally (d) further processing the extracted gelatin produced in step (c). Also described is the use of a gelatin, prepared by or preparable by a method according to this invention, in the manufacture of capsules for food, cosmetic and/or pharmaceutical applications; particularly soft gelatin capsules.

Description

Improved Acid Process for the Preparation of Type A Fish Gelatin

The present invention relates to a method for the manufacture of gelatin from fish collagen, the gelatin thereby produced and its uses, particularly as wall material for capsules. In particular, this invention relates to an improved acid process for the production of gelatin having advantageous physical characteristics.

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 cannot satisfy. Thus, people who have strict preferences for Kosher or Halal ingredients (such as for foodstuffs) will have had some difficulty, since pigskins, 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 1 19

Glycine 333 345 343

Alanine 112 107 121

Valine 20.1 19 17

Methionine 5.5 13 9.5

Isoleucine 12 1 1 8

Leucine 23.1 23 23

Tyro sine 1.5 3.5 3

Phenylalanine 12.3 13 12

Histidine 4.5 7.5 9.5

Lysine 27.8 25 25

Arginine 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, glutamine 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 (pi) 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 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 1 016 347. 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 capsules, whereby the fish gelatin may be used in forming the walls of the capsules. The use of gelatin in capsule manufacture 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 ²/₃% 60°C); for the production of soft capsules, a typical gelatin specification may be Bloom 155-180 with viscosity 3.6-4.2 mPas 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. WO96/20612

Both gelling and non-gelling fish gelatins may be hydrolysed to produce gelatins with relatively low viscosity and low molecular weights. These hydrolysed fish gelatins may be used to advantage in micro-encapsulation as well as in tabletting and many other applications. The use of hydrolysed 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 A fish gelatin, having a high iso-ionic pH, such as in the range of from 6.3-9.2 and preferably at least 8.0, can be obtained.

Accordingly, the present invention provides a process for producing gelatin from fish collagen, which process comprises:

(a) treating the fish skins with a 0.1-1 % w/w solution of a base for a period long enough to allow swelling of the skins but less than 5 days;

(b) acidifying with a mineral acid the material produced in step (a) to a pH in the range of from 1 to 6; and

(c) acid-extracting the gelatin from the prepared skins; and, optionally,

(d) further processing the extracted gelatin produced in step (c).

Preferably, the base, such as sodium hydroxide, is present as a 0.2-0.8% solution (by weight), more preferably around 0.2% w/w. Step (a) is carried out in order to pre-condition the skins in the base (eg NaOH) solution without any significant de-amidation of amide residues. Without such a pre- conditioning step, the product gelatin may retain a fishy odour and be of poorer clarity. Preferably, the sodium hydroxide and the fish skins are kept in admixture in step (a) for a period in the range of from 6 to 48 hours, such as around 24 hours.

More preferably, step (a) is preceded and/or immediately followed by washing steps. Especially preferred is when the step (a) is preceded by a wash including an oxidising agent, such as H₂O₂.

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

Especially preferred is when the acidification step (b) is carried out using undiluted mineral acid, such as sulphuric acid, preferably of pH about 1 to 3, which is added to the aqueous mixture of skins, to result in an equilibrium pH of the solution in the range of from 3 to 5, such as about 3.5 to 4. The 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 the physical properties, such as Bloom and viscosity, of the extracted gelatin, the extraction step (c) is carried out at acid pH, preferably in the range given above, such as about 4, especially using sulphuric acid as the mineral acid. Usually, it is not necessary to add further acid after the acidification step (b) in order to obtain the desired pH for acid extraction. The acid extraction is likewise preferably carried out in the absence of added organic acid(s) and/or in the absence of added sulphurous (SO₂) moieties. It is preferably carried out at the lowest temperature possible, typically 40-50°C, although elevation in the range of from 55° to about 70°C or higher is practicable, particularly towards the end of the extraction step to help extract all the available gelatin from the acidified skins. 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 resins to reduce the salts content of extracted liquors. The use of ion exchange resin 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.

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, 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 (H₂O₂), 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 advantageously contain such reagents. These procedures are well known in the art and are described in European patent specification no. EP 1 016 347.

In the process of this invention, the fish skins are therefore treated sequentially, preferably in the following stages: defrost the skins, wash, add sodium hydroxide to about 0.2% and hydrogen peroxide, steep for 2 - 48 hours, wash, add sulphuric acid to pH about 3, transfer to extractors; extract at 40-50°C, filter, deionise, evaporate, dry, grind and store. More preferably, the process is chosen so as to result in a gelling gelatin.

Gelling fish gelatin (Type A) produced by the acid process of this invention can be used for the manufacture of soft capsules. The gelatin is characterised as having an iso-ionic pH of 6.0 to 9.5, preferably 7.5-9.0; the Bloom value is 100-320, preferably 160-200; the viscosity is 2.0-5.5, preferably 2.7-4.2 mPas (6²/₃% 60°C); 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 2.0 - 5.5.

Accordingly, the present invention further provides:

(a) the use of a gelatin, prepared by or preparable by a method according to this invention, in the manufacture of capsules for food, cosmetic and/or pharmaceutical applications;

(b) a soft capsule comprising a gelatin according to (a);

(c) a gelatin derivative derived or derivable from a gelatin according to (a)

(d) a gelatin according to (a) or a gelatin derivative according to (c) for use in the preparation of a food, cosmetic or pharmaceutical product.

The present invention is illustrated by the following examples.

Example 1 - Process for Preparing Type A Gelatin from Tilapia Fish

Skins

Tilapia skins were defrosted and a known weight (1005g) placed in a beaker. Eight times the skins' weight of water (8 kg) was added and 25% sodium hydroxide (64g) added to produce a 0.2 % solution. This was left to stand overnight.

The skins were then washed in cold water until the pH was less than nine. Eight times the skins' weight of water (8kg) was added and 98% sulphuric acid (16g) added to produce a 0.2% solution. The skins were again left to stand overnight. Next morning, the skins were washed in cold water until the pH was between six and seven.

Four times the skins' weight of water was added (4kg), and the temperature raised to 55°C and the pH maintained between 3.5-4.0 with 98% sulphuric acid during a two hour extraction. The liquor was then filtered and the weight and gelatin concentration (measured by refractive index Rl) noted. The liquor was evaporated to 8% Rl. The concentrated liquor was filtered and then placed in plastic trays to cool and set overnight in the fridge. This was then minced and air-dried before testing.

Analysis of the fish skin raw material

As shown below, Tilapia skins can therefore be used to produce a gelatin with an excellent Bloom of 198g, and good viscosity of 4.96 mPas. The moisture and ash-free yield was 34.3%.

Analysis of fish gelatin

Example 2 - Process for Preparing Type A Gelatin from Tuna Fish Skins

Skins from yellow-finned tuna were defrosted and a known weight placed in a beaker. Eight times the skins' weight of water was added, and 25% sodium hydroxide added to produce a 0.2 % solution. This was left to stand overnight.

The skins were then washed in cold water until the pH was less than ten. Eight times the skins' weight in water was added and 98% sulphuric acid added to produce a 0.2% solution. The skins were again left to stand overnight.

Next morning the skins were again washed in cold water. The same weight of water as before was added, the temperature raised to 55°C and the pH maintained between 3.5-4.0 with 98% sulphuric acid for 2 hours. The liquor was then filtered and the weight and Rl noted. The liquor was evaporated to 15% Rl. The concentrated liquor was filtered and then placed in plastic trays to cool and set overnight in the fridge. This was then minced and air-dried before submitting for testing.

Analysis of the fish skin raw material

Analysis of fish gelatin

Tuna skins can therefore be used to produce a gelatin with a good Bloom of 186g, having good colour and clarity, and a good viscosity of 5.11 mPas. The moisture and ash free yield was also good at 45.1%.

Claims

1. A process for producing gelatin from fish collagen, which process comprises: (a) treating the fish skins with a 0.1-1 % w/w solution of a base for a period long enough to allow swelling of the skins but less than 5 days;

(b) acidifying with a mineral acid the mixture produced in step (a) to a pH in the range of from 1 to 6; and

(c) acid-extracting the gelatin from the prepared skins; and, optionally, (d) further processing the extracted gelatin produced in step (c).

2. A process according to claim 1 , wherein the pH of the mineral acid is in the range of from 1 to 3.

3. A process according to claim 1 or claim 2, wherein the acidification step (b) is carried out to an equilibrium pH of the mixture in the range of from 3 to 5.

4. A process according to any preceding claim, wherein steps (b) and (c) are carried out in the absence of added organic acid(s).

5. A process according to any preceding claim, wherein the acidification step (b) is undertaken in the absence of added sulphurous (SO₂) moieties.

6. A process according to any preceding claim, wherein the acidification step (b) is carried out using dilute sulphuric acid.

7. A process according to any preceding claim, wherein the acidification step (b) is carried out using in the order of 0.2% w/w sulphuric acid.

8. A process according to any preceding claim, wherein the acid extraction step (c) is carried out at a temperature in the range of from 55°- 70°C.

9. A process according to any preceding claim, wherein the base in step (a) is sodium hydroxide.

10. A process according to any preceding claim, wherein the base in step (a) is present as a 0.2-0.8% solution (by weight).

11. A process according to any preceding claim, wherein, in step (a), the fish skins are kept in admixture for a period in the range of from 6 to 48 hours.

12. A process according to any preceding claim, wherein, in step (a), the skins are substantially free from other fish offal, including bones, flesh or scales.

13. A process according to any preceding claim, wherein, in step (a), the fish skins are derived from fish that have evolved in warm water.

14. A process according to any preceding claim, wherein, in step (a), the fish skins are derived from tuna species, tilapia and/or Nile perch.

15. A process according to any preceding claim, wherein, in step (a), the fish skins are substantially free from the skins or other offal of aquatic mammals.

16. A process according to any preceding claim, wherein the further processing step (d) comprises one or more of the standard separation and/or purification techniques known in the art selected from filtration, ion-exchange, concentration, sterilisation and drying.

17. A gelatin produced by a process according to any of claims 1 to 16.

18. A gelatin according to claim 17, comprising a thermo-reversible gel at 5% concentration at 20°C.

19. A gelatin comprising a gelling gelatin produced by a process according to any of claims 1 to 18, which gelatin has one or more of the following characteristics: an iso-ionic pH of 6.0 to 9.5; a Bloom value of 100-320; a viscosity of 2.0-5.5 mPas (6²/₃% 60°C); and no or very little odour or taste.

20. A gelatin comprising a gelling gelatin produced by a process according to any of claims 1 to 18, which gelatin has one or more of the following characteristics: an iso-ionic pH of 7.5-9.0; a Bloom value of 160-200; a viscosity of 2.7-4.2 mPas (6²/₃% 60°C); and no or very little odour or taste.

21. The use of a gelatin according to any one of claims 17 to 20, in the manufacture of capsules for food, cosmetic and/or pharmaceutical applications.

22. A soft capsule comprising a gelatin according to any one of claims 17 to 20.

23. A gelatin derivative derived or derivable from a gelatin according to any one of claims 17 to 20.

24. A gelatin according to any one of claims 17 to 20 or a gelatin derivative according to claim 23 for use in the preparation of a food, cosmetic or pharmaceutical product.