WO2014209101A2

WO2014209101A2 - Method of obtaining gelatin from aquatic animals

Info

Publication number: WO2014209101A2
Application number: PCT/MY2014/000191
Authority: WO
Inventors: Fatimah Md. Yusoff; Jamilah Bakar; Mahiran Basri; Maznah Ismail; Mun Hoe Nicholas KHONG
Original assignee: Universiti Putra Malaysia
Priority date: 2013-06-28
Filing date: 2014-06-25
Publication date: 2014-12-31
Also published as: WO2014209101A3

Abstract

The present invention relates to a simple, cost and time effective process of producing gelatin from aquatic animal tissue, which comprises the steps of: (a) treating the cleaned and dehydrated aquatic animal tissue with salt solution, preferably of less than 1.0M; (b) treating biomass obtained after Step (a) with aqueous alkali; (c) washing the alkali-treated biomass with water until the washing water is substantially neutral; (d) treating the neutralized biomass from step (c) with aqueous acid with continuous stirring for up to 4 h; (e) washing the acid-treated biomass with water until the washing water is substantially neutral; (f) extracting with water at pH less or equal to 2 at temperatures in the range of 45°C to 65°C for 6 to 8 h; (g) dialysing the gelatin obtained from step (f) against neutral buffer or deionised distilled water for at least 24 hours; (h) removal of water from the gelatin obtained until moisture content of the resultin gelatine is less than 13%. The process provides a gelatine having excellent properties, such as quality, appearance, gel strength etc., suitable for use in pharmaceutical, cosmeceutical, food and photography products.

Description

METHOD OF OBTAINING GELATIN FROM AQUATIC ANIMALS

Background of the Invention Field of the Invention

The present invention relates to a method of obtaining gelatin from aquatic animals. Background Art

Gelatin is a natural protein of great commercial value. It produces clear thermo-reversible gels when combined with water and is an important ingredient in many types of food, in pharmaceutical formulations (e.g., capsules) and in many other fields.

Gelatin is commonly prepared form bovine hides, pig skin and animal bones. These sources bring about halal or kosher and religious complications and risk of transmitting illnesses (e.g., bovine spongiform encephalophaty).

There were attempts at producing gelatine from marine organisms, which would overcome the disadvantages of the present methods of producing gelatine.

Zhuang et al. (Zhuang, Y.L., Sun, L.P., Zhoa., X., Hou, H. & Li, B.F. (2010) Investigation of Gelatin Polypeptides of Jellyfish (Rhopilema esculentum) for Their Antioxidant Activity in vitro. Food Technol. Biotechnol. 48 (2): 222-228; Zhuang, Y., Li, B., Zhao, X., Yan, M. & Zhang, X. (2009) Extraction of Gelatin from Jellyfish by High Pressure Treatment (Abstract). Food and Fermentation Industries. http://en.cnki.com.cn Article en/CJFDTOTAL-SPFX200905032.htm) described an extraction method utilizing high pressure treatment. They also reported that the jellyfish gelatin of their method had poor solubility, negative sensory properties and weak gel strength which lead his team to produce antioxidative gelatin polypeptides instead.

Manickavasagam (US 2008/0167447 (2008)) filed a patent describing the extraction of gelatin from a marine invertebrate which is particularly referred to abalones. Only gelatin from the 2^nd extract (80°C) was reported to have gel strength in the range of 22-65 g. Up to now, no suitable method of obtaining gelatin of high quality from marine invertebrates was provided, despite several attempts.

Disclosure of the invention

The present invention provides a simple, time and cost effective process of extracting gelatin of superior yield, purity and quality from aquatic animals.

The aquatic animals are preferably zooplanktons, preferably those with nutritive edible value, including protozooplanktons such as copepods, cladocerans and rotifers; as well as metazooplanktons such as cnidarians, molluscan, crustaceans and tunicates.

The aquatic animal tissue can be obtained by, e.g., maceration, homogenization, sonication, cutting and/or slicing fresh or frozen biomass. Preferably, moisture is removed from this raw material as much as possible using methods such as freeze-thawing.

The process comprises the steps of:

(a) treating the cleaned and dehydrated aquatic animal tissue with aqueous solution of inorganic salt of metal of IA or IIA groups, preferably halide of metal of IA or IIA groups, more preferably sodium or potassium halides such as NaCl or KCl, preferably at the concentration of less than 1.0M;

(b) treating biomass obtained after Step (a) with aqueous alkali, preferably at a concentration of 0.025M to 0.25M, more preferably the alkali is NaOH or KOH;

(c) washing the alkali-treated biomass with water until the washing water is substantially neutral;

(d) treating the neutralized biomass from step (c) with aqueous acid with continuous stirring for up to 4 h;

(e) washing the acid-treated biomass with water until the washing water is substantially neutral;

(f) extracting with water at pH less or equal to 2 at temperatures in the range of 45°C to 65°C for 6 to 8 h;

(g) dialysing the gelatin obtained from step (f) against neutral buffer or deionised distilled water for at least 24 hours; (h) removal of water from the gelatin obtained until moisture content of the resulting gelatine is less than 13%.

Preferably, steps (a) to (e) and (g) and (h) are performed at room temperature or below room temperature.

In a preferred embodiment, the pH of the alkali treatment in step (b) is in the range of 9.5 to 10.5. In another preferred embodiment, the pH of the acid treatment in step (d) is in the range of 1 to 2. More preferably, the acid treatment can be performed in a continuous manner or in repetition with washings with water in between. The acid treatment can preferably be performed with one type of acid, more preferably weak acids, or with interchanging acids between strong acids and weak acids.

Preferably, the washing in steps (c) and (e) should achieve the pH of 7±1.

At most 3 times repeating of the treatments of steps (a) and (b) is preferable. The aqueous alkali and aqueous acid preferably are used in concentrations within the range of about 0.05 to about 3% wt./vol.

The alkali is preferably selected from sodium hydroxide, calcium hydroxide and magnesium hydroxide.

The weak acids are preferably selected from acetic acid, citric acid, tartaric acid, malic acid and lactic acid.

The strong acids are preferably selected from sulfuric acid and hydrochloric acid.

Preferably, the process can further comprise the steps of analyzing the gelatin, in particular amino acid analysis (namely the analysis of glycine, proline, hydroxyproline and threonine content) and gel strength. Another object of the invention is the gelatin obtainable by the process of the present invention, showing the gel strength ranging from 200 to 300 g Bloom.

The invention further includes a pharmaceutical composition comprising the gelatin obtainable by the process of the invention and a pharmaceutical product comprising such composition. The pharmaceutical product includes hard capsule, soft capsule, tablets, emulsions, surgical sponges, ointments, salves, jellies, suppositories, encapsulation material, drug carrier, film material, wound dressing, plasma substitute and natural protective coating for medicines.

The invention further includes a cosmeceutical composition comprising the gelatin obtainable by the process of the invention and a cosmeceutical product comprising such composition. The cosmeceutical product includes cream, topical formulations, lotion, eye cream, ointment or gel, sun-screen, hair care products, lipsticks and fingernail formulas, oral administration, face mask cream, antioxidant supplements, anti-inflammatory medicine, and/or anti-irritant medicine.

The invention further includes a nutraceutical composition comprising the gelatin obtainable by the process of the invention and a nutraceutical product comprising such composition. The nutraceutical product includes antioxidant and/ or anti-inflammatory supplements, active encapsulations, excipient, amino acids rich formulation, arthritis/joint care formulas, nutritional bars and protein drinks.

The invention further includes a food composition comprising the gelatin obtainable by the process of the invention and a food product comprising such composition. The food product includes beverages, dairy products, bakeries, confectioneries, meat products, chocolates, and any application in food formulation/s as an ingredient or for any functional properties. The invention further includes a photography composition comprising the gelatin obtainable by the process of the invention and a photography product. The photographic grade gelatin is used in all of the layers of a photographic product including the silver halide crystal-containing emulsion layer, coating layer, sub-coating layer, anti-halogen layers and non-curl layer.

Brief description of Drawings

Figure 1. Appearance of gelatin extracted from jellyfish at the temperature of (A) 50°C and (B) 55°C; SEM appearance of gelatin extracted from jellyfish at the temperature of (C) 50°C and (D) 55°C. Figure 2. The proximate composition of jellyfish gelatin extracted at different temperatures.

Example of carrying out the Invention

Materials and chemicals: River jellyfish (Acromitus hardenbergi) is an edible jellyfish caught extensively from a local fisherman village in Bagan Datoh, Perak. Freshly caught jellyfish weighed between 400 g to 800 g (bell) and 150 g to 350 g (oral arms) per animal. The jellyfish were stored at -20°C until used. All other chemicals used were of analytical grade. Extraction process used:

Step 1. Raw intact and unprocessed jellyfishes was transported at 4°C to the laboratory and stored at -20°C prior to extraction.

Step 2. Dewatering of jellyfish tissues was performed using freeze-thaw procedure and the resulting tissue was washed appropriately to remove excessive mucus.

Step 3. The jellyfish was cut in strips of 1-2 cm wide and weighed.

Step 4. The tissue was stirred gently in normal saline (1 :5 w/v) for 20 min at room temperature.

Step 5. The tissue was pretreated in 0.2N sodium hydroxide (1 :5 w/v) for 60 min at room temperature.

Step 6. The alkaline-treated tissues were washed with excessive tap water until pH 7 ± 1. Step 7. Pretreatment of the washed tissues continued by resuspending the tissues into 3% acetic acid (1 :5 w/v) for 3 hr at room temperature with gentle stirring.

Step 8. The acid-treated tissues was rewashed as in Step 6. Step 9. The gelatin was extracted from the blended (2 min, Waring blender), washed pretreated tissue with acidified distilled water (pH 1.0 ± 1, 1 :2 w/v) with stirring at 50 ± 5 °C for 6 hr.

Step 10. The gelatin slurry was centrifuged at 6000 xg for 10 min at room temperature. The pellet was collected for repeated extraction for 3 hr.

Step 11. The supernatant obtained (jellyfish gelatin, JG) was dialysed against 10 volumes of deionised distilled water for 24 hours with exchange of distilled water every 6 hours. Step 12. The buffer exchanged gelatin was collected and pre- frozen to -20°C prior to lyophilization.

Step 13. Dried JG was homogenized using a kitchen blender.

Step 14. The milled JG was stored for analysis.

Analysis of Lyophilized Jellyfish Gelatin:

Yield: The yields of gelatin were obtained and calculated in terms of weight and protein basis as follows:

(i) Yield (wet weight basis) = (Dry weight of gelatin/wet weight of jellyfish umbrella) x 100%

(ii) Yield (dry weight basis) = (Dry weight of gelatin)/(wet weight of jellyfish umbrella - moisture contain) x 100%

(iii) Yield (protein basis) = (Dry weight of gelatin/protein content of jellyfish umbrella) x 100%

Appearance: Note was made of the colour, odour, and texture of JG by visual inspection.

Proximate composition, colorimetry and pH: Moisture, total protein, total crude lipid, total ash and total carbohydrate contents were determined according to the methods of AOAC (Official Methods of Analysis. 17th ed. Assoc. Anal. Chem., Arlington, VA, 2000). Colorimetry was determined as Hunter values of L, a and b using a chromameter while pH by a well calibrated pH meter of a 6.67% JG. Gel strength: The gel strength, or Bloom, was determined according to the procedure of ISO 9665 to 6.67% JG using 0.5 in. diameter cylindrical probe (radiused) measuring the force at 4 mm penetration. Amino acids composition: Amino acids composition was determined as PITC (phenylisothiocyanate) derivatives using HPLC-DAD (High-Performance Liquid Chromatography equipped with Diode- Array Detector).

Scanning electron microscopy: Ultrastructural examination of gelatin was performed using scanning electron microscopy (SEM). Lyophilized samples were mounted on a stub and gold-sputtered (Bal-Tec, SCD005, Balzers, Germany) prior to microscopic analysis. Observations and imaging were done with a scanning electron microscope (JEOL, JSM- 6400, Japan) at an acceleration voltage of 20 kV. The EM was utilized to study the ultrastructure as well as the surface appearance of JG.

The results of the analysis are summarized in tables 1 and 2 and Figures 1 and 2.

Table 1. The yield and physicochemical properties of gelatin from jellyfish.

Properties Gelatin extracted at different temperature, °C

50 55

Yield, % Wet basis 0.69 0.45

Dry basis 38.50 28.03

Protein basis 73.34 53.40

Gel strength, g Bloom 253.5 282.9

pH 3.9 3.9

Appearance Snowy white Snowy white

Texture Light textured, crumb like Very fluffy and light textured, spongy-like

Odour description Slight detectable fishy Barely detectable fishy odour odour Table 2. Amino acids contents (mg/g) of the gelatins obtained from jellyfish

Gelatin extracted at different temperature Amino acids (g/ lOOg)

50°C 55°C

Asp 3.4 1.8

Glu 8.1 6.1

Hyp 5.1 5.4

Ser 2.4 2.6

Gly 17.8 19.4

His 5.2 2.2

Arg 5.1 5.5

Thr 9.6 10.9

Ala 3.8 4.3

Pro 8.5 9.4

Tyr 1.6 1.9

Val 4.4 4.8

Met 1.2 1.3

Cys 5.0 5.8

He 4.0 4.4

Leu 5.1 8.2

Phe 6.4 1.8

Lys 3.4 4.2

Total amino acid 100.0 100.0

Total imino acid 13.6 14.9

Claims

1. A process of producing gelatin from aquatic animal tissue, which comprises the steps of:

(a) treating the cleaned and dehydrated aquatic animal tissue with aqueous solution of inorganic salt of metal of IA or IIA groups, preferably of less than 1.0M;

(b) treating biomass obtained after Step (a) with aqueous alkali;

(g) dialysing the gelatin obtained from step (f) against neutral buffer or deionised distilled water for at least 24 hours;

(h) removal of water from the gelatin obtained until moisture content of the resulting gelatine is less than 13%.

2. The process of claim 1, wherein the aquatic animals are zooplanktons, preferably selected from copepods, cladocerans, rotifers, cnidarians, molluscan, crustaceans and tunicates.

3. The process of claim 1, wherein the aquatic animal tissue is obtained by maceration, homogenization, sonication, cutting and/or slicing fresh or frozen biomass.

4. The process of claim 1 , wherein moisture is removed from the raw animal aquatic tissue prior to step (a) as much as possible using methods such as freeze-thawing.

5. The process of any of the preceding claims, wherein steps (a) to (e) and (g) and (h) are performed at room temperature or below room temperature.

6. The process of any of the preceding claims, wherein the pH of the alkali treatment in step (b) is in the range of 9.5 to 10.5.

7. The process of any of the preceding claims, wherein the pH of the acid treatment in step (d) is in the range of 1 to 2.

8. The process of any of the preceding claims, wherein the acid treatment is performed in a continuous manner or in repetition with washings with water in between.

9. The process of any of the preceding claims, wherein the acid treatment is performed with one type of acid, more preferably weak acid, or with interchanging acids between strong acids and weak acids.

10. The process of any of the preceding claims, wherein the washing in steps (c) and (e) achieves the pH of 7± 1.

1 1. The process of any of the preceding claims, wherein the aqueous alkali and aqueous acid are used in concentrations within the range of about 0.05 to about 3% wt./vol.

12. The process of any of the preceding claims, wherein the alkali is selected from sodium hydroxide, calcium hydroxide and magnesium hydroxide.

13. The process of any of the preceding claims, wherein the weak acids are selected from acetic acid, citric acid, tartaric acid, malic acid and lactic acid.

14. The process of any of the preceding claims, wherein the strong acids are selected from sulfuric acid and hydrochloric acid.

15. The process of any of the preceding claims, wherein the process further comprises the steps of analyzing the gelatin, in particular amino acid analysis and gel strength.

16. A gelatin obtainable by the process of the present invention, showing the gel strength ranging from 200 to 300 g Bloom.

17. A pharmaceutical composition comprising the gelatin of claim 16.

18. A pharmaceutical product comprising the composition of claim 17, selected from hard capsule, soft capsule, tablets, emulsions, surgical sponges, ointments, salves, jellies, suppositories, encapsulation material, drug carrier, film material, wound dressing, plasma substitute and natural protective coating for medicines.

19. A cosmeceutical composition comprising the gelatin of claim 16.

20. A cosmeceutical product comprising the composition of claim 19, selected from cream, topical formulations, lotion, eye cream, ointment or gel, sun-screen, hair care products, lipsticks and fingernail formulas, oral administration, face mask cream, antioxidant supplements, anti-inflammatory medicine, and/or anti-irritant medicine.

21. A nutraceutical composition comprising the gelatin of claim 16.

22. A nutraceutical product comprising the composition of claim 21, selected from antioxidant and/ or anti-inflammatory supplements, active encapsulations, excipient, amino acids rich formulation, arthritis/joint care formulas, nutritional bars and protein drinks.

23. A food composition comprising the gelatin of claim 16.

24. A food product comprising the composition of claim 22, selected from beverages, dairy products, bakeries, confectionaries, meat products, chocolates, and any application in food formulation/s as an ingredient or for any functional properties.

25. A photography composition comprising the gelatin of claim 16.

26. A photography product comprising the composition of claim 25 in any layer selected from silver halide crystal-containing emulsion layer, coating layer, sub-coating layer, anti- halogen layers and non-curl layer.