WO2021229271A1

WO2021229271A1 - Process for preparation of product containing mucopolysaccharides from clam tissue

Info

Publication number: WO2021229271A1
Application number: PCT/IB2020/054547
Authority: WO
Inventors: Porntip VIJARNPREECHA; Surapong PINITGLANG; Ratchanee SAIPRAJONG
Original assignee: Gold Crown Co., Ltd.; Genius Foods Co.,Ltd.
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2021-11-18

Abstract

Provided is a process for preparation of a product containing mucopolysaccharides from clam tissue, said process comprises contacting the clam tissue with a liquid solvent at a temperature of 80-90°C for not less than 1 hour to obtain a starting solution; modifying properties of the starting solution, comprising steps of separating solid impurities from the starting solution using a method selected from centrifugation or microfiltration, and effectively separating mucopolysaccharides having a molecular weight of more than 150,000 Da from the starting solution using ultrafiltration method; contacting the starting solution with enzymes comprising at least one type of enzyme selected from the group of cysteine protease with an enzyme activity of 2-10 units per milligram of protein at a temperature of 50-60°C, pH of 6.5-7 for 3-6 hours to obtain a digested solution; heating the digested solution to a temperature at which the enzyme activity is effectively prevented; effectively recovering mucopolysaccharides having an average molecular weight of not more than 4,000 Da from the solution using ultrafiltration method; and improving stability of the recovered mucopolysaccharides.

Description

SPECIFICATION

TITLE OF THE INVENTION

Process for Preparation of Product Containing Mucopolysaccharides from Clam

Tissue

1. Field of the invention

The present invention relates to the preparation of food or pharmaceutical products, and particularly to the preparation of such products which contain mucopolysaccharides.

2. Background of the invention

Mucopolysaccharides, also known as glycosaminoglycans (GAGs), are polymeric carbohydrates which contain both sugar molecules and proteins as repeating units in the same macromolecular chain. Mucopolysaccharides may be divided into 5 subtypes, namely chondroitin sulfate, keratan sulfate, dermatan sulfate, heparan sulfate, and hyaluronan (Handley, et al, 2006).

Mucopolysaccharides have many important functions in the human body. The functions specified in various reports include moisture retention, joint lubrication, antioxidation, anti-inflammation, facilitating the process of exchanging compounds between cells, etc. The amount of mucopolysaccharides in the human body decreases with aging; therefore, the elderly need more mucopolysaccharides intake than that needed by younger people to compensate for such decreased amount. It is well known in the art that in case of consumption of mucopolysaccharides, if the consumed mucopolysaccharides have low molecular weight (that is, having short macromolecular chains), the body will be able to digest, absorb, and utilize the consumed mucopolysaccharides more efficiently.

Certain reports specify that mucopolysaccharides may be found in animal tissues. Animals with a particularly high level of mucopolysaccharides often have many mucous glands, particularly aquatic animals, and more particularly mollusks.

The patent application publication No. CN 1746191 (A) of the People's Republic of China suggests a method of extraction and preparation of mucopolysaccharides from comb scallop comprising weighing the scallop’s meat, putting the meat in water, preparing a uniform viscous solution, adding protease enzyme into said solution, heating, carrying out enzymatic hydrolysis reaction, centrifugation, deposit separation, adding secondary alcohol solution to the deposit, deposition, adding distilled water to the separated deposit, preparing solution, heating, adding into activated carbon, cooling, centrifugation, collecting supernatant, extracting and filtering to obtain a purified liquid, re-filtering to obtain a product having a molecular weight in the range of 5,000-10,000, and freezing and drying the obtained product.

3. Summary of the invention

One objective of the invention according to this application is to provide a process for preparation of a product containing mucopolysaccharides from clam tissue, which contains more mucopolysaccharides than other raw materials used in the background art.

Another objective of the invention according to this application is to provide a process for preparation of the above-mentioned product that is simple, uses less inorganic chemicals, and requires reduced operating costs.

Another objective of the invention according to this application is to provide a process for preparation of the above-mentioned product, whereby the preferable product, in which the mucopolysaccharides have a low average molecular weight, can be prepared.

In one embodiment, the process for preparation of product containing mucopolysaccharides from clam tissue comprises contacting clam tissue with a liquid solvent at a temperature of 80-90°C for not less than 1 hour to obtain a starting solution; modifying the properties of the starting solution comprising steps of separating solid impurities from the starting solution using a method selected from centrifugation or microfiltration, and effectively separating mucopolysaccharides having a molecular weight of more than 150,000 Da from the starting solution using ultrafiltration method; contacting the starting solution with enzymes comprising at least one type of enzyme selected from the group of cysteine protease with an enzyme activity of 2-10 unit per milligram (U/mg) of protein at a temperature of 50-60°C, pH of 6.5-7 for 3-6 hours to obtain a digested solution; heating the digested solution to a temperature at which the enzyme activity is effectively prevented; effectively recovering mucopolysaccharides having an average molecular weight of not more than 4,000 Da from the solution using ultrafiltration method; and improving stability of the recovered mucopolysaccharides.

In one embodiment, said clam includes Ridged venus clam and Pacific yellow cockle.

In one embodiment, said clam is a member of the family Veneridae.

In one embodiment, said clam is a member of the genus Meretrix.

In one embodiment, said enzymes have an enzyme activity of 10 units per milligram (U/mg) of protein.

In one embodiment, the at least one type of enzyme selected from the group of cysteine protease is papain enzyme.

In one embodiment, the starting solution is contacted with the enzymes at a temperature of 55°C, pH of 6.5-7 for 6 hours.

In one embodiment, the separation of mucopolysaccharides separates the mucopolysaccharides having a molecular weight of more than 20,000 Da from the starting solution.

In one embodiment, the improving of stability of the recovered mucopolysaccharides is carried out by spray-drying method.

In one embodiment, the spray-drying method is carried out at an inlet air temperature of 120 °C and an outlet air temperature of 95°C.

In addition, the inventor also appreciates that the process according to various embodiments according to the present invention can also be applied to tissue of abalone, wing shells, oysters, mussels, surf clams or other mollusks which have similar natures. Although it is not specifically specified, those who have ordinary skills in the art, once read the specification of the invention in this application, will understand that the use of tissue of abalone, wing shells, oysters, mussels, surf clams, or other mollusks which have similar natures, will result in the similar properties and functions, and produce the results which are similar to a process according to the present invention. 4. Brief description of the drawings

Figure 1 shows the steps in the process for preparation of product containing mucopolysaccharides from clam tissue according to one embodiment of the present invention.

Figure 2 shows the result of molecular weight analysis of the product containing mucopolysaccharides from clam tissue, the product being prepared using the process according to one embodiment of the present invention when used with a commercially available enzyme mix.

Figure 3 shows the result of molecular weight analysis of the product containing mucopolysaccharides from clam tissue, the product being prepared using the process according to one preferable embodiment of the present invention when used with papain enzyme.

5. Detailed description of the invention

The above-mentioned content including the description of the embodiments of the invention which will be discussed in detail below will not limit the scope of the invention to only various embodiments that are disclosed in this application but provide examples of the various embodiments of the present invention for better understanding of the concept of the invention.

Moreover, the characteristics, advantages, and features of the described embodiment may be combined in any suitable manners in one or more embodiments. A person skilled in the art will understand from the explanation of this application that this invention can be practiced without certain characteristics or advantages of the exemplary embodiments accompanying specific explanation. In other cases, additional characteristics and advantages may be perceived in certain embodiments that may not be present in all exemplary embodiments of the invention.

Hereinafter, the details of the various embodiments of this invention that are shown in the accompanying figures will be referred to. It should be noted that the figures including the text that appear in the figures will be in a simple form for ease of explanation, and not be limited to the meanings mentioned herein. In addition, specifying a quantity of one as used in the description would mean “at least one”.

“Approximately” or words with similar meaning in this application when used for describing a value or amount that may be measured, such as length, time, temperature, concentration, coefficient, and the like, would include variations in said value or amount in the range of ±20%, ±10%, ±5%, ±1. %, and ±0.1% from the exact specified value as appropriate.

In this application, various embodiments according to the invention may be presented in a range format. Therefore, it should be understood that the description in the range format is for convenience and conciseness. It was not intended and should not be interpreted in a way that would strictly limit the scope of the invention of this application. Therefore, the description of ranges should be considered to cover every sub-range and every single value that fall in that range. For example, specifying a numeric range of from 1 to 6 should be considered to include sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and any single numeric values such as 1, 2, 2.7, 3, 4, 5, 5.3, 6, etc. as well. Such considerations shall be considered valid regardless of the width of the specified range.

In this application, unless otherwise specified “microfiltration” means a method for separating the impurities which generally means solid particles from a liquid by physical filtering, by which said liquid is passed through a porous filtering medium which generally can trap particles that are not smaller than the predetermined pore size. In general, said predetermined pore size is approximately 0.1 to 1.0 micron. This does not depend on other filtering details e.g. parameters such as temperature, pressure, etc., design options such as materials or thickness of the porous medium, etc. and other devices such as liquid pumps, etc. which an ordinarily skilled person would be able to use such ordinary skills to select or specify as appropriate.

In this application, unless otherwise specified “ultrafiltration” means a method for separating the impurities from a liquid in a way similar to microfiltration, which is commonly used in the art to filter macromolecular compounds. Said method has at least one characteristic, that is, its porous medium being a membrane material or more particularly a semipermeable membrane, and said material’s predetermined pore size is approximately 0.01 to 0.1 micron.

As known in the art, the pore size of the porous medium for the above “microfiltration” or “ultrafiltration” method may optionally be represented in the form of molecular weight cut-off (MWCO).

Figure 1 shows the steps in the process for preparation of product containing mucopolysaccharides from clam tissue according to one embodiment of the present invention, in which, according to said process, the clam is a member of the family Veneridae and genus Meretrix and the liquid solvent is water, preferably, at least water that is acceptable in the food industry such as water which has been filtered by reverse osmosis (RO) or water with less impurities, and at least one type of enzyme selected from the cysteine protease group is papain enzyme.

The process according to Figure 1 begins with the step wherein the clam tissue is contacted with a liquid solvent (10) at the temperature in the range of approximately 80 to 90°C for 1 hour. Preferably, the contact is carried out such that most or all of clam tissue are submerged in the liquid solvent to obtain a starting solution; and more preferably, said contact should be performed together with stirring, beating, agitating, shaking, spinning, or other similar mechanical agitation. Said mechanical agitation may or may not intend to make the starting solution homogeneous, but preferably, the starting solution is intended to become homogeneous substantially or entirely.

Then, according to the process according to Figure 1, the starting solution will undergo property modification (20) comprising at least 2 sub-steps, which are separation of solid impurities from the starting solution (22) and the effective separation of mucopolysaccharides having a molecular weight of more than approximately 150,000 Da from the starting solution (24).

Preferably, the separation of the solid impurities from the starting solution (22) is performed before the effective separation of mucopolysaccharides having a molecular weight of more than approximately 150,000 Da from the starting solution (24). Preferably, before the above sub-steps (22), (24), the starting solution should be filtered through a pre-fdter material. More preferably, the pre-filter material has a pore size of approximately 100 microns.

The separation of the solid impurities from the starting solution (22) may be performed using any one of centrifugation and microfiltration or a combination thereof. Preferably, at least microfiltration is included.

More preferably, said microfiltration should be performed using a filter material with a pore size of approximately 0.2 to 0.45 micron. Optionally, a plurality of said filter materials having pore sizes in said range may be arranged in series. Even more preferably, said filter material should be a tubular membrane made of ceramic or the like. Preferably, during the filtering operation, the temperature should be controlled to be in the range of approximately 60 to 70°C, or more preferably in the range of approximately 60 to 65 °C. Preferably, the transmembrane pressure during said filter operation should be controlled to be in the range of approximately 0.2 to 1.4 bar, most preferably approximately 1.2 bar.

The starting solution that passed the step of separation of solid impurities from the starting solution (22) is clear and may be yellowish. The solid impurities are separated and removed from the process.

For said effective separation of mucopolysaccharides having a molecular weight of more than approximately 150,000 Da from the starting solution (24), preferably, it is performed using ultrafiltration, preferably using a filter material with a pore size which is represented by molecular weight cut-off (MWCO, see the description above) of approximately 150,000 Da. Most preferably, the filter material with a pore size which is represented by molecular weight cut-off of approximately 20,000 Da is used. Optionally, a plurality of said filter materials having said pore size may be arranged in series. More preferably, said filter material is a tubular membrane made of ceramic or the like. Preferably, during the filter operation, the temperature should be controlled to be in the range of approximately 50 to 70°C, or more preferably in the range of approximately 50 to 65°C. Preferably, during said filter operation, the transmembrane pressure should be controlled to be in the range of approximately 0.5 to 2.5 bar, more preferably in the range of approximately 1.5 to 2.5 bar, most preferably in the range of approximately 2.0 to 2.5 bar.

The starting solution obtained from the second step (20) is clear and may be yellowish. Mucopolysaccharides with a molecular weight more than approximately 150,000 Da, or most preferably more than approximately 20,000 Da are effectively separated from the starting solution and removed from the process.

Then, according to the process according to Figure 1, the starting solution obtained from the second step (20) is contacted with enzymes (30), preferably the above-mentioned enzymes. Preferably, this step should be performed in a batch operation in at least one container or reactor. Preferably, the enzymes inside the container or reactor should have an enzyme activity of approximately 2 to 10 units per milligram of protein, more preferably approximately 10 units per milligram of protein. Preferably, the contact of the starting solution with the enzymes should be performed at the temperature in the range of approximately 50 to 60°C, more preferably approximately 55°C. Preferably, during this step, the pH of the solution is controlled to be in the range of 6.5 to 7. Preferably, this step should be performed for approximately 3 to 6 hours, most preferably approximately 6 hours. The solution obtained from the step of contacting with enzymes (30) is referred to as a digested solution.

After that, according to the process according to Figure 1, the digested solution is heated (40) to a temperature at which the enzyme activity is effectively prevented. Preferably, the temperature at which the enzyme activity is effectively prevented is approximately 90°C or more, most preferably approximately the boiling point of the digestedsolution. Preferably, the digested solutions should be heated (40) to the temperature at which the enzyme activity is effectively prevented, and should be maintained at such temperature for at least 10 minutes.

Then, according to the process according to Figure 1, the digested solution is then subjected to effective recovery (50) of mucopolysaccharides having an average molecular weight of not more than approximately 4,000 Da from the solution. Preferably, this should be performed using the ultrafiltration method. Preferably, this should be performed using a filter material with a pore size represented by molecular weight cut-off (MWCO, see the description above) of approximately 4,000 Da. Optionally, a plurality of said filter materials of said pore size can be arranged in series. More preferably, the filter material should be a tubular membrane made of ceramic or the like. Preferably, during said filter operation, the temperature should be controlled to be in the range of approximately 45 to 55°C. Preferably, during said filter operation, the transmembrane pressure should be controlled to be in the range of approximately 1.0 to 3.0 bar, more preferably in the range of approximately 1.5 to 2.0 bar.

Once passed the recovery process (50), mucopolysaccharides having an average molecular weight of more than 4,000 Da are effectively separated from the digested solution and removed from the process.

The digested solution which has passed the recovery step (50) contains mucopolysaccharides having an average molecular weight of not more than approximately 4,000 Da. Preferably, in the digested solution obtained from the recovery step (50), the mucopolysaccharides having the highest molecular weight intensity (i.e. the number of macromolecules) are mucopolysaccharides having a molecular weight of approximately 2,600 Da or less, or more preferably, said mucopolysaccharides having the highest molecular weight intensity are mucopolysaccharides having a molecular weight of approximately 1,300 Da or less.

Then, the recovered mucopolysaccharides from the recovery step (50) is subjected to stability improvement (60). In this stability improvement step (60), the recovered mucopolysaccharides from the recovery step (50) in the above paragraph will be dried to preserve the condition. Preferably, said step should be performed using freeze-drying method or spray-drying method, more preferably the spray-drying method. Most preferably, said step is performed using the spray-drying method carried out at an inlet temperature of approximately 120 °C and an outlet air temperature of approximately 95 °C.

Examples and analyses

Figure 2 shows analysis results of the molecular weight of the product containing mucopolysaccharides from clam tissue, said product being prepared using the process according to one embodiment of the present invention with a commercially available mixed enzymes in the trade name “Protease MF 102” distributed by Angel Yeast Co., Ltd.

Figure 3 shows analysis results of the molecular weight of the product containing mucopolysaccharides from clam tissue, said product being prepared using the process according to a preferable embodiment of the present invention when papain enzyme is used.

The molecular weight analyses according to Figures 2 and 3 above were carried out using Matrix- Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) from Bruker Daltonics, Germany, Autoflex P model, using nitrogen laser having a wavelength of 337 nanometers and a voltage of 20 kV. The samples were mixed with a matrix solution (a-cyano-4-hydroxycinnamic acid) for analysis of 1 mΐ sample, and the results were analyzed using Flex Analysis software from Bruker Daltonics, Germany.

The details and other variables of the process used to prepare the product containing mucopolysaccharides from clam tissue analyzed according to Figures 2 and 3 above were controlled to be the same or substantially similar and in accordance with the most preferred characteristics, ranges, or approximate values as mentioned above.

From Figure 2, it can be seen that in the product obtained from the process according to this embodiment, mucopolysaccharides having the highest molecular weight intensity were mucopolysaccharides having a molecular weight of approximately 2,583.348 Da.

From Figure 3, it can be seen that in the product obtained from the process according to this embodiment, mucopolysaccharides having the highest molecular weight intensity were mucopolysaccharides having a molecular weight of approximately 1,264.622 Da.

The products according to Figures 2 and 3 above were analyzed for antioxidant capacity using DPPH (2,2-diphenyl- 1 -picrylhydrazyl) protocol as described in details in Van Amsterdam et al. (1992) which is incorporated in the present application by reference. The standard solutions used in the analysis according to this application were 1 L of ascorbic acid solution in distilled water at concentrations of 6.25, 12.5, 50, 100, 200, 300, 400, 500, and 600 mM mixed with DPPH solution at a concentration of 100 mM in pure ethanol, left at room temperature in the dark for 30 minutes. Then, the light absorbance at a wavelength of 517 nm was measured. The samples were tested in triplicate.

After analyzing antioxidant capacity of the product according to Figures 2 and 3using the above-mentioned methods, it was found that the product according to Figure 2 exhibited an antioxidant value of 27.75 ± 1.36 percent, while the product according to Figure 3 exhibited an antioxidant value of 44.25 ± 0.50 percent.

6. The best mode of the invention

As described in the detailed description of the invention.

Claims

1. A process for preparation of a product containing mucopolysaccharides from clam tissue comprising contacting the clam tissue with a liquid solvent at a temperature of 80-90°C for not less than 1 hour to obtain a starting solution; modifying properties of the starting solution, comprising steps of separating solid impurities from the starting solution using a method selected from centrifugation or microfiltration, and effectively separating mucopolysaccharides having a molecular weight of more than 150,000 Da from the starting solution using ultrafiltration method; contacting the starting solution with enzymes comprising at least one type of enzyme selected from the group of cysteine protease, with an enzyme activity of 2-10 units per milligram of protein at a temperature of 50-60°C, pH of 6.5-7 for 3-6 hours to obtain a digested solution; heating the digested solution to a temperature at which the enzyme activity is effectively prevented; effectively recovering mucopolysaccharides having an average molecular weight of not more than 4,000 Da from the solution using ultrafiltration method; and improving stability of the recovered mucopolysaccharides.

2. The process according to claim 1 wherein the clam is Ridged venus clam or Pacific yellow cockle.

3. The process according to claim 1 or 2 wherein the clam is a member of family

Veneridae.

4. The process according to claim 1, 2, or 3 wherein the clam is a member of genus Meretrix.

5. The process according to any of the preceding claims wherein the enzymes have an enzyme activity of 10 units per milligram of protein.

6. The process according to any of the preceding claims wherein the at least one type of enzyme selected from the group of cysteine protease is papain enzyme.

7. The process according to any of the preceding claims wherein the starting solution is contacted with the enzymes at a temperature of 55°C, at pH of 6.5-7 for 6 hours.

8. The process according to any of the preceding claims wherein the separation of mucopolysaccharides separates mucopolysaccharides having a molecular weight of more than 20,000 Da from the starting solution.

9. The process according to any of the preceding claims wherein the improvement of stability of the recovered mucopolysaccharides is carried out using spray-drying method.

10. The process according to claim 9 wherein the spray drying method is carried out at an inlet air temperature of 120 °C and an outlet air temperature of 95°C.