WO2009022900A2

WO2009022900A2 - A process to produce polypeptides from soybean

Info

Publication number: WO2009022900A2
Application number: PCT/MY2008/000080
Authority: WO
Inventors: La Sheng Yin; Chyi Lin Tiong; Chan Kok Lee
Original assignee: Mci Bio-Peptide Sdn.Bhd.
Priority date: 2007-08-14
Filing date: 2008-08-13
Publication date: 2009-02-19
Also published as: MY150934A; WO2009022900A3

Abstract

A process to extract peptides from soybean protein source comprises the steps of reacting soybean protein source paste with neutral protease via a first hydrolysis reaction to form a first hydrolysate; terminating the first hydrolysis reaction; reacting the first hydrlysate with pancreatin via a second hydrolysis reaction to form a second hydrolysate; terminating the second hydrolysis reaction; and filtering the second hydrolysate through layers of kaolin and activated carbon to acquire a filtrate containing the peptides free of bitterness taste.

Description

A PROCESS TO PRODUCE POLYPEPTIDES FROM SOYBEAN

FIELD OF THE INVENTION

The present invention relates to a simplified process to extract peptides from soybean which is free of bitterness taste. In more specific, the disclosed method employs a single filtration step in removing the bitterness taste of the extracted peptides thus greatly simplify the overall process.

BACKGROUND OF THE INVENTION Currently, the methods of processing soybean protein source are by acidic process, alkaline process or enzymatic hydrolysis process to convert the larger protein molecules in the protein into fragments of polypeptides which can be more readily absorbed by the human body upon ingestion and play an important role in nutrition and bio-function. Among all the mentioned conventional methods, enzymatic hydrolysis process is wider in usage as both acidic and alkaline process are deemed to be too drastic which may degrade the quality of the end products as well as render unwanted chemical residues in the end products.

Still, the polypeptides acquired from the enzymatic hydrolysis process generally carry with some bitterness thereby affecting the taste of the final products and hence causing the market acceptability and commercialization in large quantity not viable. Intensive research and development are conducted in order to seek a solution for removing the bitterness taste. Nevertheless, some of the offered solutions are time- consuming which entails few extra procedures thus renders higher labor cost. From the view point of manufacturing, it would be ideal to have the process simplify yet capable of producing the extracted polypeptides free of bitterness taste.

SUMMARY OF THE INVENTION

The present invention aims to disclose a process to extract polypeptides from soybean protein source free of bitterness. Thus enable the incorporation of the extracted polypeptides into health promoting products without adding any sweeteners and reduce the cost of the health promoting products.

Still it is another object of the present invention in disclosing a simplified yet efficient process in extracting polypeptides from the soybean protein source.

Further object of the present invention is to provide a cost effective process to extract polypeptides from soybean protein source as the disclosed process can greatly reduce the labor cost due to its simplified steps.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiment of the present invention includes a process to extract peptides from soybean protein source comprises the steps of reacting soybean protein source paste with neutral protease via a first hydrolysis reaction to form a first hydrolysate; terminating the first hydrolysis reaction; reacting the first hydrlysate with pancreatin via a second hydrolysis reaction to form a second hydrolysate; terminating the second hydrolysis reaction; and filtering the second hydrolysate through layers of kaolin and activated carbon to acquire a filtrate containing the peptides free of bitterness taste.

Further embodiment of the disclosed process involves a step to concentrate the filtrate to obtain peptides in powder form after the filtering.

In the preferred embodiment of the present invention, the soybean protein source paste is prepared by grinding soybean protein source with distilled waster in a ratio of 1 : 6-16 by weight percentage.

Preferably the first hydrolysis reaction is conducted within a temperature of 50⁰C to 55⁰C for the neutral protease to function optimally in catalyzing the soybean protein source into smaller protein fragments, while the reaction pH preferably ranges from 7.0 to 7.9. In the preferred embodiment, the termination of the first hydrolysis reaction is conducted by heating the hydrolysate to a temperature of 80⁰C to 90⁰C for 10 to 30 minutes to deform the neutral protease which carry out the hydrolysis process.

On the other hand, the second hydrolysis reaction is preferably conducted in a temperature of 45⁰C to 55⁰C and at a pH ranges from 7.1 to 7.8 for the reaction to perform at its optimum condition.

Owing to the properties of the polypeptides produced in the second hydrolysis reaction, the termination of the second hydrolysis reaction is conducted by heating the hydrolysate to a temperature of 60⁰C to 90⁰C for 15 to 45 minutes at a pH of 4 to 6.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a flow chart showing the work flow of the disclosed process in producing polypeptides free of bitterness from soybean protein source.

DETAILED DESCRIPTION OF THE INVENTION

It is to be understood that the present invention may be embodied in other specific forms and is not limited to the sole embodiment described herein. However modification and equivalents of the disclosed concepts such as those which readily occur to one skilled in the art are intended to be included within the scope of the claims which are appended thereto

Generally, the present invention entails a process to extract peptides from soybean protein source comprises the steps of reacting soybean protein source paste with neutral protease via a first hydrolysis reaction to form a first hydrolysate; terminating the first hydrolysis reaction; reacting the first hydrlysate with pancreatin via a second hydrolysis reaction to form a second hydrolysate; terminating the second hydrolysis reaction; and filtering the second hydrolysate through kaolin and activated carbon to acquire a filtrate containing the peptides free of bitterness taste. It is important to be noted that the soybean protein source employed in the present invention refers to soybean powder, pre-treated soybean, isolated soybean protein and the like. Nonetheless, it is most preferred to use purified soybean protein source such as isolated soybean protein so that the polypeptides produced may contain less contamination from other substances. The isolated soybean protein utilized preferably contains total nitrogen content not less than 11% by weight. Furthermore, the soybean protein source is preferably prepared into a paste form in the disclosed process to enhance the enzymatic reaction to be carried out in the subsequent steps. Preferably, the soybean protein source paste is produced by grinding the soybean protein source with water, most preferred distill water. The concentration of the soybean protein in the prepared paste can be crucially affect the subsequent process especially in terms of reaction time thus most likely to be maintained in a ratio of soybean protein source to distilled waster is 1 : 6-16 by weight percentage upon the types of soybean protein source used.

One skilled in the art shall appreciate the fact that flavor of the polypeptides produced mainly depends on the molecular structure of the polypeptides produced. It is known in the art that if the produced polypeptides being cleaved at the site of hydrophobic amino acids, such fragment shall be tasted bitter instead of appetizing. On the other hand, exposure of hydrophobic amino acids on the molecular surface of the folded polypeptides may also confer the corresponding polypeptides tasted bitter. Likewise, free hydrophobic amino acids also tasted bitter if being ingested. Consequently, it is crucial in selecting correct proteolytic catalysts to be employed in preparing the polypeptide thus avoiding the production of the polypeptides with bitter taste. Therefore, the present invention employs two different categories of catalyses to attain the set forth objects. Applying two different categories of proteolytic enzymes produce polypeptides in smaller fragments which improve absorbability of these polypeptides in the human body. It is possible to perform the enzymatic hydrolysis by using these two types of catalysts together in a same reaction, but such reaction is not favor in producing polypeptides free of bitterness as the catalysts may compete for catalytic sites thus lead to production of undesired polypeptides. Thus conducting the catalytic reaction in two different steps is much preferable in the present invention.

According to the preferred embodiment of the present invention, the first hydrolysis process involves react the soybean protein source paste with the neutral protease. In order to have the first enzymatic hydrolysis performs at its optimum condition, preferably the temperature of reaction is 50⁰C to 55⁰C and the pH of 6.5 to 7.9. Moreover, the amount of neutral protease used in the reaction is more preferable in 2% to 8% by weight in reference to the total weight of the substrate, the soybean protein source paste. It is found by the inventors that the neutral protease performs optimally under this condition, and the reaction may continue for 3 to 8 hours relying upon the amount of the substrate as well as the condition employed. It should be known by one skilled in the art that the pH adjustment can be achieved by using different basic materials or solvent before carrying out the first hydrolysis reaction. Ammonia is utilized in the preferable embodiment due to its weak base property as strong base may degrade the protein structure contained in the paste.

After hydrolyzing the soybean protein source paste to the desired level to form the first hydrolysate, the first hydrolysis reaction is terminated by deactivating the neutral protease used in the reaction. It is well known in the art that deactivation of biological catalysts, particularly enzyme, can be attained by applying heat to the catalysts or subjecting the catalysts to pH shift to deform it. In respect to the preferred embodiment of the present invention, the first hydrolysis is terminated by heating the hydrolysate up to a temperature range 80⁰C to 90⁰C for 10 to 30 minutes. It is possible to degrade the neutral protease at higher temperature with shorter heating duration or vice versa, such modification shall not depart from the scope of the present invention.

Then the first hydrolysate is subjected to second hydrolysis reaction by reacting with pancreatin to further catalyse the polypeptides in the first hydrolysate to smaller fragments which are more readily to be absorbed upon ingestion. Preferably, the second hydrolysis reaction is performed at a temperature of 45⁰C to 55⁰C and pH of 7.1 to 7.8. As in the foregoing description, the reaction time can range from 3 to 6 hours reliance upon the condition and substrates used until a satisfied degree of hydrolysis achieved in the second hydrolysate. In the most preferred embodiment, calcium hydroxide is employed as the solvent to adjust the pH of the first hydrolysate before and during the second hydrolysis reaction. It is the inventor interest that the calcium ions in the calcium hydroxide can greatly enhance the pancreatin enzymes activity, thus being chosen to be employed in the present invention.

Before proceed with further process to acquire the polypeptides contained inside the second hydrolysate, the second hydrolysis is terminated by deactivating the pancreatin enzymes via pH change. In the preferred embodiment of the present invention, termination of the second hydrolysis reaction is conducted by heating the hydrolysate to a temperature of 60⁰C to 90⁰C for 15 to 45 minutes at a pH of 4 to 6. Similarly, different acidic solvent can be used to attain the pH change in the second hydrolysate, preferably hydrochloric acid with concentration of 18% to 24% (w/w) is used. The pH of 4 to 6 is known to be the pH level which the pancreatin enzymes will be degraded. Moreover, strong acids like hydrochloric acids are useful in further hydrolyzing un- catalyzed protein molecules in the hydrolysate to smaller fragments of polypeptides.

Though it is possible to use the polypeptides in the second hydrolysate without further processing it, the disclosed process preferably additionally comprise a single purification step to remove the chemical residues, non-hydrolyzed substrates, and the bitterness taste of the polypeptides produced. The purification step involves filtering the second hydrolysate through a filtering means having filtering layers made of kaolin and activated carbons. In order to significantly sieve off the suspended solids derives from the non-hydrolysed substrates; layers of mechanical screen filter can be used in the filtering means. Throughout the filtering process, kaolin in the filtering means is capable of adsorbing free amino acids, which normally tasted bitter if hydrophobic, available in the hydrolysate. On the other hand, activated carbon used in the filtering means is able to bind on the polypeptides with the hydrophobic site exposed on it molecule surface, thus removing these polypeptides from the hydrolysate and rendering the remaining polypeptides in the second hydrolysate free of bitterness taste. Besides of that, activated carbon also capable of removing chemical residues in the second hydrolysate thus improving the safety of the produced polypeptides upon ingestion.

In one of the embodiments, the filtrate is ready to be consumed therefore can be directly incorporated into food or drinks in promoting healthcare. Yet another embodiment involves concentrating the filtrate to obtain the produced polypeptides in powder form after the filtering step. Different approaches can be applied in order to get rid of the solvent in the filtrate such as applying heat, vacuum concentration, centrifugal concentration and the like. The acquired polypeptides in powder form is subjected to the user preference in combining with food, drink or even medicine in promoting healthcare.

It is important to be noted that during both first and second hydrolysis reaction, the pH of hydrolysate is preferably monitored closely to ensure the whole process is carried out at its optimum condition. Shifting of the pH of the hydrolysate from alkaline to acidic is common in the process as free amino acids are released from the larger molecules of protein thus contributes acidity to the hydrolysates. If both the first and second hydrolysis reaction are not preserved in the preferred pH range, higher acidity not only degrades the catalysts used but also changes the conformation of the proteins or polypeptides structure which may lead to production of undesired products.

Example 1

20kg isolated soybean protein having nitrogen content not less than 11% was ground with 170 liter of distilled water until acquiring an isolated soybean protein paste. Then, the isolated soybean protein paste was deposited into a reactor and heat up to a temperature of 52⁰C with the pH adjusted to 7.3 with ammonia. Subsequently, the pre-treated isolated soybean protein paste is mixed with 1.2kg of neutral protease for conducting a first hydrolysis reaction. During the continuous 3 hours process of first hydrolysis reaction, the pH in the reactor was monitored closely and any shift of pH was immediately adjusted to 7.3 with ammonia. The hydrolysate obtained from the first hydrolysis reaction was then heated up to 90⁰C for 18 minutes to deactivate the neutral protease thus terminating the first hydrolyisis reaction. After the deactivation step, the temperature of the first hydrolysate was brought down to 55⁰C and the pH was adjusted to 7.6 with 30% (w/w) calcium hydroxide solution. The pre-treated second hydrolysate was then mixed with 0.78kg of pancreatin for conducting a second hydrolysis reaction. Likewise, any pH changes during the second hydrolysis reaction were rectified by using calcium hydroxide solution and the reaction continued for about 4 hours to obtain the second hydrolysate. In order to terminate the second hydrolysis reaction, the second hydrolysate was subjected to a pH change by using concentrated acid. The pH of the second hydrolysate was adjusted to 5.5 and heated up to mild boil for 20 minutes to deactivate the pancreatin. The second hydrolysate was passed through layer of filtering means, layers of kaolin and layers of activated carbon to remove the suspended solids in the hydrolysate as well as the polypeptides tased bitterness. Finally, the filtrate was subjected to drying to obtain the polypeptides in powder form.

The present disclosure includes as contained in the appended claims, as well as that of the foregoing description. Although this invention has been described in its preferred form with a degree of particularity, it is understood that the present disclosure of the preferred form has been made only by way of example and that numerous changes in the details of construction and the combination and arrangements of parts may be resorted to without departing from the scope of the invention.

Claims

CLAIMS:

1. A process to extract polypeptides from soybean protein source comprising the steps of (a) reacting soybean protein source paste with neutral protease via a first hydrolysis reaction to form a first hydrolysate;

(b) terminating the first hydrolysis reaction;

(c) reacting the first hydrlysate with pancreatin via a second hydrolysis reaction to form a second hydrolysate; (d) terminating the second hydrolysis reaction; and

(e) filtering the second hydrolysate through kaolin and activated carbon to acquire a filtrate containing the peptides free of bitterness taste.

2. A process according to claim 1 further comprises the step of concentrating the filtrate to obtain peptides in powder form after the filtering.

3. A process according to claims 1 and 2, wherein the soybean protein source paste is produced by grinding soybean protein source with water.

4. A process according to claim 3, wherein ratio of soybean protein source to distilled waster is 1: 6-16 by weight percentage.

5. A process according to claims 1 and 2, wherein the first hydrolysis reaction is conducted within a temperature of 50⁰C to 55⁰C.

6. A process according to claims 1 and 2, wherein the first hydrolysis reaction is conducted within a pH ranges from 7.0 to 7.9.

7. A process according to claims 1 and 2, wherein the termination of the first hydrolysis reaction is conducted by heating the hydrolysate to a temperature of

80⁰C to 90⁰C for 10 to 30 minutes.

8. A process according to claims 1 and 2, wherein the second hydrolysis reaction is conducted within a temperature of 45⁰C to 55⁰C.

9. A process according to claims 1 and 2, wherein the second first hydrolysis reaction is conducted within a pH ranges from 7.1 to 7.8.

10. A process according to claims 1 and 2, wherein the termination of the second hydrolysis reaction is conducted by heating the hydrolysate to a temperature of

60⁰C to 90⁰C for 15 to 45 minutes at a pH of 4 to 6.