KR102563473B1 - Method for manufacturing biosugar from seaweed and biosugar using the same - Google Patents

Abstract

The present invention relates to a method for producing biosugar using seaweed and biosugar manufactured using the same, and more particularly, to preparing a liquid mixture by adding seaweed to an aqueous hydrogen peroxide solution and mixing while maintaining a constant temperature, the liquid mixture preparing a saccharification solution by adding and mixing agarase into the saccharification solution, separating and refining to remove particulate impurities from the saccharification solution, distilling the saccharification solution that has undergone the separation and purification step under reduced pressure to prepare a concentrate, and concentrate A process technology for easily separating and producing biosugar, which is used as an important intermediate material for biochemical material production, is provided.

Description

Seaweed-derived biosugar manufacturing method and biosugar manufactured using the same {METHOD FOR MANUFACTURING BIOSUGAR FROM SEAWEED AND BIOSUGAR USING THE SAME}

The present invention relates to a method for producing biosugar using seaweed, and more specifically, to a method for producing biosugar by saccharifying seaweed using hydrogen peroxide and an agarase enzyme to obtain biosugar, and biosugar produced using the same It is about.

Galactose is one of the sugars that are constituents of seaweed, and is a very important candidate material that is expected to have useful functionality in the future development of raw materials for chemical reactions and physiologically active substances and pharmaceutical applications. As a starting material used in the chemical synthesis and production of adipic acid, a raw material of resin, it is a very important material that will become a raw material of bio-nylon in the future.

Galactose is mainly contained in seaweed and is produced through a saccharification process of hydrolysis using seaweed as a raw material. In most cases, the saccharification of seaweed is saccharified through an oxidation reaction using an acidic substance such as hydrochloric acid or sulfuric acid.

However, in the case of the conventional method using an oxidation reaction, seaweed has a problem in that carbohydrates cannot be completely saccharified. In addition, in the case of the method of adding an acidic substance, in order to proceed with the enzyme input treatment and saccharification process after acid treatment, additional neutralization treatment must be performed in advance. It is fair. Therefore, the conventional saccharification method using an oxidation reaction has a disadvantage that is a major obstacle to improving the economic feasibility of bioshuga manufacturing technology.

In addition, the acidic material treatment method has a problem in that the saccharification yield cannot be maximized. That is, when the concentration of the acidic substance is increased to decompose the cell walls of seaweed as much as possible, the structure of the biosugar produced at the same time as the reaction is deformed by the acidic substance, so the concentration of by-products increases.

Galactose, which is a saccharification product produced during the saccharification of seaweed, is a monosaccharide, and it is very difficult to obtain it directly as particles in a liquid saccharification liquid state. This is because seaweed contains protein components and impurities originally present in seaweed, and at the same time, acidic chemicals introduced during saccharification exist.

As such, the current research and development of seaweed saccharification is mainly focused on producing fuel materials such as bioethanol through fermentation after saccharification with acidic chemicals, neutralization, and fermentation. .

Meinita, M.D.H., Marhaeni, B., Winanto, T., Jeong, G.T., Khan, M.N.A.K., Hong, Y.K., 2013. Comparison of agarophytes (Gelidium, Gracilaria, and Gracilariopsis), as potential resources for bioethanol production. J. Appl. Phycol. 25 (6), 1957-1961. Jang, S. S., Yoshihito, S., Motoharu, U., Minato, W., 2012. Production of mono sugar from acid hydrolysis of seaweed. Afr. J. Biotechnol. 11, 1953-1963. Wu, F. C., Wu, J. Y., Liao, Y. J., Wang, M. Y., Shih, I. L., 2014. Sequential acid and enzymatic hydrolysis in situ and bioethanol production from Gracilaria biomass. Bioresour. Technol. 156. 123-131. Gurvan Michel, Pi Nyval-Collen. Tristan Barbeyron, Mirjam Czjzek, William Helbert, 2006. Bioconversion of red seaweed galactans: a focus on bacterial agarases and carrageenases. Appl Microbiol Biotechnol. 2006 Jun;71(1):23-33

Considering the above points, an object of the present invention is to provide a technology that is simple, economical, and industrially very useful compared to conventional acidic methods. Specifically, the method for producing biosugar derived from seaweed of the present invention A method in which seaweed, a resource, is first structurally dismantled using hydrogen peroxide, and then biosugar is produced by a technology of secondary saccharification using an agarase enzyme, and then the biosugar is separated and granulated. is to provide

In order to achieve the above object, as shown in FIG. 1 , the biosugar manufacturing method of the present invention includes preparing a liquid mixture by adding and mixing seaweed in an aqueous hydrogen peroxide solution (S110), and agarase in the liquid mixture. ) to prepare a saccharification solution (S120), a separation and purification step of removing particulate impurities from the saccharification solution (S130), distilling the saccharification solution that has undergone the separation and purification step under reduced pressure to prepare a concentrated solution (S140), and It is characterized in that it comprises a biosuga obtaining step (S150) in which alcohol is added to the concentrate and mixed to obtain biosuga galactose particles.

In the step of preparing the liquid mixture (S110), seaweeds are contacted and decomposed with an aqueous hydrogen peroxide solution in a liquid state, thereby disaccharides including galactose from the seaweed cell walls, tetrasaccharides, and other oligosaccharide-level substances such as oligosaccharides. Prepare a liquid mixture to

In the step of preparing the liquid mixture (S110), it is preferable to mix at a rate of 5 g to 15 g of seaweed per 500 cc of the hydrogen peroxide aqueous solution while maintaining the temperature at 40 ° C to 90 ° C.

And the concentration of the hydrogen peroxide aqueous solution is 2 jungnang% to 5% by weight, preferably 3.5% by weight can be used.

In one embodiment, the liquid mixture preparation step is a commercially available reagent, 35% by weight of hydrogen peroxide is mixed with distilled water, and finally, 5g to 15g of seaweed is added to 500 cc of 3.5% by weight hydrogen peroxide aqueous solution at a temperature of 40 ° C to 90 ° C. It can be mixed while holding. Here, the seaweed is preferably dried and pulverized to a size of about 0.1 to 10 mm before preparing the liquid mixture, and the weight of the seaweed is the weight of the seaweed thus pulverized.

In the step of preparing the liquid mixture (S110), if the amount of seaweed exceeds the mixing ratio of the aqueous hydrogen peroxide solution and the seaweed presented above, the decomposition of the seaweed may be insufficient and the effectiveness of the subsequent process may be reduced. When the amount is small, impurities in addition to galactose, which is the final biosugar, are increased due to excessive decomposition of seaweed, resulting in a decrease in biosuga yield.

In addition, when the mixing temperature in the step of preparing the liquid mixture is out of the suggested temperature range of 40 ° C to 90 ° C, the decomposition performance of seaweed rather deteriorates and the reaction safety deteriorates.

Then, in the step of preparing a saccharification solution (S120), 1 g to 2 g of agarase per 500 cc of the prepared liquid mixture is added and mixed at a temperature of 30 ° C to 45 ° C for 72 hours to 96 hours, thereby oligosaccharides included in the liquid mixture A saccharification solution containing galactose, a monosaccharide, may be prepared by hydrolyzing a sugar substance of the same level.

When the amount of agarase, the saccharification enzyme, is less than 1 g as the lower limit, the amount of agarase is relatively small, and the efficiency of hydrolysis is reduced. There is a problem that the process cost increases.

In the step of preparing the saccharification solution, the reaction temperature is about 30 ° C to 45 ° C, and preferably the reaction is performed at 35 ° C to 40 ° C. If the temperature is out of the above suggested temperature range, the activity of the agarase enzyme is greatly reduced, and in particular, when the temperature exceeds 45° C., the protein enzyme, the agarase enzyme, is denatured and the enzyme activity is lowered.

In addition, the reaction time is appropriately 72 hours to 96 hours, and if it is less than 72 hours, the performance of the enzyme is not utilized to the maximum, and if it exceeds 96 hours, there is a problem that excessive process time is required compared to the saccharification efficiency.

The agarase used as the saccharification enzyme is preferably obtained by culturing Saccharophagus degradans 2-40, which is a marine-derived strain. However, it is not particularly limited thereto.

Saccharophagus degradans 2-40 used in the present invention can be purchased commercially (Saccharophagus degradans ATCC 43961), but can be obtained by various methods, not limited thereto.

The saccharification solution obtained after the step of preparing the saccharification solution (S120) is composed of a liquid part containing galactose, which is a biosugae, and a fine particulate solid part. Therefore, a separation and purification step (S130) is performed to separate galactose, which is biosugar, and remove the fraction.

In the separation and purification step (S130), the first separation and purification step (S131) of purifying particulate impurities and proteins by sequentially contacting the saccharification solution with a silica column containing silica by gravity or pressurization at room temperature, and the first separation and purification step (S131) and a second separation and purification step (S132) of purifying remaining fine particulate impurities by passing the saccharification solution that has undergone the purification step through a membrane filter.

The first separation and purification step (S131) and the second separation and purification step (S132) as described above are repeated once or twice or more to remove components such as particulate impurities and proteins in the saccharification solution.

In the separation and purification step, it is preferable to use silica particles having a diameter of 100 μm to 500 μm and a pore size of the membrane filter of 0.45 μm. However, it is not limited thereto, and the desired liquid bioshu can be changed within a range capable of separating only galactose.

For example, at a room temperature of about 20 ° C. to 30 ° C., the saccharification solution is 100 μm to 500 μm in diameter at a flow rate of 0.1 mL / min to 20 mL / min using a method such as gravity or pressurization. It is purified by passing it through a silica column filled with phosphorus silica particles (S131), and then the liquid material passed through the column is passed through a membrane filter having a pore size of 0.45 μm to remove fine solid particulate impurities (S132).

The step of preparing a concentrate (S140) is a step of concentrating the saccharified solution from which particulate impurities have been removed through the separation and purification step by distillation under reduced pressure, and specifically, using a rotary vacuum distillation apparatus at a temperature of 30 ° C to 60 ° C, 10 mbar Water in the saccharification solution is evaporated under reduced pressure under a pressure condition of from 150 mbar to 150 mbar to prepare a concentrate concentrated to a volume level of about 1/10 to 1/30 of the volume of the saccharification solution before the vacuum evaporation.

By concentrating the purified saccharification solution in this way to reduce the volume, it is possible to facilitate the process of obtaining biosugar obtained by granulating bioshu thereafter, and accordingly, manufacturing time and cost can be reduced. That is, if the concentrate is concentrated to a level lower than 1/30 volume, the volume of the concentrate increases correspondingly, so the amount of alcohol added in the subsequent process increases and the efficiency of obtaining biosugar may decrease. Conversely, if the concentration exceeds 1/10 of the volume, the yield of biosugar may also be lowered because stirring is not well performed in the subsequent process and biosugar is not well precipitated.

In the step of obtaining biosugar (S150), alcohol at a temperature of -10°C to 25°C is added to the concentrate and mixed to precipitate biosugar galactose particles, and the precipitated galactose particles are filtered and dried.

As the alcohol, it is preferable to use at least one selected from methanol, ethanol, propanol, and butanol. Of course, it is not limited thereto, and an aliphatic alcohol represented by the general formula ROH (where R is an alkyl group having 1 to 6 carbon atoms) may be used.

In this way, in the step of obtaining biosugar (S150), the low-temperature alcohol is added in an amount of 10 to 30 times the volume of the concentrate and stirred to precipitate biosugar galactose particles. The precipitated galactose particles can be filtered through a membrane filter and then dried by a conventional drying method to obtain solid galactose particles.

On the other hand, in the present invention, it is preferable to use red algae containing a large amount of galactose among seaweeds. The red algae are Chondrus, Eucheuma, Gigartina, Pterocladia, Hypnea, Iridaea, and Kappaphycus. , Gellidium, or Gracilaria seaweed, but is not limited thereto.

Biosugar can be produced by the biosugar manufacturing method as described above, and this biosugar is galactose and can be used in the bioplastics field as a starting material for producing adipic acid, a raw material for bionylon.

In the conventional method, it was very difficult to obtain biosugar particles in a saccharified liquid state, but the biosugar manufacturing method of the present invention performs saccharification by using a hydrogen peroxide aqueous solution and an agarase enzyme in combination, and then separates and refines biosugar through a purification process. Since it can be produced by a simple process of obtaining granules, biosuga galactose can be mass-produced at low cost using seaweed, which is a non-food resource.

In addition, since bioshu, which is used as an important intermediate material for biochemical material production, can be granulated into galactose, it is an industrially useful technology by providing a raw material that enables the production of biomaterials such as bionylon more effectively.

1 is a flowchart illustrating a method for manufacturing biosugar according to an embodiment of the present invention.

Hereinafter, examples and comparative examples of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. However, the present application may be implemented in many different forms and is not limited to the examples and comparative examples described herein.

Terms used herein are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this application, the terms "include" or "have" are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

As used throughout this specification, the terms "about", "substantially", and the like are used at or close to the value when manufacturing and material tolerances inherent in the referenced meaning are given, and the understanding of the present invention To help prevent exploitation by unscrupulous infringers of the disclosed disclosure, exact or absolute figures are used.

In addition, “biosugar” means a raw material that is the basis of biochemistry as a sugar substance obtained from biomass. The biosugar produced by the production method of the present invention is preferably galactose, and throughout this specification, terms such as “biosugar”, “galactose” or “biosugar galactose” will be used. can

Hereinafter, the biosugar manufacturing method of the present invention will be described in more detail using Examples, Comparative Examples, and Experimental Examples.

Example 1 is prepared by drying and pulverizing red algae sea string collected from the coast of Wando, Jeollanam-do, Korea, to a size of about 0.1 mm to 10 mm. Then, 450 cc of distilled water and 50 cc of hydrogen peroxide (concentration: 35% by weight, manufactured by Daejung Chemical Co., Ltd.) were mixed in the flask, and 10 g of pulverized string was added, followed by stirring at 50 ° C. for 24 hours. After stopping stirring of the solution and allowing it to stand at room temperature for 24 hours, only the liquid mixture of the upper liquid phase excluding the precipitate is taken out.

Thereafter, agarase as an enzyme is added to the liquid mixture and mixed to prepare a saccharification solution. The agarase is obtained by culturing Saccharophagus degradans 2-40 The obtained enzyme and its specific production method are as follows.

Since the activity of Saccharophagus degradans 2-40 ( Saccharophagus degradans 2-40) is first stored in a refrigerator or storage, the activity of the bacteria is reduced. 2-40) in an autoclave-sterilized medium (50mM Tris-HCl, 2.3% (w/v) Instant Ocean Sea Salt (Aquarium Systems), 0.1% (w/v) Yeast extract, 0.05% (w/v) /v) ammonium chloride, 0.2% (w/v) agar, pH 7.4) and perform pre-incubation in a shaking incubator at 30 ° C and about 200 rpm for 16 hours. After transferring 1 mL of the pre-culture medium obtained by pre-culture to 100 mL of a new medium, incubate again at 30 ° C for 72 hours. Afterwards, as the strain cells grow, the agarase enzyme is released to the outside, so the agarase is obtained after culturing in this way.

In the step of preparing the saccharification solution, 2 g of the agarase prepared in the above manner is added to the liquid mixture and stirred for 72 hours at a temperature of 37 ° C. so that the liquid mixture and the agarase are evenly contacted, followed by saccharification to prepare a saccharification solution.

Then, the saccharification solution of the above process is separated and purified by passing through a silica column filled with neutral silica particles having a size of 100 μm to 500 μm. At this time, the volume of silica filled in the column is about 100 cc. Thereafter, the saccharification solution passed through the silica column is separated and purified using a membrane filler having a pore size of 0.45 μm.

In Examples 2 to 5, biosugar was prepared in the same manner as in Example 1, but there were changes in some process conditions such as temperature conditions for preparing the liquid mixture, amount of agarase enzyme and type of alcohol, which are shown in Table 1 below. appears in

Comparative Example 1 was performed in the same manner as in Example 1 except that hydrogen peroxide was not used.

Comparative Examples 2 to 5 were performed in the same manner as Comparative Example 1, but there were changes in some process conditions, such as the amount of agarase enzyme input, the type of alcohol, and the temperature conditions for preparing the saccharification solution, which are shown in Table 1 below.

division Example 1 Example 2 Example 3 Example 4 Example 5 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 Comparative Example 5 A(cc) 500 500 500 500 500 0 0 0 0 0 B(g) 10 10 10 10 10 10 10 10 10 10 C(℃) 50 55 60 45 50 50 50 50 50 50 D(g) 2.0 2.0 1.8 1.9 1.7 0 0 0.5 4.0 0.1 E(℃) 37 37 37 37 37 50 50 50 50 50 passing silica column you you you you you you you you you you membrane filter treatment you you you you you you you you you you treated alcohol methanol ethanol propanol methanol methanol methanol ethanol propanol methanol methanol Get bio sugar ○ ○ ○ ○ ○ X X X X X A: Amount of hydrogen peroxide aqueous solution
B: Amount of red algae seaweed (from Wando, Korea) input
C: mixing temperature of aqueous hydrogen peroxide solution and flirting
D: Agarase enzyme dosage
E: Mixing temperature of the liquid mixture and the agarase enzyme

In Experimental Example 1, the melting point of the particles precipitated in Examples 1 to 5 was determined by using Differential Scanning Calorimetry (DSC) to determine whether biosugar was a galactose component. ) was measured, and its physical properties are shown in Table 2 below.

division Particle Generation After Concentration melting point
(melting point, ℃) Examples 1-5 Bioshoe created 167 Comparative Example 1 A sticky substance is created
No particulate matter produced 65 Comparative Example 2 A sticky substance is created
No particulate matter produced 66 Comparative Example 3 A sticky substance is created
No particulate matter produced 50 Comparative Examples 4 to 5 A sticky substance is created
No particulate matter produced 45

As shown in the results of Table 2, all of the biosugars produced according to the examples of the present invention (Examples 1 to 5) were prepared in the form of solid particles, and galactose was produced with a melting point of 167 ° C. was able to confirm

However, in Comparative Examples 1 to 5, no particulate material was produced, and it was confirmed that galactose was not generated with a melting point of 45°C to 66°C.

In the conventional process for producing biosugar from general biomass, there are problems in that very strong and toxic acidic substances such as hydrochloric acid or sulfuric acid are used, the production yield is very low, and it is difficult to granulate biosugar. However, according to the biosugar manufacturing method of the present invention as described above, a large amount of biosugar particles can be produced from seaweed, which is one of the non-food marine resources, through a much simpler process than conventional manufacturing processes at low cost and high yield. .

Claims (13)

preparing a liquid mixture by adding 5 g to 15 g of seaweed per 500 cc of hydrogen peroxide aqueous solution and mixing while maintaining the temperature at 40 ° C to 90 ° C;
preparing a saccharification solution by adding agarase to the liquid mixture and mixing;
Separation and purification step of removing particulate impurities from the saccharification solution;
preparing a concentrate by distilling the saccharified solution subjected to the separation and purification step under reduced pressure; and
and a biosugar obtaining step in which alcohol is added to the concentrate and mixed to obtain particles of galactose, which is biosugar. According to claim 1,
The separation and purification step,
A first separation and purification step of contacting and purifying the saccharified solution with a silica column in a gravity method or a pressurized method; and
and a second separation and purification step of purifying the saccharified solution that has passed through the first purification step by passing it through a membrane filter. According to claim 1,
The method for producing biosugar, characterized in that the agarase is an enzyme obtained by culturing Saccharophagus degradans 2-40. According to claim 1,
The seaweed is red algae,
The red algae are Chondrus, Eucheuma, Gigartina, Pterocladia, Hypnea, Iridaea, and Kappaphycus. , A method for producing biosugar, characterized in that at least one selected from Gellidium and Gracilaria. According to claim 1,
Wherein the alcohol is at least one selected from methanol, ethanol, propanol and butanol. delete delete According to claim 1,
The method for producing biosugar, characterized in that the concentration of the aqueous hydrogen peroxide solution is 2% to 5% by weight. According to claim 1,
In the step of preparing the saccharification solution, 1 g to 2 g of the agarase is added and mixed per 500 cc of the liquid mixture. According to claim 1,
In the step of preparing the saccharification solution, the agarase is added to the liquid mixture and mixed at a temperature of 30 ° C to 45 ° C for 72 hours to 96 hours. According to claim 1,
In the step of preparing the concentrated solution, the saccharified solution that has undergone the purification step is concentrated to a volume of 1/10 to 1/30 at a pressure of 10 mbar to 150 mbar at a temperature of 30 ° C to 60 ° C. According to claim 1,
In the step of obtaining biosugar, low-temperature alcohol at -10 ° C to 25 ° C is added to the concentrate and mixed to precipitate biosugar galactose particles, and the precipitated galactose particles are obtained by filtering and drying. . Claims 1, 2, 3, 4, 5, 8, 9, 10, 11, and 12, prepared by any one of the biosugar manufacturing methods biosugar.

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