TWI399432B - Glucosamine production using microwave heating - Google Patents

Description

Method for producing glucosamine and acetaminophen using microwave technology

The present invention relates to a method for producing glucosamine and acetaminophen, and more particularly to a method for producing glucosamine and acetoglucosamine by hydrolyzing chitin or chitosan using a microwave device as a heat source for hydrolysis reaction.

Glucosamine (GlcN), or 2-amino-2-deoxy-D-Glucose, is one of the components of articular cartilage, which provides nutrients for joint tissues. Increase the synovial fluid to restore lubrication, promote the regeneration of degenerative joints, and thus effectively reduce the pain caused by bone friction and prevent the deterioration of arthritis symptoms. The human body can synthesize glucosamine by itself, but with the increase of age, the rate of synthesis of glucosamine in the human body is not as fast as the decomposition of glucosamine, so it is easy to produce glucosamine deficiency in the body and joints, and affect the metabolism of cells in the joint.

Acetylated glucosamine or a derivative thereof - acetyl glucosamine (N -acetyl glucosamine, GlcNAc) having: (1) stimulation of new cartilage mother cells, promote the metabolism, bone supplying nutrition, reduce inflammation, pain; ( 2) Protect the chondrocytes from drugs and external forces, and prevent the degradation of bones and joints; (3) Increase the amount of lubricating fluid and its viscosity, promote joint lubrication, improve bone and joint function; (4) Improve back pain and other effects characteristic. In Europe, glucosamine has been widely used to treat osteoarthritis; after consumption of glucosamine, the body can be quickly absorbed and transported to various tissues in the body for use. It has been confirmed by acute toxicity test and microbial mutation test in rats that glucosamine is a safe and non-toxic health food. Early supplementation of glucosamine can prevent arthritis.

Due to the various medical functions and advantages mentioned above, glucosamine and acetaminophen have many health foods or supplements containing glucosamine as the main component. The research and development of glucosamine process is also the focus of many pharmaceutical companies. One; since glucosamine and acetoglucosamine are monomers that make up chitin and chitin, in the traditional industry, the method of producing glucosamine and acetoglucosamine is to hydrolyze chitin or a few with acid or enzyme. Butan; the procedure is: soak the shrimp shell powder or crab shell powder in a hydrogen chloride solution to remove calcium carbonate, and then boil it with alkali to remove fat and protein, and the product is chitin; Soaking chitin in a hydrogen chloride solution until the chitin is dissolved into a liquid phase, adding activated carbon to remove color and heavy metals, after concentration, crystallization, washing with alcohol, and vacuum drying at low temperature, the white crystalline powder is obtained. Glucosamine.

However, in the above manufacturing method, shrimp shell powder or crab shell powder of different sources may affect the purity of glucosamine, and if the source of shrimp and crab contaminated with poison is used, the produced glucosamine may also be toxic; Before the hydrolysis of shrimp shells and crab shells, it is necessary to wash the shells and crab shells to avoid foul odor; while glucosamine is not the only product in the hydrolysis of shrimp shells, it still needs to be purified to separate glucosamine and other by-product.

In addition to the above-mentioned use of shrimp shell powder or crab shell powder as a source of chitin or chitosan to produce glucosamine or acetaminoglucosamine by hydrolysis, microorganisms are currently used to produce glucosamine or acetaminoglucosamine. Method: The characteristics of chitin or chitosan are produced in the cell wall of the fungus. These fungi are used as a source of chitin or chitosan, and the fungal cells are broken and chitin or chitosan is taken out. The hydrolysis reaction is carried out with hydrochloric acid to obtain a glucosamine or acetaminoglucosamine product.

In the above hydrochloric acid hydrolysis reaction procedure, the reaction temperature and the concentration of hydrochloric acid play an important role; the length of the reaction time and the yield of the glucosamine or acetaminoglucosamine product may vary depending on the concentration of the hydrochloric acid and the reaction temperature; In general, the reaction temperature is between 60 ° C and 100 ° C; the hydrochloric acid concentration is between 10 ^and 40% (w/w), and the reaction time varies from tens of minutes to 24 hours.

Hsieh, JW, HS Wu, YH Wei, and SS Wang. 2007. Determination and kinetics of producing glucosamine using fungi, Hsieh et al. (2007) published in the Biotechnology Progress Journal. Biotechnol. Prog ., 23:1009-1016):

The hydrochloric acid reaction conditions disclosed in U.S. Patent Publication No.: US 2008/0188649 A1 to Cao et al. are:

The hydrochloric acid reaction conditions disclosed in U.S. Patent No.: US Pat. No. 6,486,307 B1 to G.

Deng et al., U.S. Patent Publication No.: US 2004/0091976 A1, the reaction rate of chitin hydrochloride and the reaction temperature, the concentration of glucosamine or acetaminophen in solution, and the hydrochloric acid solution. Concentration-related; successful reaction conditions include a reaction temperature of 60 ^~ 100 ° C, the reaction time varies depending on the reaction temperature and hydrochloric acid concentration, the reaction time of high concentration hydrochloric acid and high reaction temperature is 10 minutes, low concentration of hydrochloric acid and low reaction The reverse time of the temperature ranges from 3 hours to 24 hours.

It is known from the above-mentioned various prior art that the time required for the acidification hydrolysis reaction for the production of glucosamine or acetoglucosamine varies from tens of minutes to 24 hours; it is well known that in the industrial mass production process, the reaction of the production process The longer the time, the more people you need, and the lower the productivity, which will not help reduce production costs.

It can be seen that there are still many shortcomings in the above-mentioned methods for producing glucosamine and acetaminophen glucosamine, which is not a good designer and needs to be improved.

In view of the shortcomings derived from the above-mentioned conventional methods, the inventors of the present invention have improved and innovated, and after years of painstaking research, finally successfully developed a method for producing glucosamine and acetaminophen using microwave technology.

The object of the present invention is to provide a method for producing glucosamine and acetaminophen by using microwave technology; at present, the industrial production of glucosamine is still hydrolyzed by chitin or chitosan for hydrolysis, but the production process is quite expensive. When the energy is wasted, it is necessary to wait for the completion of the hydrochloric acid reaction to proceed to the next step; for this reason, the object of the present invention is to provide a time-saving and energy-saving hydrochloric acid reduction process capable of reducing the hydrochloric acid reaction for several hours to Completed in 3 minutes, saving energy and reducing production costs.

In order to achieve the above object, the inventors of the present invention utilized electromagnetic energy of microwaves as thermal energy to promote chemical reactions efficiently and rapidly. Microwave technology can help engineers significantly reduce costs, accelerate reaction rates, increase production, and more. Therefore, in the present invention, microwave technology is utilized to hydrolyze fungal biomass to produce glucosamine or acetaminoglucosamine, which reduces acidification reaction time in an acidic environment, and saves energy and production costs.

A method for producing glucosamine and acetaminophen using microwave technology to achieve the above object includes the following steps:

Step 1: provide a source of chitin or chitosan;

Step 2: adding an acid solution to the chitin or chitosan source to form a reaction solution;

Step 3: The reaction solution is heated in a microwave device to carry out a hydrolysis reaction to hydrolyze chitin or chitosan to produce glucosamine or acetaminoglucosamine;

Wherein the chitin or chitosan source is a microorganism capable of producing chitin or chitosan; the microorganism is Rhizopus oligosorus BCRC 31996, Monascus purpures BCRC 31499, Monascus pilosus BCRC 31527 or Aspergillus sp. BCRC 31742; In a preferred embodiment, the microorganism is Aspergillus sp. BCRC 31742.

Wherein the acid solution is a hydrochloric acid solution or a sulfuric acid solution; in a preferred embodiment, the acid solution is a hydrochloric acid solution. The concentration of the hydrochloric acid solution is from 2N to 6N; in a preferred embodiment, the concentration of the hydrochloric acid solution is 6N.

The power of the microwave device is 700 watts to 2100 watts; in a preferred embodiment, the power of the microwave device is 1120 watts to 1400 watts; in a more preferred embodiment, the power of the microwave devices is 1400 watts.

The heating time of the third step is 90 seconds to 270 seconds; in a preferred embodiment, the heating time is 180 seconds to 270 seconds; in a preferred embodiment, when the concentration of the hydrochloric acid solution is 6N, The heating time is 180 seconds.

Example 1 Preparation of chitin or chitosan source

In this embodiment, the fungus which produces chitin or chitosan is cultured in a large amount by shaking flask fermentation, and these fungi are used as a source of chitin or chitosan, and then the fungal cells are broken and taken out. Butyl or chitosan is finally hydrolyzed with hydrochloric acid to obtain a glucosamine or acetaminoglucosamine product.

1.1 test strain

In the present example, Aspergillus sp. BCRC31742 was used as a strain for producing fungal biomass, and the strain was purchased from the Food Industry Development Research Institute (Taiwan, Hsinchu).

1.2 shake flask fermentation

The Aspergillus sp. BCRC31742 was first cultured in a solid medium of PDA (Potato Dextrose Agar: 200 g/L Diced potatoes, 20 g/L Glucose, 15 g/L Agar) at 30 ° C for 7 days, and then a single colony was inoculated. Sterilize PDB (Potato Dextrose Broth: 20g/L Diced potatoes, 4g/L Glucose, 150ml) in liquid medium at 30°C for 7 days at 200rpm for secondary activation; then shake flask culture, after activation The strain was inoculated into sterilized GP medium (glucose peptone medium: 25 g/L Glucose, 20 g/L Peptone, 0.5 g/L KH ₂ PO ₄ , 0.5 g/L MgSO 4‧7H ₂ O, 0.1 g/L CaCl ₂ ‧2H ₂ O), cultured at 30 ° C for 7 days at 200 rpm. After shaking flask culture, the obtained fungal mash (fungal biomass) is separated from the culture solution by vacuum filtration, and after washing several times with sterile water, the fungus is placed in an oven to be dried, and then dried. The fungal biomass was resuspended in 10 ml of sterile water, followed by breaking the fungal cells with a homogenizer and used as a chitin or chitosan source for the Example 2 hydrolysis test.

Example 2 Hydrochloric acid hydrolysis reaction test

The hydrochloric acid hydrolysis reaction test procedure is shown in Figure 1. The fungal biomass obtained in the first embodiment is used as a source of chitin or chitosan. After adding hydrochloric acid, the hydrolysis reaction is carried out under different reaction conditions to obtain glucose. The amine or acetaminoglucosamine product was added to the 1-naphthyl isothiocyanate pyridine (1-NITC) solution for derivatization, and the glucosamine content was determined by High Performance Liquid Chromatography (HPLC).

2 .1 using a conventional oven as a heat source test

10 ml of fungal cell homogenate was used as the test material in each sample, and 10 ml of 2N, 4N, 6N hydrochloric acid (HCl) solution were added separately, mixed and placed in a 100 ° C conventional oven to heat the reaction 1, 2, 3, 4, respectively. 6, 8, 12, 16, 20, 24 hours, then add 10ml of sterile water to terminate the reaction; after the solution is cooled to 30 ° C, the reaction solution is neutralized to pH value with 12N sodium hydroxide (NaOH) solution 7.0, and then filtered through a 45 μm filter, 0.1 ml of the filtered reaction solution was subjected to a derivatization reaction, and the glucosamine content was measured by HPLC. The results are shown in FIG.

2.2 Using a microwave oven as a heat source test

10 ml of fungal cell homogenate was used as the test material in each sample, and 10 ml of 2N, 4N, 6N hydrochloric acid (HCl) solution were added separately, mixed and placed in a microwave oven, and the reaction was heated at 100% power (1,400 watt), respectively. 120, 150, 180, 210, 240, 270 seconds, each 10 ml of sterile water was added to terminate the reaction; after the solution was cooled to 30 ° C, the reaction solution was neutralized to a pH value with a 12 N sodium hydroxide (NaOH) solution. 7.0, and then filtered through a 45 μm filter, 0.1 ml of the filtered reaction solution was subjected to a derivatization reaction, and the glucosamine content was measured by HPLC. The results are shown in FIG.

2.3 Different microwave power tests

Each sample was treated with 10 ml of fungal cell homogenate as test material, each 10 ml of 6N hydrochloric acid (HCl) solution was added, mixed and placed in a microwave oven at 80% power (1120 watts), 90% power (1260 watts), and 100% power ( 1,400 watt) heating reaction for 90, 120, 150, 180 seconds, and then adding 10 ml of sterile water to terminate the reaction; after the solution is cooled to 30 ° C, the reaction solution is neutralized to pH value with 12N sodium hydroxide (NaOH) solution. It was 7.0, and then filtered through a 45 μm filter. 0.1 ml of the filtered reaction solution was subjected to a derivatization reaction, and then the glucosamine content was measured by HPLC. The results are shown in FIG.

2.4 Derivatization reaction

The above-mentioned 2.1, 2.2, and 2.3 tests were carried out, and the reaction liquid obtained after the hydrolysis reaction under different conditions was subjected to a derivatization reaction, 0.1 ml of each reaction liquid was taken, and 0.3 ml of a 40 mol/m ³ 1-NITC solution was added thereto at 50 ° C. The reaction was carried out at 100 rpm for 1 hour to form a glucosamine hydrochloride derivative. After the reaction, 0.1 ml of HPLC internal standard [0.1% (wt) 3,5-dinitrobenzonitrile dissolved in acetonitrile solution] was added, and 10 μL of the filtrate was taken after filtration. Glucosamine content determination was performed.

2.5 Determination of Glucosamine Content by High Performance Liquid Chromatography (HPLC)

The glucosamine content was determined by high performance liquid chromatography (HPLC). The HPLC analysis conditions were as follows:

HPLC pump: Shimadzu LC-10AS

Column: 100 RP-18 (5μm, 4mm id × 250mm)

Column temperature: 40 ° C

Mobile phase: water / acetonitrile (87 / 13), water and acetonitrile are HPLC grade

Flow rate: 1.3ml/min

Detector: Uv-Vis Detector SPD-10A, 0.0100 AUFS (Simadzu, Japan)

Pressure: 130 ^~ 150kgf

UV detection wavelength: 230nm

UV detection time: 40 minutes

According to the peak area ratio of the glucosamine hydrochloride derivative to the internal standard, the glucosamine hydrochloride calibration line with the ratio of the peak area of the glucosamine hydrochloride derivative to the internal standard was substituted, and the glucosamine was obtained by interpolation. The grams are converted to the glucosamine content (GluN Content); the glucosamine content measured in each of 2.1, 2.2, and 2.3 is shown in Figures 2, 3, and 4, respectively.

From the results of Figure 2, when the conventional oven is used as the heat source, the sample with 6N HCl solution has the highest content of glucosamine (0.22 g/g dry weight cells) after heating for 4 hours; the sample with 4N HCl solution is After heating for 24 hours, the content of glucosamine in the sample reached the highest (0.22 g / g dry cell); while the sample added with 2N HCl solution also reached the highest content of glucosamine in the sample after heating for 24 hours (0.14 g / g Dry weight cells).

As can be seen from the results in Figure 3, when a 100% power (1400 watt) microwave oven is used as the heat source, the sample with 6N HCl solution is heated for 180 seconds (3 minutes), and the sample has the highest content of glucosamine (0.22 g/g dry weight cells). The sample added to the 4N HCl solution reached the highest level of glucosamine in the sample (about 0.10 g/g dry weight cells) after heating for 270 seconds (4.5 minutes); the sample added to the 2N HCl solution was also heated for 270 seconds. After (4.5 minutes), the content of glucosamine in the sample reached the highest (0.06 g/g dry weight cells).

In addition, as shown in FIG. 4, the hydrolysis reaction was carried out with a 6N HCl solution, and when a microwave oven of different power was used as a heat source, the content of glucosamine in the sample was heated by microwave heating at 80% power (1120 watt) for 180 seconds (3 minutes). The highest (about 0.18 g / g dry cell); the same, after heating at 90% power (1260 watt) microwave for 180 seconds (3 minutes), the sample has the highest content of glucosamine (about 0.19 g / g dry cell) After heating at 100% power (1400 watts) for 180 seconds (3 minutes), the sample contained the highest level of glucosamine (about 0.22 g/g dry weight cells).

It can be seen from the test results of the present examples that the present invention provides a method for producing glucosamine by microwave technology, which can shorten the time of hydrochloric acid hydrolysis reaction from several hours to three minutes. The comparison between the conventional hydrochloric acid method and the hydrochloric acid method of the present invention is as follows. From this, it is apparent that in the present invention, the use of microwave technology for the hydrochloric acid reaction can effectively shorten the time and reduce the energy consumption cost.

The method for producing glucosamine and acetaminophen using microwave technology provided by the invention has the following advantages when compared with other conventional techniques:

1. The difference between the present invention and the traditional hydrochloric acid is that the conventional hydrochloric acid process is carried out in an oven at a high temperature (60 ° C ^~ 100 ° C) for several hours (generally about 4 ^~ 24 hours); and the screen of the present invention is removed from the oven, and the microwave oven is used instead. The hydrochloric acid reaction can effectively shorten the reaction time, and it takes only about 3 ^to 10 minutes to achieve the same effect as the conventional method.

2. The hydrochloric acid reaction method provided by the invention has simple operation, few steps and short time (3 ^to 10 minutes can be completed), and if the process is applied to industrial large-scale production of glucosamine or acetaminoglucosamine, Reducing the cost of heating energy costs in the production process can also significantly shorten the process time, increase production capacity, and reduce the production cost of glucosamine or acetaminophen.

The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

In summary, this case is not only innovative in terms of method, but also can improve the above-mentioned multiple functions compared with the conventional methods. It should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply in accordance with the law. You are requested to approve the invention. Patent application, in order to invent, to the sense of virtue.

Figure 1 is a flow chart showing the test for producing glucosamine or acetaminoglucosamine by hydrochloric acid hydrolysis reaction.

Figure 2 shows the analysis of the glucosamine content produced by the hydrolysis of a sample with different concentrations of HCl solution by heating (100 °C) with a conventional oven as the heat source device; (O) 6N HCl, (▽) 4N HCl, (□ ) 2N HCl.

Figure 3 shows the analysis of the glucosamine content produced by the hydrolysis of a sample with different concentrations of HCl solution (100% power, 1400 watts) using a microwave oven as a heat source device; (O) 6N HCl, (▽) 4N HCl, (□) 2N HCl.

Figure 4 shows the analysis of the glucosamine content produced by heating the sample with 6N HCl solution by heating the microwave oven with different power as the heat source device; (O) 80% power (1120 watts), (▽) 90% power ( 1260 watt), (□) 100% power (1400 watts).

Claims (2)

A method for producing glucosamine and acetaminophen using microwave technology, comprising the steps of: Step 1: providing a source of chitin or chitosan selected from microorganisms of one of the following groups: Rhizopus oligosorus BCRC 31996, Monascus purpures BCRC 31499, Monascus pilosus BCRC 31527 or Aspergillus sp. BCRC 31742; Step 2: adding an acid solution of one of the group consisting of a hydrochloric acid solution and a sulfuric acid solution to the chitin or chitosan source, Forming a reaction liquid; and Step 3: placing the reaction liquid in a microwave device, heating at a power of 1120 watts to 2100 watts for 90 seconds to 270 seconds to carry out a hydrolysis reaction to make chitin or chitin Hydrolysis of the glycan produces glucosamine or acetaminophen. A method for producing glucosamine and acetaminophen using microwave technology as described in claim 4, wherein the concentration of the hydrochloric acid solution is 6N.