KR102577777B1 - High Efficiency Enzymatic Synthesis of Pyridoxine Laurate with Emulsifying Properties Using Gas-Assisted Solvent-Free System - Google Patents

Abstract

The present invention relates to a highly efficient enzymatic synthesis method of pyridoxine laurate using a gas-assisted solvent-free reaction system, which enables enzymatic synthesis of pyridoxine and lauric acid without using an organic solvent and increases the dispersion efficiency of pyridoxine. Accordingly, pyridoxine low rate can be synthesized with high efficiency.

Description

High Efficiency Enzymatic Synthesis of Pyridoxine Laurate with Emulsifying Properties Using Gas-Assisted Solvent-Free System}

The present invention relates to a method of synthesizing pyridoxine laurate using pyridoxine and lauric acid as substrates, and involves enzymatically synthesizing pyridoxine laurate using a gas-assisted solvent-free reaction system. It's about method.

Enzymatic synthesis is a synthetic method that utilizes the site specificity of an enzyme for its substrate. The enzymatic synthesis method induces a selective reaction of a specific structure, so it can produce substances with higher purity than the chemical synthesis method, and also has the advantage of easy separation and purification of the product.

Enzymatic synthesis methods are generally carried out in an organic solvent reaction system. When an organic solvent reaction system is used, the enzyme may be inactivated due to protein denaturation by the organic solvent during the reaction, and the toxicity of the product may be problematic due to the toxicity of the organic solvent. Problems that require additional treatment of wastewater due to the use of organic solvents may occur.

Accordingly, attempts are being made to use a solvent-free reaction system to overcome the shortcomings of the organic solvent reaction system. The solvent-free reaction system is environmentally friendly in that it does not use solvents, and there is no concern about the toxicity of the product. In addition, since an excess amount of substrate can be added by using one substrate as a reaction medium, there is an advantage in increasing production per volume compared to the organic solvent reaction system. However, if the substrate is not dissolved in the reaction medium and has low dispersibility, there is a problem of very low productivity.

Republic of Korea Patent No. 10-1115283 (2012.02.06.) describes a method for producing a complex of omega-3 unsaturated fatty acid and ascorbic acid. Republic of Korea Patent No. 10-0605055 (2006.07.19.) describes a method for producing 1,3-diglyceride containing medium-chain fatty acids using lipase in a reaction system that does not use organic solvents.

The present invention seeks to overcome the disadvantages of the enzymatic synthesis method using a conventional organic solvent reaction system.

The present invention seeks to provide an enzymatic synthesis method that can synthesize pyridoxine low rate with high efficiency.

The present invention is a method for producing pyridoxine laurate, characterized in that pyridoxine, lauric acid, and lipase are placed in a reactor, and an enzyme reaction is induced by spraying nitrogen gas. provides.

In the present invention, preferably, the pyridoxine is in powder and the lauric acid is in a molten state.

In the present invention, the nitrogen gas is preferably injected into the reactor through a porous glass filter at the bottom of the reactor.

In the present invention, the pyridoxine and lauric acid preferably have a molar ratio of pyridoxine:lauric acid of 0.05 to 0.3:1.

In the present invention, the enzyme reaction is preferably performed at 50 to 100°C.

Additionally, the present invention provides an emulsifier composition containing pyridoxine laurate.

In the present invention, the pyridoxine low rate preferably exhibits uniform particle distribution at pH 2.0 to 4.0.

The present invention provides a method for enzymatically synthesizing pyridoxine and lauric acid without using an organic solvent.

The present invention can synthesize pyridoxine low rate with high efficiency by increasing the dispersion efficiency of pyridoxine.

Figure 1 is a diagram showing a method for enzymatically synthesizing pyridoxine low rate of the present invention.
Figure 2 is a diagram showing an example of the synthesis process of the present invention.
Figure 3 (a) is a diagram showing a reaction curve according to the substrate molar ratio of pyridoxine and lauric acid, and Figure 3 (b) is a diagram showing a reaction curve according to reaction temperature.
Figure 4 is a diagram comparing the pyridoxine low rate productivity of an organic solvent reaction system, a solvent-free reaction system, and a gas-assisted solvent-free reaction system.
Figure 5 is a diagram comparing the enzyme stability of an organic solvent reaction system, a solvent-free reaction system, and a gas-assisted solvent-free reaction system.
Figure 6 (a) is a diagram showing the results of LC-ESI-MS analysis of pyridoxine low rate, (b) is a diagram showing the results of ¹ H NMR, and (c) is a diagram showing the results of ¹³ NMR.
Figure 7 is a diagram showing the interfacial tension reduction ability of pyridoxine low rate.
Figure 8 (a) is a diagram showing the average particle size and uniformity by pH of the emulsion to which pyridoxine low rate was added, and (b) is a diagram showing the zeta potential measurement results for each pH of the emulsion to which pyridoxine low rate was added.
Figure 9 shows the results of visual observation of an emulsion containing pyridoxine lowate with a pH of 2.0 to 8.0 immediately after preparation.
Figure 10 shows the results of visual observation of an emulsion containing pyridoxine lowate with a pH of 2.0 to 8.0 3 hours after preparation.
Figure 11 shows the results of visual observation of an emulsion containing pyridoxine lowate with a pH of 2.0 to 8.0 24 hours after preparation.
Figure 12 shows the results of visual observation of an emulsion containing pyridoxine lowate with a pH of 2.0 to 8.0 60 hours after preparation.
Figure 13 shows the results of visual observation of an emulsion containing pyridoxine lowate with a pH of 2.0 to 8.0 336 hours after preparation.
Figure 14 is a diagram showing the results of visual observation of emulsions at different concentrations of pyridoxine low rate 12 hours after preparation.
Figure 15 (a) is a diagram showing the particle size of the emulsion according to the concentration of pyridoxine low rate, and (b) is a diagram showing lecithin, tween 20, and saponin at the same concentration as pyridoxine low rate. This is a diagram comparing the particle sizes of the emulsions contained in each.

The present invention provides a method for producing pyridoxine laurate, which is characterized in that pyridoxine, lauric acid and lipase are placed in a reactor and an enzyme reaction is induced while nitrogen gas is sprayed.

The present invention synthesizes pyridoxine laurate by inducing a selective (chemoselective) ester bond between pyridoxine and lauric acid. Amphipathic pyridoxine laurate can be prepared by enzymatically synthesizing hydrophilic pyridoxine and hydrophobic lauric acid. there is.

The present invention uses lauric acid as a reaction medium when synthesizing pyridoxine laurate. Lauric acid is a saturated fatty acid with 12 carbon atoms and exists as a white solid at room temperature. Loric acid is a natural product found in coconuts, plums, etc., and is used as a raw material for detergents, surfactants, and plasticizers.

In order to use lauric acid as a reaction medium in the present invention, it is preferable to heat and melt lauric acid, and the melting of lauric acid should be done within a temperature range that can maintain the stability of the lipase enzyme used in the present invention. In the case of the lipase used in the present invention, enzyme stability can be maintained at 100°C or lower, and the melting point of lauric acid is 43.5°C, so melting of lauric acid is preferably performed at 45 to 100°C. Accordingly, the synthesis of pyridoxine low rate of the present invention is preferably performed at 45 to 100°C, and for more efficient synthesis of pyridoxine low rate, it is preferably performed at 50 to 80°C.

Pyridoxine is a type of water-soluble vitamin, and hydrophilic pyridoxine does not dissolve or mix in the hydrophobic lauric acid reaction medium, which reduces substrate dispersibility, which may lower the production efficiency of pyridoxine laurate. However, the present invention was able to increase the synthesis efficiency and significantly increase the conversion rate by dispersing pyridoxine by spraying nitrogen gas.

The present invention relates to a method for enzymatically synthesizing amphipathic pyridoxine laurate by synthesizing hydrophilic pyridoxine and hydrophobic lauric acid (Figure 1). Generally, enzymatic synthesis uses an organic solvent reaction system, but there are concerns that the enzyme may be inactivated by the organic solvent or the toxicity of the organic solvent may be problematic, so the present invention used a solvent-free reaction system.

However, there is a problem in that hydrophilic pyridoxine is difficult to mix with hydrophobic lauric acid. Accordingly, the present invention maximized the dispersion efficiency of pyridoxine by using nitrogen gas, an inert gas, and as a result, was able to increase the enzymatic synthesis conversion rate of pyridoxine low rate.

Meanwhile, in existing solvent-free reaction systems, physical energy such as magnetic stirring must be applied to increase reactivity, which causes the enzyme to separate from the immobilized support. However, in the gas-assisted solvent-free reaction system of the present invention, the enzyme was not separated from the immobilized support, and physical damage to the enzyme was found to be significantly reduced. This means that the immobilized enzyme can be used for many batches, and considering the cost of the immobilized enzyme in the entire production process, this can explain why the present invention is very economical.

The enzymatic synthesis method of pyridoxine lowate of the present invention can be carried out in a reactor. In order to introduce high purity (99.9% or more) nitrogen gas into the reactor, it is advisable to control the flow rate of nitrogen, preferably spraying nitrogen gas at a flow rate of 1.5 to 2.5 L/min.

In addition, it is preferable to further improve the dispersibility of pyridoxine by finely spraying nitrogen into the reactor through a porous glass filter. In addition, the porous glass filter is more preferably installed at the bottom of the reactor.

In addition, in the present invention, the substrate molar ratio of pyridoxine and lauric acid is preferably 0.05 to 0.3. If it is outside the above range, the synthesis efficiency of pyridoxine low rate may be minimal.

The present invention uses lipase to enzymatically synthesize pyridoxine and lauric acid. The present invention used an immobilized lipase. When using an immobilized enzyme, it has the advantage of cost reduction as it can be reused, and because it is insoluble, it can be used in a continuous reactor and reduces the sensitivity of the enzyme to changes in external conditions. Therefore, the reaction can proceed stably.

In particular, the present invention used Novozym® 435 ( Candida antarctica lipase B), and this enzyme was immobilized and used in the present invention. It is advisable to add the immobilized lipase appropriately considering the substrate, reaction volume, etc., and preferably 20 to 30 mg/mL per reaction volume.

In addition, it is advisable to set the reaction time so that pyridoxine and lauric acid can sufficiently react, but since it may vary depending on the reaction volume and amount of substrate, it can be appropriately adjusted depending on the progress of the reaction. However, it is economical to carry out the reaction until it reaches equilibrium.

The present invention can be mass-produced by utilizing a gas-assisted solvent-free reaction system capable of high-efficiency production, and can be constructed in a packed bed reactor (packed bed enzyme reactor) mainly used on an industrial scale, so it is expected to be easily applied industrially. Accordingly, it is believed that the pyridoxine low rate of the present invention can effectively replace chemically synthesized emulsifiers that can cause problems such as toxicity, environmental pollution, etc. In addition, as in the present invention, enzymatic synthesis in a gas-assisted solventless reaction system It can be applied to bioconversion processes such as microbial fermentation processes.

Additionally, the present invention provides an emulsifier composition containing pyridoxine lowate. As described above, the pyridoxine low rate of the present invention, synthesized by an enzymatic synthesis method using two natural products as substrates, has emulsifying properties due to its amphiphilic nature and can be used as an emulsifier. The emulsifier composition containing pyridoxine lowate of the present invention can be used in the food industry and chemical industry as a natural product-based low-molecular-weight emulsifier.

It was shown that the emulsifier composition containing pyridoxine lowate of the present invention can produce particles (droplets) of uniform size at a pH level of 2.0 to 4.0. The emulsion containing pyridoxine low rate of the present invention had an average particle size of about 300 nm at pH 2.0 to 4.0, and it was confirmed that an emulsion with small, uniform particles distributed at a uniformity index (PDI) of 0.18 was formed. . Meanwhile, the average particle size at pH 8.0 was found to be about 550 nm, and the uniformity index (PDI) was also at the level of 0.26, confirming the formation of an emulsion with relatively small and uniform particles distributed.

Meanwhile, it was confirmed that the emulsifier composition containing pyridoxine lowate of the present invention stabilizes the emulsion for a long period of time at pH 2.0~3.0 and 8.0.

In addition, as a result of comparing the particle size of the emulsifier composition containing pyridoxine low rate of the present invention with the conventionally commercialized low molecular weight emulsifiers lecithin, Tween 20, and saponin, the particle size of the emulsifier composition containing pyridoxine low rate of the present invention was found to be It was confirmed that it was formed in a small size, and accordingly, it was confirmed that the emulsifier composition containing pyridoxine low rate of the present invention can be used as a low molecular weight emulsifier.

Hereinafter, the contents of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of the technical idea equivalent thereto.

[Experimental Example 1: Derivation of optimal reaction conditions for enzymatic synthesis of pyridoxine lowate in a gas-assisted solvent-free reaction system]

This experiment attempted to derive optimal enzymatic synthesis conditions by deriving a reaction curve according to the substrate molar ratio and reaction temperature when synthesizing pyridoxine lowate.

The pyridoxine low rate synthesis process according to this experiment is shown in Figure 2. The synthesis process consists of a nitrogen generator, a reactor, and a constant temperature circulation water tank. The purity of nitrogen gas was controlled to 99.9% by controlling the flow rate, and the dispersibility of pyridoxine was improved by finely spraying nitrogen into the reactor through a porous glass filter installed at the bottom of the reactor. A constant temperature circulating water bath was constructed to keep the reaction temperature constant. Novozym® 435 ( Candida antarctica lipase B) immobilized with lipase was used as a catalyst. At this time, the enzyme input amount was set at 26.4 mg/mL, the reaction volume was 15 mL, and the reaction was conducted for 12 hours.

(1) Substrate molar ratio (pyridoxine/lauric acid)

In this synthesis process, we sought to confirm the synthesis efficiency according to the substrate molar ratio of pyridoxine:lauric acid, 0.05~0.3:1. The reaction temperature was set at 70°C. The reaction curve according to reaction time is shown in (a) of Figure 3. As shown in Figure 3 (a), the highest synthesis efficiency was found when the substrate molar ratio of pyridoxine:lauric acid was 0.1:1.

(2) Reaction temperature

In this synthesis process, we sought to confirm the synthesis efficiency according to the reaction temperature (50~80℃). At this time, the substrate molar ratio of pyridoxine:lauric acid was set at 0.1:1 based on the above experimental results. The reaction curve according to reaction time is shown in Figure 3 (b). As shown in (b) of Figure 3, when the reaction temperature was 70°C, the synthesis efficiency was found to be the best.

From the above experimental results, it was determined that the pyridoxine lowate synthesis process of the present invention would have the best synthesis efficiency when the substrate molar ratio of pyridoxine:lowic acid was set to 0.1:1 and the reaction temperature was 70°C.

Meanwhile, in the following, at the optimal substrate ratio and reaction temperature derived above, 'gas-assisted solvent-free reaction system' (Example 1 below), 'organic solvent reaction system' (Comparative Example 1 below), and 'solvent-free reaction system' (comparison below) After operating Example 2), their reaction results (conversion yield, volumetric productivity) were compared in Experimental Example 2.

[Example 1: Synthesis of pyridoxine lowate in a gas-assisted solvent-free reaction system]

The synthesis of pyridoxine lowate was performed in the gas-assisted solvent-free reaction system of Example 1 under the conditions shown in Table 1 below.

reaction volume 15mL reaction temperature 70℃ Substrate molar ratio
([Pyridoxine]/[Louric acid]) 0.1 Enzyme dosage 26.4 mg/mL Nitrogen injection flow rate 2.0 L/min

As shown in Table 1, the total reaction volume was 15 mL, and as the substrate molar ratio of pyridoxine:lauric acid was set to 0.1:1, 6.59 mmol of pyridoxine and 65.89 mmol of lauric acid were used. Nitrogen was finely injected into the reactor through a porous glass filter at the bottom of the reactor, and nitrogen gas was injected at 2.0 L/min to maintain purity of over 99.9%.

6.59 mmol (1.115 g) of pyridoxine, 65.89 mmol (13.199 g) of lauric acid, and 26.4 mg/mL (396 mg) of Novozym® 435 ( Candida antarctica lipase B) were added to the reactor, the temperature of the reactor was set to 70°C, and reaction was carried out. was derived. The reaction appeared to reach equilibrium at 9 hours (Figure 4).

[Comparative Example 1: Synthesis of pyridoxine lowate in organic solvent reaction system]

Pyridoxine lowate was synthesized using a reaction system using acetonitrile as a solvent. The total reaction volume was 15 mL, and considering the solubility of pyridoxine in the solvent acetonitrile, 0.3 mmol, which is the maximum amount of pyridoxine that can be added, was used. By setting the substrate molar ratio of pyridoxine:lauric acid to 0.1:1, 0.3 mmol of pyridoxine and 3 mmol of lauric acid were used, 26.4 mg/mL (396 mg) of Novozym® 435 ( Candida antarctica lipase B) was added, and 70 The synthesis of pyridoxine low rate was induced by magnetic stirring at 300 rpm at °C. The reaction appeared to reach equilibrium in 1.5 hours (Figure 4).

[Comparative Example 2: Synthesis of pyridoxine lowate in a solvent-free reaction system]

Pyridoxine lowate was synthesized using a solvent-free reaction system. The total reaction volume is 15 mL, and 6.59 mmol of pyridoxine, 65.89 mmol of lauric acid, and 26.4 mg/mL (396 mg) of Novozym® 435 ( Candida antarctica lipase B) were added to the reactor and magnetically stirred at 300 rpm at 70°C to obtain pyridoxine low. The synthesis of rate was induced. The reaction appeared to reach equilibrium at 12 hours (Figure 4).

[Experimental Example 2: Comparison of productivity of pyridoxine low rate by reaction system of Example 1 and Comparative Examples 1 and 2]

The productivity of pyridoxine low rate of each reaction system according to Example 1 and Comparative Examples 1 and 2 was compared by calculating conversion yield (%) and volumetric productivity (mmol/L/h). The conversion yield and volumetric productivity were calculated as follows when the reaction in each reaction system reached equilibrium.

▶ Conversion yield (%) = [Concentration of pyridoxine low rate after reaction (mmol/L) / Concentration of initial pyridoxine (mmol/L)] × 100

▶ Volumetric productivity (mmol/L/h) = [Concentration of pyridoxine low rate after reaction (mmol/L) / reaction time (h)]

reaction system Conversion yield (%) Volumetric productivity (mmol/L/h) Comparative Example 1
(Organic solvent reaction system) 79.56 ± 2.33 11.10 ± 0.33 Comparative Example 2
(Solvent-free reaction system) 54.29 ± 1.35 19.86 ± 0.49 Example 1
(Gas-assisted solvent-free reaction system) 84.56 ± 5.90 41.24 ± 2.89

The reaction curve for comparing productivity for each reaction system is shown in Figure 4. As shown in Table 2, the conversion yield of the gas-assisted solvent-free reaction system of the present invention was similar to that of the organic solvent reaction system of Comparative Example 1, but the volumetric productivity of the gas-assisted solventless reaction system of the present invention was about 3.7 times higher. appear. That is, the amount of substrate added in Example 1 is about 20 times higher than that of Comparative Example 1, but considering that Example 1 reaches reaction equilibrium after 9 hours and Comparative Example 1 reaches reaction equilibrium after 1.5 hours, the volumetric productivity is As a result of the comparison, it was determined that the volumetric productivity of the gas-assisted solventless reaction system of the present invention was significantly higher than that of Comparative Example 1.

In addition, the conversion yield of the gas-assisted solvent-free reaction system of the present invention was found to be about 1.6 times higher than that of the solvent-free reaction system of Comparative Example 2, and the volumetric productivity was also found to be about 2.1 times higher.

[Experimental Example 3: Evaluation of enzyme stability for each reaction system of Example 1 and Comparative Examples 1 and 2 ]

According to Example 1 and Comparative Examples 1 and 2, the time for each reaction system to reach reaction equilibrium was set as 1 batch, and each reaction was repeated 6 batches to compare enzyme stability (operational stability). Enzyme stability was calculated as follows.

▶ Enzyme stability (%) = [Concentration of pyridoxine low rate produced in the nth batch (mmol/h) / Concentration of pyridoxine low rate produced in the 1st batch (mmol/h)] × 100

As shown in Figure 5, the organic solvent reaction system of Comparative Example 1 showed enzyme stability of 82.7% when repeated 6 times, and the solvent-free reaction system of Comparative Example 2 showed enzyme stability of 88.2% when repeated 6 times.

Meanwhile, the gas-assisted solvent-free reaction system of the present invention showed an enzyme stability of 94.5% when repeated 6 times, confirming that the enzyme stability is higher than other reaction systems.

In the organic solvent reaction system, there is a risk that the enzyme will be inactivated due to protein denaturation by the organic solvent, and in the solvent-free reaction system, there is a risk that the enzyme may be separated from the support from the immobilized enzyme by physical energy such as magnetic stirring. On the other hand, when using the gas-assisted solvent-free reaction system of the present invention, there is no concern about protein denaturation by organic solvents, and by using nitrogen gas, the dispersibility of pyridoxine can be increased without magnetic stirring, so enzyme stability is high even if batches are repeated. It was judged to be maintained.

[Experimental Example 4: Structural analysis of pyridoxine lowate produced in a gas-assisted solvent-free reaction system]

Pyridoxine low rate prepared by the enzymatic synthesis reaction in Example 1 was separated and purified to high purity (99.3%) through solvent extraction using nucleic acid and water.

Mass spectrometry was performed using LC-ESI-MS, and the analysis results are shown in (a) of Figure 6. As shown in (a) of Figure 6, it was confirmed that pyridoxine lorate, in which 1 equivalent of lauric acid was bound to 1 pyridoxine molecule, was produced through the enzyme synthesis reaction.

In addition, the results of ¹ H NMR confirmation are shown in (b) of FIG. 6, and the results of ¹³ NMR confirmation are shown in (c) of FIG. 6. As shown in (b) and (c) of Figure 6, it was confirmed that the enzymatic synthesis reaction in the gas-assisted solventless reaction system of the present invention has selectivity for 5-OH among 4-OH and 5-OH of pyridoxine. .

[Experimental Example 5: Analysis of the interfacial tension reduction ability of pyridoxine lowate produced in a gas-assisted solvent-free reaction system]

MCT (medium chain triglycerides) oil refers to oil composed of medium chain fatty acids, a type of saturated fatty acid, and is mainly contained in foods such as coconut oil, butter, cheese, palm oil, and milk.

In this experiment, the pyridoxine low rate prepared in Example 1 was dissolved in MCT oil at a concentration of 0 to 6 mM, placed in a container, dropped with water, reacted at 25°C for 15 minutes, and the shape and interface of the water drop were measured. The interfacial tension reduction ability was evaluated by measuring the angle. Water was dropped into MCT oil in which pyridoxine lowate was dissolved, and the shape of the water droplet and the angle at the interface were measured to evaluate the ability to reduce interfacial tension.

As shown in Figure 7, it was confirmed that as the concentration of pyridoxine low rate increased, the interfacial tension between oil and water decreased. In other words, it was confirmed that the pyridoxine low rate of the present invention can be used as an emulsifier.

[Experimental Example 6: Analysis of emulsification characteristics of pyridoxine lowate produced in a gas-assisted solvent-free reaction system]

In this experiment, the emulsification characteristics of pyridoxine low rate according to pH of the present invention were analyzed.

(1) Check average particle size and size distribution according to pH

Add 0.2% (w/w) of pyridoxine chlorate to 1 g of MCT oil, mix with 9 mL of citric acid-sodium citrate buffer (10 mM, pH 3.0), and homogenize with a homogenizer at 11,000 rpm for 2 minutes. An emulsion was prepared by applying ultrasonic waves for 10 minutes using an ultrasonic disperser at 30% power. To evaluate emulsification characteristics according to pH, 5.0 N of NaOH was added to adjust the pH to 2.0~8.0. The droplet size distribution of the prepared emulsion was analyzed and shown in (a) of Figure 8.

As shown in (a) of Figure 8, the average particle size was found to be about 300 nm at pH 2.0 to 4.0, and it was confirmed that an emulsion was formed in which small, uniform particles with a uniformity index (PDI) of 0.18 were distributed. Meanwhile, at pH 5.0 to 7.0, the average particle size was about 1,000 nm and the uniformity index (PDI) was about 0.4, confirming the presence of non-uniform particles. In other words, emulsion stability was judged to be low at pH 5.0~7.0. In addition, even at pH 8.0, the particle size was found to be about 550 nm, and the uniformity (PDI) was also found to be at the level of 0.26. Considering that the particle size of low-molecular emulsifiers is generally 700 nm or less (Figure 15), it was determined that the pyridoxine low rate of the present invention can be used as a low-molecular emulsifier at pH 2.0 to 4.0 and 8.0.

Meanwhile, the results of measuring the zeta potential of the prepared emulsion using a Zeta Sizer are shown in (b) of Figure 8. As shown in (b) of Figure 8, it was determined that at pH 5.0 to 7.0, agglomeration occurred between particles due to a decrease in particle surface charge, resulting in low emulsion stability.

(2) Evaluation of emulsion stability through visual observation of emulsion liquid

An emulsion was prepared in the same manner as above by adding 0.2% (w/w) of pyridoxine lowate, and the pH of the emulsion was adjusted to 2.0-8.0. The emulsion was left at 25°C for 12 hours and then visually observed. The emulsion immediately after production is shown in Figure 9, the emulsion 3 hours after production is shown in Figure 10, the emulsion after 1 day (24 hours) of production is shown in Figure 11, the emulsion after 5 days (120 days) of production is shown in Figure 12, and the emulsion after 14 days of production is shown in Figure 12. The emulsion after (336 hours) is shown in Figure 13. As shown in Figures 9 to 13, it was shown that the emulsion stabilized by pyridoxine low rate could be maintained for a long period of time at pH levels of 2.0 to 3.0 and 8.0.

(3) Visual observation of emulsion containing pyridoxine lowate at pH 3.0

An emulsion was prepared in the same manner as above by adding 0, 0.07, 0.14, and 0.28% (w/w) of pyridoxine low rate, and the pH of the emulsion was adjusted to 3.0. Figure 14 shows the results of leaving the emulsion at 25°C for 12 hours.

As shown in Figure 14, it can be seen that in the emulsion (blank) to which pyridoxine low rate was not added, water and oil were completely separated. However, separation of water and oil did not occur in the 0.07-0.28% (w/w) emulsion, and it was confirmed that the emulsion was stabilized by pyridoxine lorate.

(4) Confirmation of average particle size according to pyridoxine low rate concentration at pH 3.0

Dissolve 0~0.2% (w/w) of pyridoxine chlorate in 1 g of MCT oil, mix with 9 mL of citric acid-sodium citrate buffer (10 mM, pH 3.0), and homogenize for 2 minutes at 11,000 rpm with a homogenizer ( homogenization) and ultrasonic dispersion at 30% power for 10 minutes to prepare an emulsion. An emulsion was prepared by adding 5.0 N of NaOH to adjust the pH to 3.0.

The results of analyzing the droplet size of the prepared emulsion are shown in Figure 15. As shown in (a) of Figure 15, it was confirmed that the average particle size decreased as the concentration of pyridoxine low rate increased. This was believed to be because the amount of pyridoxine low rate located on the particle surface increased as the concentration increased. On the other hand, it was determined that at a concentration of 0.10% (w/w) or more, the amount of pyridoxine located on the particle surface was saturated and particles could no longer be formed small.

Meanwhile, in order to compare the particle size with the commercialized low-molecular-weight emulsifiers lecithin, Tween 20, and saponin, an emulsion was prepared by adding them at the same concentration (0.20%, w/w), and the average particle size was compared. As shown in (b) of 15, it was confirmed that the pyridoxine low rate of the present invention forms smaller particles compared to commercially available low-molecular-weight emulsifiers.

Claims (7)

A method for producing pyridoxine laurate, characterized in that pyridoxine, lauric acid, and lipase are placed in a reactor, and an enzyme reaction is induced by spraying nitrogen gas.
According to paragraph 1,
The pyridoxine is a powder,
A method for producing pyridoxine lorate, characterized in that the lauric acid is in a molten state.
According to paragraph 1,
The nitrogen gas is,
A method for producing pyridoxine low rate, characterized in that it is sprayed into the reactor through a porous glass filter at the bottom of the reactor.
According to paragraph 1,
The pyridoxine and lauric acid,
A method for producing pyridoxine lorate, characterized in that the molar ratio of pyridoxine: loric acid is 0.05 to 0.3: 1.
According to paragraph 1,
The enzyme reaction is,
A method for producing pyridoxine low rate, characterized in that it is carried out at 50 to 100 ° C.
delete delete