KR102085318B1 - Lactobionic Acid Manufacturing Method - Google Patents

Abstract

The present invention provides a method for preparing lactobionic acid, comprising the following steps of: (1) culturing a strain in a culture medium including water, a beef extract, peptone, a yeast extract, sodium chloride and lactose; and (2) culturing a primary culture medium, which includes water, a beef extract, peptone, a yeast extract, sodium chloride and lactose, and which has completed the first step in a solution including any one salt among Na_2CO_3, K_2CO_3, CaCO_3, NaOH and KOH. Therefore, according to the present invention, provided is a method for preparing lactobionic acid which can be commercially produced in large quantities, can be simply and conveniently prepared and produced, and can improve economic efficiency when prepared and produced.

Description

Lactobionic Acid Manufacturing Method

The present invention relates to a method for producing lactobionic acid, and relates to a method for producing lactobionic acid in which the method is improved to enable a simple and convenient commercial mass production.

Lactobionic Acid (4-O-B-D-galactopyranosyl-D-gluconic acid) consists of a molecule that attaches a molecule of Gluconic acid to Galactose through ether linkage. Cluconic acid, which is naturally produced in cells, is known as polyhydroxy AHA (PHA), which has a beneficial effect on the skin, and is now widely used in skin care products. Galactose is chemically neutral and endogenous (naturally occurring in the body) and is used to grow cells that enhance glycosaminoglycan synthesis, collagen synthesis, and wound healing.

In addition, lactobionic acid is a new type of polyhydroxy acid, which has been tested for safety, antioxidant, and moisturizing as a skin care material, and has been recognized for its functionality. In addition, lactobionic acid has an effect of blocking ions such as iron, thereby inhibiting the production of hydroxyl radicals and thus preventing oxidation in cellular tissues. As a hygroscopic compound, gel matrices are used at room temperature. It has a moisturizing function that catches moisture by forming a matrix.

In addition, lactobionic acid is used as a counter ion to mineral supplementation and intravenous erythromycin in salt form in the medical field. In addition, it has been shown to have a positive effect on collagen synthesis / agglomeration and also shows efficacy in wound healing.

On the other hand, lactobionic acid helps to effectively absorb Ca ions when used as food additives and can prevent osteoporosis. It is confirmed that the mixed use with isoflavones helps diet by reducing blood lipids and cholesterol.

Therefore, it is desirable to mass produce such lactobionic acid for a variety of applications as described above.

[references]

Publication No. 10-2016-0026042 (published Mar. 09, 2016)

Publication No. 10-2006-0123514 (published Dec. 1, 2006)

It is an object of the present invention to provide a method for producing lactobionic acid which is commercially available for mass production.

Another object of the present invention is to provide a method for producing lactobionic acid, which is simple and convenient in production and production.

In addition, another object of the present invention is to provide a method for producing lactobionic acid which can improve economical efficiency during production and production.

An object of the present invention, (1) the process of culturing the strain in a culture solution containing water, beef extract, peptone, yeast extract, sodium chloride, lactose; Water, beef extract, peptone, yeast extract, sodium chloride, lactose, including the salt of any one of Na ₂ CO ₃ , K ₂ CO ₃ , CaCO ₃ , NaOH and KOH 1 (2) process of culturing the primary culture; including, but in the process (2) the 100% by weight of the beef extract in the range of 0.1 to 2.0wt%, the peptone is in the range of 0.1 to 2.0wt%, the yeast extract is 0.1 to 2.0 wt%, the sodium chloride is 0.1 to 5.0wt%, the lactose is 20.0 to 30.0wt%, the primary culture solution is 1.0 to 10.0wt%, CaCO ₃ 1 to 5wt %, The remainder containing water, after sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 0.5 ~ 2.0vvm (volume of air added by the method for producing lactobionic acid, characterized in that the culturing the strain for 12 to 48 hours in the range (to liquid volume per minute) It is sex.

In addition, the strain (1) in the process is Pseudomonas teatrolens ( Pseudomonas teatrolens ), Acetobacter orientalis ( Burkholderia cepacia ), Halobacterium saccharovorum ( Halobacterium saccharovorum ) It is preferable to include either.

In addition, the beef extract of the 100wt% in the process (1) is in the range of 0.1 ~ 2.0wt%, the peptone is in the range of 0.1 ~ 2.0wt%, the yeast extract in the range of 0.1 ~ 2.0wt%, the sodium chloride is 0.1 ~ 5.0wt% range, the lactose is made in the range of 1.0 ~ 5.0wt% and the remainder contains water, after sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, supply The amount of air is preferably incubated with the strain for 12 to 48 hours in the range of 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute).

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In addition, it is preferable to check the conversion rate of the strain after the process (2) and to further add lactose if the factor including the oxygen concentration and pH is within or above the set range.

In addition, it is preferable to produce lactobionic acid or lactobionic acid salt through the above (2) process.

In addition, between the (1) process and the (2) process, the beef extract in the range of 100wt% is 0.1 ~ 2.0wt%, the peptone is in the range of 0.1 ~ 2.0wt%, the yeast extract is 0.1 ~ 2.0wt% In the range, the sodium chloride is in the range of 0.1 to 5.0wt%, the lactose is in the range of 1.0 to 5.0wt%, the first culture of the process (1) is made of 1.0 to 10wt% range and the rest includes water, After sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 12 ~ 48 hours in the range of 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute) It is preferable to incubate for a while.

In addition, the deactivation process of deactivating by sterilizing the activated strain after the (2) process; A cooling step of cooling after the deactivation process; A filtration process to filter out foreign matter; It is preferable to include; a purification process for recovering the precipitate or to remove the ionic material including the salt.

In addition, when the conversion rate of the strain is 80% or more, the oxygen concentration is 0 to 20%, pH is 5.5 or more, it is determined that the activity of the strain is active, it is preferable to add more lactose.

Thus, according to the present invention, it is possible to provide a method for producing lactobionic acid which can be commercially mass-produced.

In addition, it is possible to provide a method for producing lactobionic acid, which is simple and convenient to manufacture and produce.

In addition, it is possible to provide a lactobionic acid production method that can improve the economics at the time of manufacture and production.

1 is a flow chart listing the process for producing lactobionic acid according to an embodiment of the present invention,
2 is a process flow chart listing processes for preparing lactobionic acid according to another embodiment of the present invention;
3a to 3c are photographs showing the primary to the main culture process.

A method for producing lactobionic acid according to an embodiment of the present invention will be described in detail with reference to FIGS. 1 to 3C.

1 is a flowchart listing a process for preparing lactobionic acid according to an embodiment of the present invention, and FIG. 2 is a flowchart illustrating a process for preparing lactobionic acid according to another embodiment of the present invention. Figure 3c is a photograph showing the primary to the main culture process.

Method for producing lactobionic acid comprises the steps of (1) the step (S110, S210) of culturing the strain in the primary culture medium containing water, beef extract, peptone, yeast extract, sodium chloride, lactose; In the solution containing water, beef extract, peptone, yeast extract, sodium chloride, lactose and salts of any one of Na ₂ CO ₃ , K ₂ CO ₃ , CaCO ₃ , NaOH and KOH, (2) process of culturing (S120, S220); includes.

Here, the strain used in the process (1) is Pseudomonas teatrolens ( Pseudomonas teatrolens ), Acetobacter orientalis , Burkholderia cepacia , Halobacterium saccharovorum ( Halobacterium saccharovorum) It is preferable to select any one of the group consisting of

In the process (1), the beef extract in the range of 100 wt% is in the range of 0.1 to 2.0 wt%, the peptone is in the range of 0.1 to 2.0 wt%, the yeast extract is in the range of 0.1 to 2.0 wt%, and the sodium chloride is 0.1 In the range of ~ 5.0wt%, the lactose is in the range of 1.0 ~ 5.0wt% and the rest is made of water.

In addition, the (1) process is sterilized for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 0.5 ~ 2.0vvm (volume of air added to liquid volume per It is preferable to incubate the strain for 12 to 48 hours in a minute) range (see FIG. 3A).

Beef extracts are used as nitrogen sources in strain culture because they contain abundant nutrients such as amino acids, peptides, organic acids, phosphoric acid and vitamins.

Peptone is an inducible protein that is water-soluble, does not coagulate in heat, and does not precipitate by sulphite or other precipitation methods. Used as

Sodium chloride is a compound of sodium and chlorine, which is used in everyday life and industry such as seasoning and salting. In animals, it is essential to play an important role in the maintenance of osmotic pressure in the body, and is known to maintain cellular osmotic pressure to prevent cell rupture.

Yeast extract is a water-soluble extract of autolytic yeast cells, rich in amino acids, vitamin B group, etc., and used as an additive to many media.

Lactose (Lactose) is called lactose or lactose and is a component that is converted to lactobionic acid by a culture strain. For the convenience of culturing added between primary and secondary cultures ((1) and (2) processes of the present invention) In the case of 'secondary culture', a small amount is added to adapt the strain to induce good activity of the strain in the present culture.

Most of the 100wt% is occupied by water and the remaining composition is mixed and sterilized, and the strain is primarily cultured under the above-described conditions.

Including the primary culture solution that went through this process is going through the process (2).

The composition added to the process (2) is in the range of 1.0 ~ 10.0wt% of the primary culture cultured in the process (1), 0.1 ~ 2.0wt% of beef extract, 0.1 ~ 2.0wt% of the peptone, Yeast extract is in the range of 0.1 to 2.0wt%, sodium chloride is in the range of 0.1 to 5.0wt%, the lactose is in the range of 1.0 to 50.0wt%, the primary culture is in the range of 1.0 to 10.0wt%, Na ₂ CO ₃ , K ₂ CO ₃ , CaCO ₃ , NaOH, KOH any one of the group consisting of 1 to 5wt% range, the rest contains water, after sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, The rotational speed is 50 ~ 150rpm, the amount of air supplied is incubated for 12 to 48 hours in the range of 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute). After this culture, the conversion rate to lactobionic acid or lactobionic acid salt and the degree of activation of the strain are checked (see FIG. 3C).

Such conversion to lactobionic acid or lactobionic acid salt is measured by High Performance Liquid Chromatography (HPLC) (eg SHIMADZU RID-20A, JAPAN). The conditions of use are shown in parentheses (Rezex ^tm ROA-Organic Acid H ⁺ column; Mobile phase: 0.005 NH ₂ SO _4; Flow Rate 0.5 mL; Temperature: 70 ° C .; Injection: 20 μl).

After the incubation, it is possible to confirm whether lactobionic acid or lactobionic acid is generated through HPLC analysis of the culture solution. It can be seen that most of them are present as lactobionic acid salts.

The conversion rate is converted to lactobionic acid or lactobionic acid and the oxygen concentration, pH, etc. to determine the conversion rate is 80% or more, when the oxygen concentration is 0-20%, pH is 5.5 or more, the activity of the strain is active It is advisable to add more lactose (eg, about 10-40 wt%) after determining that it is losing. As microorganisms grow, organic acids such as acetic acid are produced as metabolites. The pH decreases due to these organic acids, and the activity of microorganisms is markedly lowered at pH below 5.5. It is determined that the activity of microorganism is the highest when oxygen concentration in the incubator is in the range of 0 ~ 20% under the condition that oxygen is injected and discharged normally. If more than 20% of the oxygen is present in the state of low oxygen consumption of the strain it is determined that the activity is reduced due to lack of nutrients or aging.

By adding lactose in this way, lactose can be rapidly converted to lactobionic acid or lactobionic acid by the activated strain. As a result, it can be seen through experiments that the productivity of lactobionic acid salt to lactobionic acid to be obtained is increased to increase productivity and economy. That is, the process of adding lactose in Figure 2 (S227) is preferably made if it is determined that the activated as a result of determining the activation of the strain (S225, S226).

In addition, between the (1) process and (2) process, the beef extract in the 100wt% range of 0.1 ~ 2.0wt%, the peptone is in the range of 0.1 ~ 2.0wt%, the yeast extract is 0.1 ~ 2.0wt In the range of%, the sodium chloride is in the range of 0.1 to 5.0wt%, the lactose is in the range of 1.0 to 5.0wt%, including the primary culture at 1.0 to 10wt% and the rest is made of water. Next, after sterilization for 15 minutes at 121 ℃ as in the step (1), the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 0.5 ~ 2.0vvm (volume of air added to liquid volume It is preferable to incubate the strain for 12 to 48 hours in the range per minute) (see FIG. 3B, S215) (as described above, this process is also referred to as 'secondary culture' as necessary).

If the intermediate process ('secondary culture') is further included between the processes (1) and (2), lactobionic acid salt or lactose is more smoothly used in the final process (2) (main culture) depending on the type of strain. Nonionic acids are preferred since they can be produced.

After completion of the main culture process (2) is made a process of inactivation (S130, S230). Inactivation process is a process of sterilization for 15 minutes while maintaining 121 ℃ in the fermenter.

The next cooling process is a process of cooling the state of high temperature of the deactivation process to about 50 ° C. or less (S140 and S240).

The filtration process is a process in which the solids present are coated with a filtration tank including diatomaceous earth or perlite, for example, and then filtered (S150, S250).

In the process of purification, the precipitate is recovered by reacting the alcohol in the filtrate at a ratio of 1.2 to 10 times. The recovered precipitate is dissolved in water, and then lactobionic acid can be obtained by removing 'ionic materials such as salts' using ion exchange resins (anions, cations) (S160, S260). Here, the cationic resin used is TRILITE SCR04, SCR-B, SCR10, SCR12 AMBERLITE IR-45, IR-100, IR-105, IR-112, IR-120, etc., for example, For example, TRILITE AMP16, AMP26, AMP24, AW90, AMBERLITE IRA-400, IRA-401, IRA-4B and the like. However, during the refining process, the alcohol may not be mixed to recover the precipitate as needed.

After the post-treatment including the process of concentrating, filtration, and powdering the lactobionic acid thus obtained, the final product lactobionic acid can be obtained in powder form (S170, S270). This post-treatment process is generally known in the art, and thus a detailed description thereof will be omitted.

Thus, according to the present invention, it is possible to provide a method for producing lactobionic acid which can be commercially mass-produced.

In addition, it is possible to provide a method for producing lactobionic acid, which is simple and convenient to manufacture and produce.

In addition, it is possible to provide a lactobionic acid production method that can improve the economics at the time of manufacture and production.

The present invention can be implemented in other embodiments except for the above-described embodiment. Those skilled in the art should understand that technical solutions formed by equivalent replacement or equivalent modifications fall within the protection scope of the present invention.

Claims (9)

(1) the process of culturing the strain in a culture solution containing water, beef extract, peptone, yeast extract, sodium chloride, lactose;
Water, beef extract, peptone, yeast extract, sodium chloride, lactose, including the salt of any one of Na ₂ CO ₃ , K ₂ CO ₃ , CaCO ₃ , NaOH and KOH 1 (2) process of culturing the primary culture;
In the (2) process, the beef extract in the range of 100wt% is 0.1 to 2.0wt%, the peptone is in the range of 0.1 to 2.0wt%, the yeast extract is in the range of 0.1 to 2.0wt%, and the sodium chloride is 0.1 to 5.0 In the wt% range, the lactose is in the range of 20.0 ~ 30.0wt%, the primary culture medium is in the range of 1.0 ~ 10.0wt%, CaCO ₃ is in the range of 1 ~ 5wt%, the rest includes water, 121 ℃ After sterilization for 15 minutes at, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute) range for 12 to 48 hours A method for producing lactobionic acid, comprising culturing a strain. The method of claim 1,
In the process (1), the strain is any one of Pseudomonas teatrolens , Acetobacter orientalis , Burkholderia cepacia , and Halobacterium saccharovorum. The lactobionic acid production method comprising a. The method of claim 1,
In the process (1), 100% by weight of the beef extract in the range of 0.1 to 2.0wt%, the peptone is in the range of 0.1 to 2.0wt%, the yeast extract in the range of 0.1 to 2.0wt%, the sodium chloride is 0.1 to 5.0 In the wt% range, the lactose is in the range of 1.0 to 5.0wt% and the rest includes water,
After sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 12 ~ 48 hours in the range of 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute) Method for producing lactobionic acid, characterized in that for culturing the strain. delete The method of claim 1,
The lactobionic acid production method according to claim 2, further comprising adding lactose when a factor including a conversion rate and an oxygen concentration and a pH is within or above a result of checking the conversion rate to activity of the strain. The method according to claim 1 or 5,
The lactobionic acid production method characterized in that the lactobionic acid or lactobionic acid salt is produced through the step (2). The method of claim 3,
Between the process (1) and the process (2) the 100% by weight of the beef extract in the range of 0.1 ~ 2.0wt%, the peptone is in the range of 0.1 ~ 2.0wt%, the yeast extract in the range of 0.1 ~ 2.0wt% The sodium chloride is in the range of 0.1 to 5.0wt%, the lactose is in the range of 1.0 to 5.0wt%, and the first culture of the process (1) is made in the range of 1.0 to 10wt%, and the rest includes water.
After sterilization for 15 minutes at 121 ℃, the temperature is 20 ~ 50 ℃, the rotation speed is 50 ~ 150rpm, the amount of air supplied is 12 ~ 48 hours in the range of 0.5 ~ 2.0vvm (volume of air added to liquid volume per minute) Method for producing lactobionic acid, characterized in that for culturing the strain. The method of claim 1,
An inactivation process of deactivating by sterile the activated strain after the process (2);
A cooling step of cooling after the deactivation process;
A filtration process to filter out foreign substances;
A process for preparing lactobionic acid, comprising: a purification process for recovering a precipitate or removing an ionic substance including a salt. The method of claim 5,
When the conversion rate of the strain is 80% or more, the oxygen concentration is 0 ~ 20%, the pH is 5.5 or more, it is determined that the activity of the strain is active and lactobionic acid production method characterized in that it further adds lactose.

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