KR102250428B1 - New Yeast Strain with high productivity of erythritol production method thereof - Google Patents

Abstract

The present invention is a novel yeast strain that produces erythritol isolated from peaches Candida sorbossiborans ( Candida sorbosivorans ) JM409 and an optimal culture method for increasing the production of erythritol using the same.In the present invention, the production capacity of erythritol is a novel yeast strain, Candida sorbosivorans JM409 strain was isolated from peaches, and since this strain established the optimal conditions for producing erythritol, it could be usefully used as a sweetener manufacturing technology to replace sugar.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention New Yeast Strain with High Productivity of Erythritol Production Method thereof TECHNICAL FIELD

The present invention is a novel yeast strain Candida sorbosiborans that produces erythritol isolated from peaches ( Candida sorbosivorans ) JM409 and an optimal culture method for increasing the production of erythritol using the same.

Recently, not only in Korea but also in the world, more and more people are aware of health problems such as chronic obesity and diabetes due to continuous consumption of sugars and prefer low-sugar products. In line with this, interest in mortgage is increasing in the food industry, and a movement to apply the mortgage trend to food is also actively taking place.

Among them, sugar alcohol, which is attracting attention as an alternative sugar, has the advantage of having a sweet taste equivalent to 60-80% of sugar, including a refreshing sensation, and above all, having a lower calorie than sugar.

In general, sugar alcohol has a polyol structure and is present in fruits and vegetables such as melons and grapes in nature. Currently, most of them are made through hydrogenation of carbohydrates by catalysts, but the production efficiency is low and the importance of producing sugar alcohols using microorganisms is increasing.

These sugar alcohols include xylitol, mannitol, sorbitol, and erythritol. Among them, erythritol is a sugar alcohol composed of four carbons, which is a low-calorie substance with almost zero calories, and it is not available to oral bacteria Streptococcus mutans and Streptococcus sobrinus. It is known to be effective in prevention. In addition, it has been reported that erythritol has an excellent ability to remove active oxygen and acts as an antioxidant.

The most studied strains as strains capable of producing erythritol are Candida magnolia and Yarrowia lipolytica. Representative strains used industrially because of their high efficiency are Aureobasidium sp. and Pseudozyma tsukubaensis.

Accordingly, the present inventors confirmed that a new strain isolated from peaches produced erythritol while studying a new strain producing erythritol, thus completing the present invention.

Registered Korean patent (0001) KR10-1576183

An object of the present invention is to provide a Candida sorbosivorans JM409 strain (KACC 93287P) having erythritol-producing ability.

Another object of the present invention is to provide a method for producing erythritol using the strain.

In order to achieve the above object, the present invention is Candida sorbose borance ( Candida) producing erythritol (Erythritol) sorbosivorans ) JM409 strain (KACC 93287P) is provided.

In one embodiment of the present invention, the Candida sorbosivorans JM409 strain is isolated from peaches.

In the present invention, the term "erythritol" is a white crystalline powder and has no odor and has a sweet taste. The chemical formula is C ₄ H ₁₀ O ₄ to be. It is well soluble in water, slightly soluble in alcohol, insoluble in ether. The melting point is 119~123℃. It has excellent heat resistance and low hygroscopicity, which makes it easy to form crystals. The sweetness is about 70-80% of sugar and has a refreshing sweetness. Unlike other sweeteners, it is not used as an energy source in the body and is mostly discharged, so it is a low-calorie sweetener due to its low absorption rate.

In addition, the present invention candida sorbose borance ( Candida sorbosivorans ) fermenting the strain JM409 (KACC 93287P) in a medium; And it provides a method for producing erythritol comprising the step of recovering erythritol from the culture medium or the strain according to the fermentation.

The medium used for the cultivation must meet the requirements of the specific strain in an appropriate manner. Sugar sources that can be used include sugars and carbohydrates such as glucose, saccharose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid. , Fatty acids such as linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid may be included, but are not limited thereto. These materials can be used individually or as a mixture.

In addition, the culture medium may contain metal ions as a reducing agent required for growth. Candida of the present invention sorbosivorans JM409 preferably includes Fe ^{2 +.} In addition to the substances, essential growth substances such as amino acids and vitamins may be included. In addition, precursors suitable for the culture medium may be used. The above-described raw materials may be added batchwise or continuously in a manner appropriate to the culture during the cultivation process.

Meanwhile, a basic compound such as sodium hydroxide, potassium hydroxide, or ammonia, or an acid compound such as phosphoric acid or sulfuric acid may be used in an appropriate manner to adjust the pH of the culture. In addition, foaming can be suppressed by using an antifoaming agent such as fatty acid polyglycol ester. Oxygen or oxygen-containing gas (e.g. air) is injected into the culture to maintain an aerobic condition. The temperature of the culture can usually be between 20°C and 45°C. Fermentation is continued until the maximum amount of estritol produced is obtained. For this purpose it can usually be achieved in 10 to 220 hours. Erythritol may be discharged into the culture medium or contained in the cells.

The recovery method of erythritol that can be used in the present invention may be a method known in the art, such as centrifugation, filtration, anion exchange chromatography, crystallization, and high performance liquid chromatography (HPLC), but is not limited to these examples. .

In the present invention, Candida sorbosivorans JM409 strain, a new yeast strain with erythritol production ability, was isolated from peaches, and since this strain established optimal conditions for producing erythritol, it is useful as a sweetener manufacturing technology to replace sugar. Will be able to use it.

1 is a graph showing the result of comparative analysis of erythritol production between the existing erythritol-producing strain and the new strain of the present invention: error bars represent the standard deviation of two biological replicas.
Figure 2 is a graph showing the fermentation characteristics according to the type of carbon source Candida sorbosivorans type strain (A) and Candida sorbosivorans JM409 (B):
Rhombus: fructose concentration, triangle: erythritol concentration, circle: cell density, error bars represent the standard deviation of two biological replicas.
3 is a result showing the fermentation characteristics of Candida sorbosivorans type strain (black symbol) and Candida sorbosivorans JM409 (white symbol) according to temperature conditions:
A: 30°C, B: 37°C, square: glucose concentration, triangle: erythritol concentration, circle: cell density, error bars represent the standard deviation of two biological replicates.
Figure 4 is a graph comparing the amount of erythritol production according to the glucose concentration of Candida sorbosivorans type strain and Candida sorbosivorans JM409:
A: Candida sorbosivorans type strain 100g/L glucose,
B: Candida sorbosivorans JM409 100g/L glucose,
C: Candida sorbosivorans type strain 200g/L glucose,
D: Candida sorbosivorans JM409 200g/L glucose,
Error bars represent standard deviation of two biological replicates.
Figure 5 is a graph showing the amount of erythritol production of Candida sorbosivorans JM409 strain according to the type of reducing agent:
Type of reducing agent: 5 kinds of metal ions (Ca ^{2 +} , Cu ^{2 +} ,Mg ^{2 +} , Mn ^{2 +} , Fe ^{2 +} ),
Error bars represent standard deviation of two biological replicates.
6 is a graph showing the results of scale-up fermentation of Candida sorbosivorans JM409 using a 5L bioreactor. Square: glucose concentration, triangle: erythritol concentration, circle: cell density

Hereinafter, the present invention will be described in detail.

The present invention confirmed that the Candida sorbovisorans JM409 strain, a new yeast strain isolated from peaches, was a strain capable of producing erythritol, and was consigned to the Korea Agricultural Microbial Resource Center (KACC) under the microbial accession number KACC93287P on October 24, 2017. .

In addition, in order to maximize the production capacity of erythritol of the Candida sorbovisorans JM409 strain, the fermentation characteristics of the strain were identified, and it was confirmed that the amount of erythritol production was the highest at 250 rpm, which is an aerobic condition (Table 1). It was ^{confirmed that the production amount was increased when Fe 2 +} metal ions were added (FIG. 5).

Hereinafter, the present invention will be described in more detail by examples. These examples are only

For the purpose of describing the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited to these examples.

< Example 1> Materials and methods

<1-1> Strains and media

In the present invention, a yeast strain collected and isolated from a domestic peach sample was used. The isolated yeast was first cultured in 10ml YPD medium (10g/L yeast extract, 20g/L Bacto peptone, 20g/L glucose) at 30°C and 250 rpm for 24 hours. The cultured strain was centrifuged to remove the supernatant, and only the strain was separated and _{inoculated to an OD 600} = 1.0. Each experiment was fermented under limited aerobic conditions in 20ml YPD medium using a 100ml Erelnmeyer flask. YPD medium was tested by dividing the glucose concentration into 100g/L and 200g/L, the temperature at 30℃, 37℃, and the oxygen condition at 80rpm, 200rpm, and 250rpm.

<1-2> According to the type of metal ion Erythritol Production analysis

As an experiment to compare the amount of erythritol produced according to the type of metal ion, a total of five metal ions were used. To supply Fe ^{2 +} , Mg ^{2 +} , Mn ^{2 +} , Cu ^{2 +} , and Ca ^{2 +} 0.35 g/L of Fe ₂ SO ₄ 7H ₂ O, MgSO ₄ 7H ₂ O, MnSO ₄ H ₂ O, CuSO ₄ ·5H ₂ O, CaCl ₂ ·2H ₂ O was added to 20ml YPD (100g/L), followed by fermentation at 250 rpm and 30°C.

<1-3> Scale-up fermentation

Scale-up fermentation of erythritol was carried out in a fed-batch manner at 2L YPD (100g/L glucose), 30℃, 250rpm, and pH5 in a 5L-bioreactor. The initial OD ₆₀₀ was set to 1.0 and inoculated, and the antifoam was used as a silicone antifoam from Dow Corning.

The scale-up fermentation run is generally a basic step for industrial application, and since it can produce different results from flask fermentation during mass fermentation, the scale-up fermentation results are checked after flask experiments and compared with flask experiments to optimize mass fermentation conditions. It was implemented for.

<1-4> HPLC analysis

The concentration of erythritol in the solution was analyzed by high performance liquid chromatography (HPLC, Agilent Technologies 1260 Series) using an RI detector. Rezex ROA-Organic Acid H+ (8%) (Phenomenex Inc., Torrance, CA) was used as the column, and the mobile phase was eluted at 50° C. at a rate of 0.6 mL/min using a 0.005 N sulfuric acid solution.

< Example 2> Experiment result

<2-1> Existing Erythritol With the production strain Erythritol Comparative analysis of production

The amount of erythritol production of the existing erythritol-producing strain and the Candida sorbosivorans JM409 strain of the present invention was analyzed.

Specifically, erythritol production of Candida sorbosivorans JM409 strains including Candida sorbosivorans type strain with Candida magnoliae and Pseudozyma tsukubaensis reported as erythritol producing strains was compared. Fermentation conditions were 20ml YPD (100g/L), 30℃, 250rpm, and fermented for 48 hours to confirm the production of erythritol.

As a result, it was shown that Candida sorbosivorans JM409 of the present invention produced the most at 4.35 g/L, and erythritol was produced in the order of Pseudozyma tsukubaensis and Candida sorbosivorans. Candida magnoliae did not produce erythritol (Fig. 1).

Therefore, although the preferred fermentation conditions are different for each strain, Candida sorbosivorans JM409 strain is also an erythritol-producing strain when comparing Candida magnolia, which requires a relatively high glucose concentration of 200-400 g/L, and Pseudozyma tsukubaensis, which prefers aerobic conditions of 500 rpm-850 rpm We believe that there is a potential possibility.

<2-2> According to fermentation conditions Erythritol Productivity analysis

The amount of erythritol production of Candida sorbosivorans JM409 strain according to the fermentation conditions of the present invention was analyzed.

Specifically, to investigate the fermentation characteristics of Candida sorbosivorans JM409, a new yeast strain isolated from peaches, the production of erythritol was compared while changing the carbon source, temperature, sugar concentration, and oxygen conditions. The control in each experiment was a Candida sorbosivorans type strain.

First, referring to the study that there is a strain of the genus Candida that favors fructose, fructose and glucose were compared as a carbon source. The experiment was conducted under the conditions of YPD (200g/L), 200 rpm, and 30°C.

As a result, both Candida sorbosivorans type strain and Candida sorbosivorans JM409 strain did not produce erythritol when fructose was used as a substrate (FIG. 2).

In addition, as a result of experimenting at YPD (200g/L) and 200 rpm with different temperature conditions at 30° C. and 37° C., not only did little growth at 37° C., but hardly produced erythritol (FIG. 3).

Thirdly, the amount of erythritol production according to the oxygen condition and the sugar concentration was compared and shown in Table 1 below.

Appropriate concentration (100g/L glucose) Appropriate concentration (200g/L glucose) Aeration
Aeration condition Candida sorbosvirans
type strain Candida sorvosivorans JM409 Candida sorbosvirans
type strain Candida sorvosivorans JM409 80rpm 1.8 3.1 2.6 3.2 200rpm 4.2 8.5 3.9 6.9 250rpm 2.5 6.8 4.0 7.4

As a result of fermentation, when tested at 80 rpm in YPD (100 g/L) medium, Candida sorbosivorans type strain produced 1.8 g/L of erythritol for 48 hours (Fig. 4A), and Candida sorbosivorans JM409 strain produced 3.1 g/L (Fig. 4B).

When the experiment was conducted at 250 rpm in the same concentration medium, the type strain was 2.5 g/L (FIG. 4A), and Candida sorbosivorans JM409 produced 6.8 g/L, which was about 1.7 times more than 80 rpm (FIG. 4B). When fermented at 80 rpm and 250 rpm with YPD (200 g/L) by varying the sugar concentration of the medium, the type strain was 2.6 g/L (Fig. 4C) for 48 hours at 80 rpm, and the Candida sorbosivorans JM409 strain was 3.2 g/L erythritol. Was produced (Fig. 4D). At 250 rpm, the type strain produced 4.0 g/L (Fig. 4C), and Candida sorbosivorans JM409 produced 7.4 g/L (Fig. 4D). There was little difference according to the sugar concentration in both strains, and all glucose fermentation in YPD (200g/L) was consumed over 96 hours regardless of rpm.

In addition, it was confirmed that both strains produced more erythritol at 250 rpm, which is an aerobic condition than 80 rpm.In particular, Candida sorbosivorans JM409 strain produced a greater amount of erythritol when compared to the type strain.

<2-3> Erythritol Analysis of the role of metal ions in production

The amount of erythritol production of Candida sorbosivorans JM409 according to the type of metal ions was confirmed, and the role of metal ions in erythritol production was analyzed using metal ions suitable for the strain.

Specifically, the experiment was used for the 0.35g / L concentration of _{Fe 2 SO 4 · 7H 2 O} , MgSO 4 · 7H 2 O, MnSO 4 · H 2 O, CuSO 4 · 5H 2 O, CaCl 2 · 2H 2 O . Each metal ion was added to confirm the erythritol yield.

As a result, Candida sorbosivorans JM409 strain produced 6.9 ^{g/L in Fe 2 +} when no metal ions were added, and 9.4 g/L erythritol produced 0.09 g/g glucose compared to the yield of 0.06 g/g glucose. Showed the highest yield. When ^{other metal ions except Fe 2 +} were added, the yield was lower than when no metal ions were added (FIG. 5). ^{Therefore, it was confirmed that Fe 2 +} was a suitable metal ion to act as a reducing agent in Candida sorbosivorans JM409 strain.

<2-4> 5L- bioreactor through Erythritol Production analysis

According to the results of Examples <2-1> to <2-3>, 5L-bioreactor scale-up fermentation was performed under the condition in which the most erythritol was produced. _{Glucose and Fe 2} SO ₄ ·7H ₂ O were supplied in a fed-batch manner while maintaining 250 rpm, 30° C., and pH 5 in 2L YPD (100 g/L) medium.

As a result, during the fermentation for 216 hours, OD increased to 34.2, and at this time, 11.2 g/L of erythritol was produced, and it was confirmed that the production of erythritol increased each time glucose was supplied (FIG. 6 ).

Institute of Agricultural Biotechnology KACC93287 20171024

Claims (5)

delete delete 1) fermenting Candida sorbosivorans JM409 strain (KACC 93287P) isolated from peaches in YPD medium; And
2) In the production method of erythritol comprising the step of recovering erythritol from the medium or the strain according to the fermentation,
The YPD medium contains 100 g/L glucose, 10 g/L yeast extract, 0.35 g/L Fe ₂ SO ₄ 7H ₂ O and 20 g/L Bacto peptone,
The fermentation of step 1) is characterized in that it is carried out for 10 to 220 hours under the conditions of 250 rpm, 30 °C and pH 5, the production method of erythritol. delete delete

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