KR102423524B1 - Sphingomonas azotifigens producing gellan and method for producing gellan using the same - Google Patents

Abstract

The present invention relates to Sphingomonas azotyfigens for producing gellan and a method for producing gellan using the same, and more particularly, to Sphingomonas azotyfigens in a medium containing lactose, whey or a mixture thereof as a carbon source. azotifigens ) It relates to a method for producing gellan, including the step of culturing, and Sphingomonas azotifigens GL-1 strain deposited with accession number KCTC14040BP for producing gellan.

Description

Sphingomonas azotifigens producing gellan and method for producing gellan using the same

The present invention relates to Sphingomonas azotyfigens for producing gellan and a method for producing gellan using the same. It relates to the production method of gellan.

Gellan is made by repeatedly combining two molecules of 1,4-β-D-glucose, one molecule of 1,4-β-D-glucuronic acid, and one molecule of 1,3-α-L-rhamnose. It is a natural biopolymer. Gellan has characteristics such as non-toxicity, biodegradability, and non-immunity as well as unique physical properties, and for this reason, it is widely used in various fields such as food, pharmaceuticals, and medicine.

In particular, the total amount of gellan required for food and chemical industries is imported and used because the production cost of gellan is higher than the cost of importing it from abroad. Therefore, it is necessary to dramatically improve the technology for producing gellan to further lower the production cost, or to directly lower the production cost by using a cheaper medium, but the domestic technology for this is insignificant. In this regard, Korean Patent Laid-Open No. 10-2002-0066154 A discloses a method for preparing a polymer gellan using soy sauce, which is a by-product of food processing.

On the other hand, most microorganisms start to grow in a suitable environment and a nutrient source including a carbon source such as starch or glucose and a nitrogen source such as yeast extract, bactopeptone, ammonium chloride, and waste molasses, and among them, they produce high molecular polymers necessary for our life. There are also microorganisms that Polymers produced by microorganisms include gelatinizers, emulsifiers, stabilizers, and gels such as pullulan, xanthan, curdlan, and gellan, and are widely used in the food industry and chemical industry. is being used

Among these, commercial production of gellan is mainly Pseudomonas elodea and Sphingomonas paucimobilis ), and other species are very limitedly known, and studies related to gellan production so far have been conducted to improve strains, including genetic manipulation, and medium composition and culture to increase production yield and reduce production costs. It is focused on condition optimization.

Therefore, when a new microbial species having gellan-producing ability is secured and mass production of gellan is possible using the same, it is expected that it can be utilized in various industries such as food, medicine, and agriculture.

One aspect of the present invention is to provide a method for producing gellan using Sphingomonas azotypigens.

Another aspect of the present invention is to provide Sphingomonas azotypigens that produces gellan.

According to one aspect of the present invention, Sphingomonas in a medium containing lactose, whey or a mixture thereof as a carbon source azotifigens ), comprising the step of culturing, a method for producing gellan is provided.

According to another aspect of the present invention, Sphingomonas deposited with accession number KCTC14040BP for producing gellan ( Sphingomonas ) azotifigens ) GL-1 strain is provided.

According to the present invention, using a novel Sphingomonas genus species having a gellan production ability, Sphingomonas azotifigens , using a low-cost carbon source and setting optimal conditions for culture, efficient biological material production way can be obtained. In particular, according to the present invention, it is expected that gellan can be mass-produced and used in various industries such as food, medicine, and agriculture.

1 shows the phylogenetic relationship between the GL-1 strain and other Sphingomonas strains based on the NJ (Neighbor-joining) tree analysis of the 16S rDNA gene sequence.
Figure 2 shows the effect of each culture factor on the gellan production by S. azotypigens GL-1, (a) the effect of pH, (b) the effect of temperature, (c) the effect of the nitrogen source, (d) The effect of carbon source, (e) the effect of molasses and (f) the effect of whey are shown.
3 shows a three-dimensional graph of gellan production by Sphingomonas azotypigens GL-1 in whey, (a) the effect of Na ₂ HPO ₄ and whey, (b) the effect of KH ₂ PO ₄ and whey; (c) shows the effect of Na ₂ HPO ₄ and KH ₂ PO ₄ .
4 is a ¹ H liquid NMR spectrum of gellan performed on D ₂ O and FTIR spectra of low-acyl gellan powder, (a) gellan standards; (b) purified gellan in glucose medium; and (c) gellan standard (red), glucose (pink), whey (green), and molasses (brown) based media, respectively.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a method for producing gellan using a microorganism having a newly confirmed gellan-producing ability that has not been reported to produce gellan. It is understood herein that gellan may be used interchangeably with 'gellan gum' and/or 'gellan'.

In more detail, the method for producing gellan of the present invention includes culturing Sphingomonas azotifigens in a medium.

The Sphingomonas azotyfigens is preferably a Sphingomonas azotyfigens GL-1 strain deposited with the accession number KCTC14040BP at the Korea Research Institute of Bioscience and Biotechnology Microbial Resource Center (KCTC, Korean Collection for Type Cultures), in the present specification It is understood that 'GL-1' or 'GL-1 strain' may be used interchangeably.

According to the present invention, gellan can be effectively produced by culturing Sphingomonas azotypigens, which has been newly confirmed to produce gellan.

In more detail, the culture is performed in a medium containing a carbon source and a nitrogen source.

In this case, the medium may include glucose, maltose, lactose, fructose, whey or a mixture thereof as a carbon source, but since economic feasibility tends to decrease when refined saccharides are used, preferably whey is used as a carbon source use it as

In the present invention, the component that can be a nitrogen source is not particularly limited, but for example, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), sodium nitrate, ammonium citrate, corn steep liquor (CSL), peptone, yeast extract ( It can be selected from yeast extract, YE), beef extract and ammonium chloride (NH ₄ Cl), and the inorganic nitrogen source is more preferable for gellan production and cell growth than the organic nitrogen source, and more preferably chlorinated Ammonium (NH ₄ Cl).

Furthermore, in order to produce gellan in high yield, it is preferable to further include at least one of a K ⁺ ion source and a Na ⁺ ion source in the medium, for example, the medium further contains Na ₂ HPO ₄ , or KH ₂ PO ₄ may be further included, or a combination thereof may be further included, and preferably a combination thereof. Na ₂ HPO ₄ and KH ₂ PO ₄ In the case of culturing Sphingomonas azotypigens in a medium containing additionally, the production and yield of gellan can be remarkably improved.

For example, the medium used in the present invention is a carbon source of 60 to 80 g/L, Na ₂ HPO ₄ 5-15 g/L and KH ₂ PO ₄ It may be one containing 3 to 9 g / L, preferably the medium is a carbon source 60 to 70 g / L, Na ₂ HPO ₄ 14-15 g/L and KH ₂ PO ₄ 7 to 8 g/L. If the Na ₂ HPO ₄ is out of the range of 5 to 15 g/L and KH ₂ PO ₄ 3 to 9 g/L, the production of gellan tends to decrease.

On the other hand, the culturing step is preferably carried out at a temperature of pH 5.0 to 8.5, and 25 to 30 °C, more acidic or more alkaline conditions reduce cell growth and gellan production, and the temperature is 25 °C to 30 °C As the temperature increases, both biomass and gellan production increase, but at a temperature exceeding 30° C., the production of biomass and gellan decreases. The most preferred optimum pH for gellan production is 6.5 and the temperature is 30°C.

In the present invention, the basal medium that can be used for culturing Sphingomonas azotypigens is not particularly limited, and any medium suitable for culturing microorganisms may be used, for example, the medium is M9 minimal medium.

In the step of culturing Sphingomonas azotypigens in the medium, the Sphingomonas azotypigens strain of the present invention is inoculated at a concentration of 0.5-10.0% (v/v), more preferably 2.0-5.0% (v/v) ) concentration, more preferably 2.0-3.0% (v/v) concentration.

The culture is performed aerobically, for example, at 100-300 rpm for 6-48 hours, and more preferably aerobically at 150-250 rpm for 12-24 hours.

Furthermore, the gellan production method of the present invention may further include the step of purifying the Sphingomonas azotypigens strain subsequent to the step of culturing. In this case, the purification may be performed, for example, by ethanol precipitation or ultrafiltration.

According to another aspect of the present invention, Sphingomonas deposited with accession number KCTC14040BP for producing gellan ( Sphingomonas ) azotifigens ) GL-1 strain is provided.

According to the Sphingomonas azotypigens GL-1 strain of the present invention, geran can be obtained in a high yield.

As described above, according to the present invention, a novel species of the genus Sphingomonas having gellan-producing ability, Sphingomonas azotifigens , is used to utilize a low-cost carbon source and set the optimal conditions for culturing, thereby providing an efficient organism. Material production methods can be obtained. In particular, according to the present invention, it is expected that gellan can be mass-produced and used in various industries such as food, medicine, and agriculture.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Medium Composition and Preparation of Materials Used for Experiments

Gellan-producing strains were screened using nutrient agar (NA) medium (1 g/L beef extract, 2 g/L yeast extract, 5 g/L peptone, 15 g/L NaCl and 15 g/L agar, manufactured by BD Korea). .

M9 minimal medium (20 g/L glucose, 6 g/L Na ₂ HPO ₄ , 3 g/L KH ₂ PO ₄ , 0.5 g/L NaCl, 1 g/L NH ₄ Cl, 1 mM MgSO ₄ 7H ₂ O, 0.1 mM CaCl ₂ 2H ₂ O and 10 ml/L trace element solution (100x)) were used for further screening of gellan yield strains and analysis of influence factors. The trace element solution (100x) consisted of: 100 mg/L MnCl ₂ .4H ₂ O, 170 mg/L ZnCl ₂ , 43 mg/L CuCl ₂ .2H ₂ O, 60 mg/L CoCl ₂ .6H ₂ O and 60 mg/L Na ₂ MoO ₄ .2H ₂ O.

Whey (cheese whey) manufactured by Namyang Dairy Co., Ltd. was used, and the whey contained 82.26% (w/w) lactose, 11.33% (w/w) protein, 4% (w/w) water, and 1.18% (w/w) lipid. w) and other dairy by-products were included, such as 1.23 %. On the other hand, molasses was manufactured by Rice Science Co., Ltd., and sucrose (28.96%), glucose (4.58%), and fructose (7.43%) are the main sugar components, and the rest are calcium, potassium, sodium, iron, and magnesium. It consists of sugar cane by-products containing trace elements such as copper and copper.

2. gellan Selection of production candidate strains

In August 2018, the soil attached to the roots of rice obtained from farmland in Sinjeong-dong (Jeong-eup: 35° 34-0 N, 126° 51-0 E) was gently shaken in sterile water to remove it and washed three times. The roots were crushed by mixing 9 ml of sterile water and 1 g of roots, and diluted 10-fold by adding sterile water. Aliquots (200 μl) were placed in NA medium and incubated at 30° C. for 48 hours. As a result, potential strains were obtained from the colonies, and each strain was isolated and then cultured in a flask to investigate the gellan production ability. More specifically, strains were cultured in 15 ml tubes containing 3 ml of liquid nutrient medium, and incubated at 30° C. for 24 hours with stirring at 200 rpm. The optical density of the culture was adjusted to OD600=2.0±0.1 and 2% (v/v) samples were each inoculated into 250 ml flasks containing 50 ml of fresh M9 minimal medium. The strains were then incubated at 30° C. and shaken at 200 rpm for 48 hours.

According to the colony phenotype, six different strains were isolated. The strain was tested for gellan production in M9 medium. Among them, only the strain named GL-1 produced the highest concentration of gellan gum (10.55±0.50 g/L) after 48 hours. GL-1 colonies grown on NA plates were yellow, round, smooth and shiny, identical to the genus Sphingomonas (Xie and Yokota, 2006).

3. Phylogenetic Analysis

Genomic DNA was extracted from the GL-1 strain, which is the gellan-producing strain obtained in 2. above, using the DNeasy Plant Mini kit (Qiangen, Hilden, Germany), and universal 27F (5'-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5) '-GGTTACCTTGTTACGACTT-3') primers were used to amplify the 16S rDNA gene sequence. Analysis of the 16S rDNA gene sequence was performed by Macrogen Co. Ltd (Seoul, Korea). The sequences were then manually edited using BioEdit software (Hall, 1999). Partial 16S gene sequences were used for comparison with strains of the type reported in the EzBioCloud database ( https://www.ezbiocloud.net/ ). Finally, the phylogenetic tree was constructed by the neighbor-joining method using MEGA 7.0 software (Saitou and Nei, 1987).

Comparison of the 16S rDNA gene sequences of the GL-1 strain was performed based on data available in the EzBioCloud nucleotide sequence database. As a result, the GL-1 strain was most similar to S. azotypigens NBRC 15497 (99.89%), and the phylogenetic tree based on the 16S rDNA gene sequence is shown in FIG. 1 . Strain GL-1 clustered with S. azotypigens NBRC 15497, indicating that it belongs to the S. azotypigens family.

The S. azotypigens GL-1 was deposited with the Korean Collection for Type Cultures (KCTC) as of November 20, 2019 with an accession number KCTC 14040BP.

4. gellan Check production conditions

Experiments were performed on M9 medium at various levels of pH (5.0-8.5) and temperature (25-37° C.) conditions. For subsequent experiments, the optimal initial pH and temperature were fixed. To study the effect of nitrogen sources, ammonium chloride was replaced with 1 g/L of ammonium sulfate, sodium nitrate, ammonium citrate, corn steep liquor (CSL), peptone, yeast extract and beef extract, respectively. The nitrogen source that provided the maximum production of gellan was used in subsequent experiments. Different carbon sources (glucose, sucrose, glycerol, maltose, mannose, xylose, lactose and fructose) were independently used at 20 g/L each in M9 medium to study the effect on gellan production. Culture flasks were inoculated (1% v/v, 24 hours), and different concentrations of molasses and whey (20-50 g/L) were added to M9 medium for gellan formation. All experiments were independently performed three times, and the data were analyzed after 48 hours and presented as the mean ± SD.

(1) Influence of pH and temperature

pH and temperature greatly affect the activity of enzymes in the metabolism involved in the growth of S. azotypigens GL-1 and the production of gellan. Fig. 20(a) shows the effect of initial pH on gellan production, and maximum gellan production (10.75 ± 0.50 g/L) and biomass (3.39 ± 0.16 g/L) were obtained at pH 6.5. The results in Fig. 2(b) suggest that temperature not only affects the biomass but also the synthesis of gellan. As the temperature increased from 25°C to 30°C, both biomass and gellan production increased. However, it was confirmed that biomass and gellan production decreased when the temperature was further increased at a temperature exceeding 30°C. It was confirmed that the optimum pH for gellan production was 6.5 and the temperature was 30 °C.

(2) Effect of nitrogen source

Eight nitrogen sources at a concentration of 1 g/L were investigated to identify the optimal nitrogen source for gellan production. Inorganic nitrogen sources have been found to be more favorable for gellan production and cell growth compared to organic nitrogen sources. NH ₄ Cl was found to be the most suitable nitrogen source for improving gellan production from S. azotypigens GL-1 and its biomass. The maximum value of gellan gum production reached 10.57±0.5 g/L, and the highest biomass of 3.37±0.16 g/L could be obtained using NH ₄ Cl (Fig. 2(c)). Consequently, NH ₄ Cl was selected as the nitrogen source for further study.

(3) Influence of carbon sources

The carbon source is an energy source for cell growth and a substrate for polysaccharide synthesis. Therefore, it directly affects the production yield, composition, structure and properties of gellan, and FIG. 2(d) shows that S. azotypigens GL-1 can effectively use glucose, sucrose, maltose, mannose, lactose and fructose. show that there is However, the utilization of glycerol and xylose was very poor, and the biomass and gellan production was not satisfactory. The highest biomass (3.55 ± 0.16 g/L) in M9 medium was obtained from maltose. Molasses and whey as economical carbon sources were tested for gellan production based on the results of using carbon sources. As a result, gellan production and biomass increased as molasses and whey increased at 20-50 g/L. The highest gellan concentrations for molasses and whey were 12.5 ± 0.61 g L and 16.75 ± 0.84 g/L, respectively ( FIGS. 2(e) and 2(f)). The whey and molasses were used in subsequent PBD and RSM studies.

5. Plackett - Burman Screening by design

The Plackett-Burman design (PBD) was used to screen for parameters important in gellan production. Glucose in M9 medium was exchanged independently with whey or molasses. Each variable was tested at two levels (+1 for high, -1 for low) in the 12 experiments shown in Table 1 below.

Plackett-Burman design for coded values and gellan production parameters for whey and molasses Run X _One X ₂ X ₃ X ₄ X ₅ X ₆ X ₇ Y _One Y ₂ One -One -One -One One -One One One 10.25 ± 0.45 4 ± 0.15 2 -One One -One One One -One One 10.50 ± 0.46 3.25 ± 0.10 3 -One One One -One One One One 0 0 4 -One -One -One -One -One -One -One 11.25 ± 0.52 3.5 ± 0.10 5 -One -One One -One One One -One 8.5 ± 0.43 2.62 ± 0.05 6 One One -One -One -One One -One 12.5 ± 0.55 7.37 ± 0.35 7 -One One One One -One -One -One 13.55 ± 0.45 8.62 ± 0.35 8 One -One -One -One One -One One 20.5 ± 0.60 8.12 ± 0.35 9 One One One -One -One -One One 18.75 ± 0.55 9.12 ± 0.40 10 One -One One One -One One One 27.75 ± 1.12 12.25 ± 0.50 11 One One -One One One One -One 18.5 ± 0.75 9.62 ± 0.45 12 One -One One One One -One -One 31.25 ± 1.20 14.25 ± 0.65

* Y ₁ and Y ₂ represent gellan production (g/L) from whey and molasses, respectively

* X ₁ is whey or molasses; X ₂ is NH ₄ Cl; X ₃ is Na ₂ HPO ₄ ; X ₄ is KH ₂ PO ₄ ; X ₅ is NaCl; X ₆ represents MgSO ₄ ·7H ₂ O and X ₇ represents CaCl ₂ ·2H ₂ O.

S. azotypigens GL-1 produced high yield of gellan in run numbers 10 and 12 (Table 1), and the statistical results are shown in Tables 2 and 3 below. All variables with a P value less than 0.05 had a significant effect. Whey or molasses, Na ₂ HPO ₄ and KH ₂ PO ₄ had positive coefficients for gellan production. In the whey-based medium, NH ₄ Cl and MgSO ₄ showed negative coefficients, and NaCl and CaCl ₂ had no effect on gellan production. However, all four variables showed negative coefficients in the molasses-based medium (Tables 2 and 3). Gellan production increased with increasing concentrations of whey or molasses, Na ₂ HPO ₄ and KH ₂ PO ₄ . On the other hand, NH ₄ Cl, NaCl, MgSO ₄ ·7H ₂ O and CaCl ₂ ·2H ₂ O was required to maintain a low concentration. Correlation coefficient (R ² = 0.9925, 0.9952) and adjusted coefficient of determination (R ² _adj ) = 0.9795, 0.9868), indicating that the present study model is very reliable for the repetition of the experiment (Tables 2 and 3).

The effect of each variable and its meaning were calculated using Design-Expert 8.0.6 software (Stat-Ease Inc., Minneapolis, Minnesota, USA) (Tables 2 and 3). Variables with a P-value <0.05 were considered to affect gellan gum production and were further optimized by the response surface methodology.

Actual Values and Statistical Analysis of PBD Variables for Production of Gellan from Whey factor Concentration (g/L) Mean Square Coefficient Estimation
Estimate) standard error F -value P - value I go Model 115.52 15.28 0.36 75.95 0.0004 X ₁ : Whey 20 60 471.25 6.27 0.36 309.82 <0.0001 X ₂ : NH ₄ Cl One 3 106.21 -2.97 0.36 69.83 0.0011 X ₃ : Na ₂ HPO ₄ 6 18 22.14 1.36 0.36 14.56 0.0189 X ₄ : KH ₂ PO ₄ 3 9 135.34 3.36 0.36 88.98 0.0007 X ₅ : NaCl 0.5 1.5 1.92 -0.40 0.36 1.26 0.3241 X ₆ : MgSO ₄ 1 mM 3 mM 66.74 -2.36 0.36 43.88 0.0027 X ₇ : CaCl ₂ 0.1 mM 0.3 mM 5.07 -0.65 0.36 3.33 0.1419

* R ² = 0.9925, R ² (adj) = 0.9795 , R ² (Pred) = 0.9328

Actual Values and Statistical Analysis of PBD Variables for Production of Gellan from Molasses factor Concentration (g/L) Mean
Square) Coefficient Estimation
Estimate) standard error F -value P - value I go Model 28.27 6.89 0.14 118.33 0.0002 X ₁ : Molasses 15 45 513.52 3.23 0.14 523.47 <0.0001 X ₂ : NH ₄ Cl One 3 17.52 -0.56 0.14 15.94 0.0162 X ₃ : Na ₂ HPO ₄ 6 18 60.75 0.92 0.14 42.2 0.0029 X ₄ : KH ₂ PO ₄ 3 9 204.19 1.77 0.14 257.65 0.0002 X ₅ : NaCl 0.5 1.5 18.75 -0.58 0.14 17.09 0.0144 X ₆ : MgSO ₄ 1 mM 3 mM 52.08 -0.92 0.14 42.2 0.0029 X ₇ : CaCl ₂ 0.1 mM 0.3 mM 31.69 -0.77 0.14 29.78 0.0055

* R ² = 0.9952, R ² (adj) = 0.9868, R ² (Pred) = 0.9567

6. Optimization by Response Surface Methodology

Response surface methodology (RSM) was applied with respect to whey, which has been shown to produce higher yields of gellan in the experiments described above to determine optimal concentrations of the three most notable factors identified by PBD. The face-centered central composite design (FCCD) consisted of a total of 20 experimental runs at 3 levels (-1, 0, +1) including 6 replicates at the center point (Table). 4). All experiments were performed in triplicate and the average of the maximum gellan concentrations was used as the actual value. Variables and responses were calculated using the quadratic equation (1) below.

...식(1)

...Equation (1)

where Y is the concentration of gellan; β ₀ is the intercept term; β _i is a linear coefficient; β _ij is a quadratic coefficient; β _ii is a squared term; Xi is the independent variable.

FCCD of coded form variables and actual values and responses to the production of gellan in whey. Experiment Whey (g/L) Na ₂ HPO ₄ (g/L) KH ₂ PO ₄ (g/L) Gellan (g/L) One 1(80) 0(10) 0(6) 27.50 ± 1.35 2 0 (60) -1(5) 0(6) 24.25 ± 1.20 3 -1(40) -1(5) -1(3) 10.25 ± 0.50 4 1(80) -1(5) -1(3) 15.50 ± 0.65 5 0 (60) 0(10) 0(6) 30.75 ± 1.50 6 0 (60) 0(10) -1(3) 23.00 ± 1.10 7 1(80) 1(15) -1(3) 25.25 ± 1.25 8 -1(40) -1(5) 1(9) 16.00 ± 0.80 9 1(80) -1(5) 1(9) 22.00 ± 1.00 10 0 (60) 0(10) 0(6) 30.50 ± 1.50 11 0 (60) 0(10) 0(6) 28.00 ± 1.35 12 -1(40) 1(15) 1(9) 24.50 ± 1.15 13 0 (60) 0(10) 0(6) 29.75 ± 1.40 14 -1(40) 0(10) 0(6) 23.25 ± 1.10 15 0 (60) 1(15) 0(6) 32.50 ± 1.55 16 1(80) 1(15) 1(9) 31.50 ± 1.50 17 0 (60) 0(10) 0(6) 29.00 ± 1.45 18 0 (60) 0(10) 1(9) 33.00 ± 1.60 19 -1(40) 1(15) -1(3) 20.00 ± 1.00 20 0 (60) 0(10) 0(6) 29.50 ± 1.45

As a result, Experiment 18 had the highest gellan production (33.00 ± 1.60 g/L), and Experiment 4 had the lowest gellan production (15.50 ± 0.65 g/L).

Further, the coefficients of the regression equation were calculated and the regression equation equation (2) was obtained as follows.

...식(2)

...Equation (2)

where Y is the gellan concentration and X ₁ , X ₃ and X ₄ are the coded values of whey, Na ₂ HPO ₄ and KH ₂ PO ₄ , respectively.

A 3D surface graph for gellan production while maintaining one variable at zero level is shown in FIG. 3 . The three response variables showed significant linear and negative quadratic effects. A negative secondary effect means a decrease in the yield of gellan. Therefore, it was confirmed that excessive concentrations of the three parameters were not useful for generating more gellan. For whey 68.34 g/L, Na ₂ HPO ₄ 14.58 g/L and KH ₂ PO ₄ 7.66 g/L, the maximum yield of gellan gum was 33.75 g/L.

7. gellan analysis

The method reported by Arockiasamy and Banik (Arockiasamy and Banik, 2008) was used to determine cell biomass and isolate gellan from fermentation broth. The gellan recovery process is described in Bajaj et al. (Bajaj et al., 2007). The gellan concentration and molecular weight (Mw) were measured using an Agilent 1100 high performance liquid chromatography (HPLC) system and a refractive index detector with a PL Aquagel-OH gel permeation chromatogram (GPC) column (300x7.5 mm; Polymer Laboratories Ltd., UK). RID). The mobile phase was ultrapure water, the flow rate was 1 ml/min, and the injection volume was 50 μl. The amount of gellan was calculated from the peak area of the GPC measurement. A standard curve was constructed using purified gel (Sigma-Aldrich Korea) and polyethylene oxide standard (Agilent Technologies, Inc., UK).

To analyze the resulting gellan carbohydrate composition and the degree of acyl groups, a certain amount of natural gellan was dissolved in pure water, the pH was adjusted to above 10.0 with 2.0 M NaOH, and then the solution was stirred to completely remove the acyl groups. It was held at 100° C. for 30 minutes. Then, three volumes (v/v) of cold ethanol were added and centrifuged at 9500 rpm for 20 minutes to separate the acyl gellan. The supernatant was dried at 80 °C, dissolved in pure water, and adjusted to pH 6.0 with 1.0M HCl. After hydrolysis with 0.5 MH ₂ SO ₄ (100 °C for 16 h), the alkaline hydrolyzate of acyl gellan and the glucose, rhamnose and glucuronic acid contents were measured using a refractive index detector (RID) and an Aminex HPX-87H column (300 x 78 mm; Bio-Rad, Hercules, CA, USA). The mobile phase was 0.5 mM H ₂ SO ₄ , and the flow rate was 0.6 ml/min. The column and cell temperatures were 65 and 45 °C, respectively.

The chemical structure of the purified gellan was confirmed by nuclear magnetic resonance (1H NMR, JNM-ECA500, JEOL, Tokyo) and Fourier transform infrared spectroscopy (FTIR, Vertex 70, UK) (Silva-Correia et al., 2011), The results are shown in FIG. 4 .

A group of gellan structures was identified by 1H-NMR analysis, and H-1 and H-6 of the α-anomer of the L-rhamnopyranosyl residue appeared at 5.00 ppm and 1.18 ppm. In addition, it was confirmed that the signals of 4.72 ppm and 4.80 ppm of D-glucopyranosyl and D-glucuropyranosyl residues almost overlap with the peak positions of the gellan standard, respectively (Silva-Correia et al., 2011).

On the other hand, typical gellan absorption bands were present in both the standard gellan and the resulting gellan. FTIR spectra are peaks at 3420 cm ^-1 (OH stretch peak), 2920 cm ^-1 (CH stretch), 1618 cm ^-1 (asymmetric COO ^- stretch), 1412 cm ^-1 (symmetric COO ^- stretch) and 1037 cm ^-1 (Silva-Correia et al., 2011).

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Claims (10)

In a medium containing 60 to 80 g/L of carbon source, Na ₂ HPO ₄ 10 to 15 g/L and KH ₂ PO ₄ 6 to 9 g/L Sphingomonas azotifigens ) Including the step of culturing A method of producing gellan.
The method according to claim 1, wherein the Sphingomonas azotyfigens is a Sphingomonas azotyfigens GL-1 strain deposited with accession number KCTC14040BP.
The method of claim 1, wherein the medium comprises glucose, maltose, sucrose, mannose, lactose, fructose, whey or a mixture thereof as a carbon source.
delete delete delete According to claim 1, wherein the medium is a carbon source of 60 to 70 g / L, Na ₂ HPO ₄ 14-15 g/L and KH ₂ PO ₄ A method for producing gellan, comprising 7 to 8 g/L.
The method according to claim 1, wherein the culturing is performed at a temperature of pH 5.0 to 8.5, and 25 to 30°C.
The method according to claim 1, wherein the medium is an M9 minimal medium.
Sphingomonas azotifigens GL-1 strain deposited with accession number KCTC14040BP to produce gellan.

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