KR100739022B1 - Method for continuous production of pullulan with feeding of the sucrose - Google Patents

Abstract

A method for continuous production of pullulan by feeding of sucrose is provided to improve production yield of pullulan by continuously feeding medium containing sucrose as a carbon source, reduce production costs by replacing yeast extract as a nitrogen source with the by-product from soybean sauce fermentation, and increase quality of pullulan by increasing molecular weight. The method for continuous production of pullulan by feeding of sucrose comprises the steps of: culturing a pullulan-producing microorganism, Aureobasidium pullulans, in a medium containing 20 g/l of glucose, 2.5 g/l of yeast extract and inorganic salt at 25-35 deg.C and 150-200 rpm for 24-48 hours to prepare seed culture medium; inoculating 3-5%(v/v) of the seed culture medium into a medium containing sucrose, yeast extract and inorganic salt and culturing it at 25-35 deg.C and 150-200 rpm for 3-5 days; and continuously feeding a mixture containing 100-200 g/l of sucrose, 2.5g/l of yeast extract, 3-5 g/l of K2HPO4, 0.01-1 g/l of MgSO4-H2O, 0.3-1.5 g/l of (NH4)2SO4 and 0.5-3.0 g/l of NaCl into the cultured medium in a dilution rate of 0.01-0.04 h^-1 and harvesting 0.5-1.5%(v/v)/h of the cultured medium.

Description

Method for continuous production of pullulan with sugar solution {Method for Continuous production of pullulan with feeding of the sucrose}

Figure 1 is a graph showing the growth of cells and production of pullulan over time using the Aureobasidium pullulan HP-2001 strain in a medium prepared using sugar and yeast extract.

Figure 2 after incubating 72 hours of the Aureobasidium pullulan HP-2001 strain in a medium prepared using sugar and yeast extract, half of the culture was collected and the same amount of sugar, yeast extract and inorganic salt, etc. It is a graph showing the production of pullulan obtained by the addition.

Figure 3 is a dilution rate of the growth of the cells and production of pullulan when culturing the Aureobasidium pullulan HP-2001 strain in the medium prepared using the sugar and yeast extract while culturing continuously by adding the medium of the same component It is a graph showing the effect on.

Figure 4 is a pullulan produced when the cultivation of the Aureobasidium pullulan HP-2001 strain in a medium prepared using a sugar and yeast extract while the continuous culture while adding the medium of the same component at a dilution rate of 0.025h ^-1 Is a graph showing the effect on the molecular weight, average molecular weight and degree of dispersion.

FIG. 5 shows the concentration of sugar as a carbon source when continuously culturing at a dilution rate of 0.025h ⁻¹ by adding the same medium while culturing Aureobasidium pullulan HP-2001 strain in a medium prepared using a sugar and yeast extract. It is a graph showing the effect on the production of pullulan.

The present invention relates to a continuous production method of pullulan (pullulan) using a sugar liquid, and more particularly, a liquid polymer of the Aureobasodium pullulans strain while continuously adding the sugar liquid is a functional polymer polymer The present invention relates to a continuous production method of pullulan (pullulan) using a sugar liquid, characterized in that to improve the concentration and productivity of pullulan by producing pullulan (pullulan) in a continuous process.

Pullulan (pullulan) is a polysaccharide, a secretion secreted by Aureobasodium pullulans belonging to an incomplete fungus out of the cell, a polymer of edible natural polysaccharides with no acute toxicity, chronic toxicity, mutagenicity, etc. Polymer. Pullulan with this property is used for various non-polluting materials such as food industry and film, etc. It includes colloids, emulsifiers, stabilizers and gels, and its use in a wide range of fields such as food industry and chemical industry It is being sought. In particular, imports are expected to continue to increase as they are indispensable to improve the quality of foods in the food industry. However, the cost of producing pullulan as a domestic technology is high. Since it is more expensive than the cost of importing from abroad, it is still almost entirely imported from Japan.

Accordingly, the present inventors have a method for manufacturing a polymer polymer pullulan using a food processing by-product containing a high concentration of sugar components of the Republic of Korea Patent Publication No. 10-339715 (registered May 23, 2002) as a way to solve the above problems And the Republic of Korea Patent Publication No. 10-414952 (registered on Dec. 30, 2003) has been completed the invention of a method for producing a polymeric polymer pullulan using soybean meal as a by-product of food processing, but in the case of the above patents, pullulan The concentration of pullulan produced by the process and the improvement of its productivity were limited, and the characteristics such as molecular weight of pullulan produced by batch culture were not constant.

Therefore, it is necessary to lower the production cost by developing a technology that increases productivity in the technology of producing pullulan, or to lower the production cost by using a cheaper medium, but domestic technology for this is insignificant.

Accordingly, the present inventors have made intensive studies to develop a method for producing pullulan economically, and as a result, after inoculating the strain of pullulan, Maureobasidium pullulan strain, on the production medium of pullulan, the sugar solution was continuously As a result, the process of continuously producing pullulan was added.

When the pullulan is produced by the continuous process according to the present invention, it is possible to continuously produce pullulan having an increase of about 3 times the concentration of pullulan. In addition, compared to pullulan produced by batch culture, pullulan produced in continuous culture has a uniform molecular weight, so that it is possible to produce pullulan of excellent quality with improved physical properties, thereby completing the present invention.

After all, the main object of the present invention is to provide a production method capable of continuously producing high quality pullulan by continuously adding a sugar liquid to the existing batch production process with low productivity of pullulan.

Method for producing pullulan according to the present invention for achieving the above object is a medium to which 20 micrograms of microorganisms producing pullulan are added as a carbon source and 2.5 g / l yeast extract and an inorganic salt as a nitrogen source Inoculated to, incubated at 25-35 ° C. for 150-200 rpm for 24 to 48 hours, and the seed culture was added with 1) 100 g / l sugar as carbon source and 2.5 g / l yeast extract and inorganic salt as nitrogen source. 2) 100 g / l to 200 g / l sugar and 2.5 g / l while inoculating 3 to 5% (v / v) in the prepared medium and incubating at 25 to 35 ° C. for 3 to 5 days at 150 to 200 rpm. And a step of purifying the pullulan by continuously collecting a predetermined amount of the culture solution while continuously adding the mixed solution to which the yeast extract and the inorganic salt of is added to the culture solution at a dilution rate of 0.01 to 0.04 h ⁻¹ .

In the mixed solution to which 100 g / l to 200 g / l sugar, 2.5 g / l yeast extract, and inorganic salt are added, the amount of the culture solution is continuously added by adding the same amount to the culture solution continuously. It should be possible to maintain a constant amount.

And the amount of the culture solution continuously collected in the above is preferably 0.5% (v / v) / h to 1.5% (v / v) / h of the total culture amount. When the amount of the culture medium collected is less than 0.5% (v / v) / h, the production rate of pullulan may decrease, and when the amount of the collected culture medium exceeds 1.5% (v / v) / h, the production rate of pullulan is Although temporarily improved, the dilution rate of the cells is high, and there is a fear that all of the pullulan producing bacteria are gradually released.

Hereinafter, the method for preparing pullulan by continuous addition of the sugar liquid of the present invention will be described in detail by dividing step by step.

Step 1: Cultivation of Pullulan Production Strains

Aureobasidium pullulans HP-2001 strain producing pullulan was inoculated into a medium containing 20 g / l of glucose as a carbon source and 2.5 g / l of yeast extract and an inorganic salt as a carbon source and 150 at 25 to 35 ° C. The spawn culture medium incubated for 24 to 48 hours at 200 rpm to 3 to 5% (v /) in medium to which 2.5 g / l yeast extract and inorganic salt were added as carbon source and 0 to 300 g / l sugar and nitrogen source. v) and inoculated at 3 to 5% (v / v) in culture at 150 to 200 rpm at 25 to 35 ° C. and incubated at 150 to 200 rpm at 25 to 35 ° C. for 3 to 5 days to obtain a culture solution. . Kinds and concentrations of the inorganic salts added to the medium were 3 to 5 g / l K ₂ HPO ₄ , 0.01 to 1 g / l MgSO _{4 ·} 7H ₂ O, 0.3 to 1.5 g / l (NH ₄ ) ₂ SO ₄ and 0.5-3.0 g / l of NaCl.

As shown in [Table 1], as the concentration of sugar as a carbon source increased, the concentration of the cells producing pullulan and the production concentration of pullulan increased, but the highest sugar to pullulan when the concentration of sugar was 100 g / l. Conversion rate is shown.

Effect of Sugar Concentration on the Production of Pullulan when Pullulan Production Using Arureobasidium Pullulan Strain HP-2001 Concentration of sugar (g / l) pH Dry cell (g / l) Pullulan (g / l) Pullulan conversion 0 6.8 1.3 0.9 - 20 4.4 6.2 5.9 0.30 50 3.4 11.3 17.5 0.35 75 3.4 12.5 28.7 0.38 100 3.2 13.5 40.0 0.40 150 3.2 14.5 47.6 0.32 200 3.2 15.1 54.2 0.27 300 3.3 19.9 53.0 0.07

For reference, Figure 1 shows the time when producing pullulan using the Aureobasidium pullulan HP-2001 strain in a medium prepared using 100 g / l sugar as a carbon source and 2.5 g / l yeast extract as a nitrogen source. As a graph showing the growth of the cells and the production of pullulan, it can be seen that the amount of added sugar decreases as the production of pullulan increases. In Fig. 1,? Indicates hydrogen ion concentration,? Indicates dry cell weight,? Indicates dissolved oxygen,? Indicates pullulan, and? Indicates residue.

Second Step: Extraction of Pullulan from Culture Medium and Preparation of Pullulan

The obtained supernatant was extracted by adding an organic solvent to the supernatant obtained by centrifugation, and the extract was centrifuged to obtain a precipitate: centrifugation was carried out for 10 to 30 minutes in the range of 5000 to 9000 × g, The organic solvent uses isopropanol or ethanol.

The electric precipitate is dissolved in distilled water, followed by dialysis to obtain an internal dialysis fraction, and the electrodialysis internal fraction is vacuum lyophilized: wherein dialysis is carried out using distilled water for 3 to 5 days using an exclusion molecular weight of 10,000 to 15,000. To 7 replacements.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention more specifically, it will be apparent to those skilled in the food industry that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Analysis of Prepared Pullulan

Pullulan produced through the first, second and third processes was analyzed using thin layer chromatography (TLC) and gas chromatography (GC). The pullulan prepared in the above Examples and Comparative Examples was hydrolyzed at 100 ° C. for 2 hours using 1N HCl, and then subjected to thin layer chromatography, using commercial pullulan (Sigma Co., USA) as a control. It was. As a developing solvent of the thin layer chromatography, a solution containing butanol, pyridine and distilled water in a ratio of 6: 4: 3 (v / v / v) was used, and 1% of aniline and diphenylamine, respectively, were used. The developed pullulan was identified using a solution prepared by mixing a solution of acetone (5 ml) and an 85% phosphoric acid solution (1 ml) at a concentration of (v / v) as a color developing reagent. In addition, gas chromatography (GC Model 5890, Hewlett-Packard, U.S.A.) used helium as a mobile phase, the injection rate was 1.2ml / min, the pressure was 16.5 atm. The analysis was maintained at 200 ° C. for 3 minutes at the time of sample injection, and increased at 8 ° C. per minute at 280 ° C., and was confirmed using a commercial pullulan (Sigma Co., U.S.A.) as a control.

Step 3: increase productivity of pullulan by replacing pullulan production medium

Aureobasidium pullulans HP-2001 strain producing pullulan was inoculated into a medium containing 20 g / l of glucose as a carbon source and 2.5 g / l of yeast extract and an inorganic salt as a carbon source and 150 at 25 to 35 ° C. 3 to 5% (v / v) of the spawn culture medium incubated for 24 to 48 hours at 200 rpm to 2.5 g / l yeast extract and inorganic salt with 0 to 300 g / l sugar and nitrogen source as carbon source Inoculated at 3 to 5% (v / v) in culture at 150 to 200 rpm at 25 to 35 ° C., incubated at 150 to 200 rpm at 25 to 35 ° C. for 3 days, and then half of the culture was Collect and separate pullulan and purify. 1) 100 g / l sugar liquor, 2) 100 g / l sugar and 2,5 g / l yeast extract and 3) 100 g / l sugar, 2.5 g / l corresponding to the volume of culture collected The yeast extract and the mixed solution containing the inorganic salts of each were added and cultured to compare the production of the cells and the production of pullulan.

As a result of observing the effect on the production of pullulan by adding a mixture of different components, as shown in FIG. 2, a medium prepared using 100 g / l sugar as a carbon source and 2.5 g / l yeast extract as a nitrogen source Inoculated with the Aureobasidium pullulan HP-2001 strain incubated for 72 hours, half of the culture was collected and the same amount of 100 g / l sugar (■), 100 g / l sugar and 2.5 When g / l yeast extract (▲) and 100 g / l sugar, 2.5 g / l yeast extract and inorganic salt (○) were added, respectively, 100 g / l sugar and 2.5 g / l yeast extract And the addition of the mixture of the inorganic salt (○) showed the highest increase in productivity, the composition and concentration of this mixture was consistent with the composition and concentration of the production medium of pullulan. That is, inoculate the pullulan production strain into the pullulan production medium and incubate for 72 hours, and then collect half of the culture solution and incubate with the same volume of fresh medium, if the medium is not replaced, only the carbon source is added, It showed higher productivity compared to the case of adding a carbon source and a nitrogen source. In each case, the concentration of the cells and the productivity of pullulan were not added when 72 hours of incubation was performed as shown in Table 2 below, when a mixture of carbon source, carbon source and nitrogen source or carbon source, nitrogen source and inorganic salt was added. The total amount of pullulan produced was 2.1 times as compared to the above. Therefore, it was confirmed that the productivity of the pullulan was the highest when the mixed solution added in the case of continuously replacing the medium to produce pullulan was added with the same component as the production medium of pullulan.

Influence of Medium Substitution on the Production of Pullulan in Production of Pullulan Using Arureobasidium Pullulan HP-2001 Strain in 7L Bioincubator with 5L Production Medium division Component of Substitution Medium ¹⁾ Me ^{2) 3) 4)} Incubation time (h) 72 120 120 120 pH 3.4 3.4 3.5 3.3 Dry cell 16.7 14.2 17.9 20.5 Pullulan 46.7 49.6 65.1 74.9 Pullulan conversion 0.47 0.49 0.59 0.65 Pullulan Total Production 186.7 291.9 353.9 392.0

Third Process: Continuous Production of Pullulan at Optimal Rate of Adding Pullulan Production Medium

Microorganisms producing pullulan were inoculated in a medium to which 20 g / l glucose was added as a carbon source and 2.5 g / l yeast extract and an inorganic salt as a nitrogen source, and then for 24 to 48 hours at 150 to 200 rpm at 25 to 35 ° C. The seed culture was inoculated at 3 to 5% (v / v) in a medium to which 100 g / l of sugar as a carbon source and 2.5 g / l of yeast extract and an inorganic salt were added as a carbon source, and then at 25 to 35 ° C. While incubating at 150 to 200 rpm, 100 g / l of sugar, 2.5 g / l of yeast extract and inorganic salts were added to the culture solution continuously at a dilution rate of 0.01 to 0.04 h ⁻¹ . The culture was harvested and the pullulan was purified from the culture. Figure 3 is a continuous culture by adding a medium of the same component while culturing the Aureobasidium pullulan HP-2001 strain in a medium prepared using 100 g / l of sugar as a carbon source and 2.5 g / l yeast extract as a nitrogen source When the dilution rate affects the growth of the cells and the production of pullulan, the graph shows the increase of the cells up to the dilution rate of 0.005h ^-1 and the dilution rate of 0.015h ^- as shown in FIG. ^{At 1} day, the production concentration of pullulan was highest. In Figure 3 ● indicates the growth of cells, ■ indicates the production of pullulan.

Fourth Step: Continuous Production of Pullulan by Continuous Addition of Pullulan Production Medium Containing Sugar

Microorganisms producing pullulan were inoculated in a medium to which 20 g / l glucose was added as a carbon source and 2.5 g / l yeast extract and an inorganic salt as a nitrogen source, and then 24 to 48 hours at 150 to 200 rpm at 25 to 35 ° C. Incubated at 3 to 5% (v / v) in a medium to which 100 g / l of sugar as a carbon source and 2.5 g / l of yeast extract and an inorganic salt were added as a carbon source, and 25 to 35 ° C. 100-200 g / l sugar, 2.5 g / l yeast extract and inorganic salts were added to the culture solution at a dilution rate of 0.015 h ⁻¹ continuously while incubating at 150 to 200 rpm. The culture solution was collected, and pullulan was purified from the culture solution. Of Figure 4 is the brother 0.025h Leo medium of the same composition and cultured Bridge Stadium pullulans HP-2001 strain in a culture medium prepared with 2.5 g / l of yeast extract as a nitrogen source of sugar and 100 g / l a carbon source ^-1 It is a graph showing the effect on the molecular weight, average molecular weight and dispersion of the produced pullulan when the continuous culture by the addition of the medium of the same component of pullulan produced when the continuous culture while adding at a dilution rate, Figure 4 As shown in Fig. 2, the average concentrations of pullulan produced were 69.3, 86.3, and 113.5 g / l when the concentration of sugar, the carbon source of the pullulan production medium added, was 100, 150, and 200 g / l. When the concentration of sugar, which is the carbon source of the added pullulan production medium, was 200 g / l compared to 100 g / l, the productivity was improved by 1.6 times. In Figure 4, ● represents the molecular weight, 은 represents the average molecular weight, □ represents the degree of dispersion.

5 is added to the same medium while culturing the Aureobasidium pullulan HP-2001 strain in a medium prepared using 100 g / l of sugar as a carbon source and 2.5 g / l of yeast extract as a nitrogen source, and adding 0.025h ⁻¹ The graph shows the effect of the concentration of sugar as a carbon source on the production of pullulan when continuously cultured at a dilution rate of. The molecular weight of pullulan produced by continuously adding pullulan production medium containing sugar to the culture medium and The average molecular weight is uniform, it can be seen that the quality is improved compared to the conventional method produced. In Fig. 5, the concentration of sugar indicates that l is 100 g / l, l is 150 g / l, and is 200 g / l.

As described and demonstrated in detail above, the present invention inoculates a strain producing pullulan into a pullulan production medium and incubated at a constant temperature while continuously adding pullulan production medium containing sugar to the culture medium to pullulan with high productivity. It provides a method for producing continuously. According to the present invention, since the yeast extract used as a nitrogen source in the production of pullulan is replaced with soy sauce, a byproduct of the fermented food manufacturing process, about 60% of the direct production cost can be reduced, and in particular, the productivity of pullulan It can improve about 65%. Since the molecular weight of the pullulan prepared using soy sauce is 3 to 6 times larger than that of the pullulan prepared using the yeast extract, which is a nitrogen source of the existing medium, it was confirmed that excellent quality pullulan can be prepared. In the manufacture of pullulan, since soybean meal, a by-product of the fermented food manufacturing process, is used as a nitrogen source, it is possible to reduce the treatment cost for the purification of environmental pollutants.

Claims (5)

Microorganisms producing pullulan were inoculated in a medium to which 20 g / l glucose was added as a carbon source and 2.5 g / l yeast extract and an inorganic salt as a nitrogen source, and then 24 to 48 hours at 150 to 200 rpm at 25 to 35 ° C. The seed culture was inoculated at 3 to 5% (v / v) in a medium containing sugar as a carbon source, yeast extract as a nitrogen source, and an inorganic salt, and then 3 to 5 days at 150 to 200 rpm at 25 to 35 ° C. After culturing for a period of time, pullulan was prepared by continuously collecting a predetermined amount of the culture solution while continuously adding the mixed solution containing the sugar and yeast extract and the inorganic salt to the culture solution at a dilution rate of 0.01 to 0.04 h ⁻¹ . In the method, The mixed solution is 100 g / l to 200 g / l sugar and 2.5 g / l yeast extract and 3 to 5 g / l K ₂ HPO ₄ , 0.01 to 1 g / l MgSO ₄ H ₂ O, 0.3 to 1.5 g / l of (NH ₄ ) ₂ SO ₄ and Using an inorganic salt of 0.5 to 3.0 g / l NaCl, The amount of the mixed solution is continuously added to the culture medium in the same amount as that of the culture medium collected continuously, And the amount of the culture solution collected is 0.5% (v / v) / h to 1.5% (v / v) / h of the total culture solution. delete delete delete delete

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