TWI450966B - Production methods of microbial exopolysaccharides - Google Patents

Description

Method for producing microbial extracellular polysaccharide

The present invention relates to a method for producing a microbial exopolysaccharide (EPS), and in particular to a microbial exopolysaccharide produced by a microbiological method.

Extracellular polysaccharides produced by microorganisms are biopolymers, which have good biocompatibility and biodegradability, so if they can be specifically applied, The deteriorating global environment will have many benefits. In addition, because these biopolymer-producing microbial strains have a special environment (dryness, temperature, pressure, salinity, acidity), they are in the process of industrial production. Even in the extreme conditions of processing (temperature, salinity, and pH), the biopolymers produced by these microorganisms retain their own properties and are not susceptible to extreme conditions generated by the process.

Due to the versatile physicochemical properties and rheological properties of biopolymers, extracellular polysaccharides have been widely used in, for example, textiles, detergents, adhesives, microbial oil recovery (microbial flooding), wastewater treatment, Biomaterials in industrial fields such as brewing, downstream processing, beauty, pharmacology and food additives, and recent studies have pointed out that EPS produced by microorganisms has the activity of scavenging superoxide radicals and scavenging activity against hydroxyl radicals (Liu et al . 2009), it is expected that the potential value and development potential of extracellular polysaccharides will have a great impact on future life.

Exopolysaccharide is a carbohydrate polymerization produced by the process of microbial fermentation. Xie products, and nowadays in the microbial fermentation, sucrose, glucose or other carbohydrates are used as carbon/nitrogen sources, and the secretion of microbial exopolysaccharides is very susceptible to external culture conditions, such as carbon sources. , nitrogen source, pH, aeration, temperature and culture time, etc., if the lack of appropriate conditions, will cause the strain to not achieve optimal growth efficiency, and will further affect the output of these metabolites.

In order to achieve the above object, the present inventors have devoted themselves to the development of the efficiency of the production of microbial exopolysaccharide by the relevant strains using biological waste materials, in order to enhance the production of extracellular polysaccharides and to achieve the purpose of properly utilizing biological resource waste, and finally invented the present invention. Method for producing microbial exopolysaccharide.

The object of the present invention is to provide a method for producing microbial exopolysaccharide (EPS), which utilizes the culture conditions for regulating microorganisms to increase the yield of EPS. In the present invention, the use is deposited in the Hsinchu Biological Resource Conservation and Research Center. The Paenibacillus sp. strain (BCRC910490 strain), which is numbered BCRC910490, is a microbial strain mainly producing microbial exopolysaccharide, and the squid pen powder (hereinafter referred to as SPP) is used as a carbon for fermentation. At the nitrogen/source, the BCRC 910490 strain will produce a higher yield of extracellular polysaccharide than in the conventional carbon/nitrogen source of conventional sugars. In one embodiment, the SPP is added in an amount of 0.5% to 1.5% in the culture solution. Further added is cultured with 0.1% potassium phosphate (K ₂ HPO ₄ ) and 0.05% magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O). In a preferred embodiment, the BCRC 910490 strain is at 37 ° C. Under the condition of culture temperature, the yield of extracellular polysaccharide can be better, and the culture time is at least four days. After the culture solution of the cultured BCRC910490 strain is obtained, purification and separation can be obtained. Extracellular polysaccharides.

In order to make the reviewer more aware of the present invention, the following examples are set forth.

Example 1: Demonstrating the effect of carbon and nitrogen source species on microbial production of microbial exopolysaccharide

In this embodiment, the Bacillus sp. Paenibacillus sp. strain (hereinafter referred to as BCRC910490 strain) deposited in the Hsinchu Bioresource Conservation and Research Center No. BCRC910490 is used to investigate various bio-aquatic wastes as carbon/nitrogen sources. The strain produces the microbial exopolysaccharide. These aquatic wastes include shrimp head powder (SHP) and squid cartilage powder (hereinafter referred to as SPP). The culture is supplemented with 0.1% K ₂ HPO _{4 .} And 0.05% MgSO ₄ . A 100 mL liquid medium of an inorganic salt such as 7H ₂ O was cultured in a shake flask at 37 ° C for 1 to 5 days.

Please refer to Figure 1 for the production curve of microbial exopolysaccharide after using SPP and SHP as carbon/nitrogen for culture of BCRC910490 strain, when SPP squid cartilage powder is used as carbon/nitrogen source. When the strain was cultured, the content of extracellular polysaccharide was gradually increased, and the highest yield was about 3200 μg per ml on the fourth day of culture, and the use of SHP shrimp shell powder did not significantly increase the yield of extracellular polysaccharide.

Example 2: Explain the effects of various culture parameters on microbial production of microbial exopolysaccharide

(1) Carbon/nitrogen source concentration: BCRC910490 strain culture was carried out under conditions of 0.5, 1.0 and 1.5% SPP as carbon and nitrogen sources. Referring to Fig. 2, the marked lines 3, 4 and the total sugar amount change line when the SPP concentrations are 0.5, 1.0, and 1.5%, respectively, are generated when the added SPP concentration is 1.0% (marked line 4). The total amount of extracellular polysaccharide is very similar to that of 1.5% SPP (line 5). Therefore, when BCRC910490 strain is cultured, 1.0% SPP can be used.

(2) Effect of culture volume: The ability of BCRC910490 strain to produce extracellular polysaccharides in different volumes of culture medium was compared under the condition of 1.0% SPP as carbon/nitrogen source, please refer to Figure 3, where the marking 6 to 9 respectively represent the volume of the culture medium of 25, 50, 100 and 200 ml, respectively. It can be clearly seen from the figure that when the volume of the culture medium is small, the extracellular polysaccharide can reach a higher yield, which is clearly observed under aeration conditions. In a better state, the BCRC910490 strain may have a better exopolysaccharide production capacity, and in the culture of 50 ml of the culture solution, the extracellular polysaccharide can be optimally produced.

(3) Effect of temperature: 1.0% SPP is used as carbon/nitrogen source, and 50 ml culture medium is used to change different temperatures. The fixed culture temperature is 25 °C (marking line 10) and 30 °C (marking line 11). The effect of 37 ° C (line 12), please see Figure 4, the production efficiency of extracellular polysaccharide is not ideal at low temperature, and it does not help to increase the production of extracellular polysaccharide, but culture at 37 ° C At 4 days, the maximum yield of extracellular polysaccharide was about 3500 μg/ml.

(4) Effect of pH value: under the condition of using 1.0% SPP as carbon/nitrogen source and 50 ml culture medium at 37 °C, it is not adjusted The pH value of the whole culture medium or the pH value of the medium was adjusted to pH 2~12 to culture the BCRC910490 strain. Please refer to Fig. 5, the production efficiency of the extracellular polysaccharide of BCRC910490 strain in alkaline environment is slightly better than that of the acidic environment. And without adjusting the pH value of the culture medium, the production of extracellular polysaccharide can reach 4000 μg/ml.

Example 3: Preparation method of extracellular polysaccharide

Recovery: In the present example, the supernatant collected after the fermentation of the BCRC910490 strain using the various conditions obtained in Example 2 was heated at 121 ° C for 20 minutes to completely dissolve the EPS in the liquid, and then added twice the volume. The methanol was decolorized, and the precipitate was recovered by centrifugation (13420 × g ), remelted with a small amount of deionized water and lyophilized, and a crude EPS of about 1.7631 g per 1000 ml of the culture solution was obtained.

Deproteinization: Take 500 mg of crude EPS and dissolve it in 100 ml of distilled water. After completely dissolving, add four volumes of absolute ethanol, and place at 4 ° C overnight, and centrifuge (13420 × g , 15 minutes) to obtain a precipitate. Re-dissolve with a small amount of distilled water, add 1/5 volume of Sewag reagent (Sevag, CHCl ₃ -BuOH, v / v = 5 / 1) and repeat the above steps several times to obtain deproteinized EPS, freeze-drying Approximately 125 mg of deproteinized EPS was obtained.

Hydrolysis: 25 mg of deproteinized EPS was dissolved in 50 ml of distilled water, and 0.5 U/mL of α-amylase (at 20 ° C, pH 6.9) was added for hydrolysis. After hydrolysis, the water was dialyzed and the solution outside the dialysis membrane was collected. Freeze-drying was carried out to obtain about 7.75 mg of lyophilized powder.

Example 4: Composition of extracellular polysaccharide produced by BCRC910490 strain

The EPS obtained after the decolorization, deproteinization and hydrolysis steps in Example 3 was subjected to thin layer chromatography (TLC) using phosphomolybdic acid reagent (2.4% (w/v) molybdenum phosphate, 5% (v/v) sulfuric acid, 1.5% (v/v) phosphoric acid) or Ninhai ninhydrin reagent for coloration. When using Ninghai quasi-staining, only glucose (R _f = 0.45) and maltose were found (R _f value = 0.38), and using phosphomolybdic acid staining, the coloration position (R _f =0.4) of the sample was found to be similar to maltose; further 10 mg of EPS was dissolved in heavy water (D ₂ O), so EPS and maltose were further subjected to nuclear magnetic resonance. Spectroscopy (Nuclear Magnetic Resonance, NMR), the hydrogen spectrum and carbon spectrum results show that the extracellular polysaccharide produced by the BCRC 910490 strain has the chemical structure of maltose.

In summary, the present invention provides a method for producing a biological exopolysaccharide, which can effectively increase the yield of the biological extracellular polysaccharide, and the operation method of the production method is very simple, and the bio-extracellular polysaccharide is used as a follow-up. Application and development.

1‧‧‧Microbial extracellular polysaccharide production curve with squid cartilage powder as carbon/nitrogen source

2‧‧‧Microbial extracellular polysaccharide production curve with shrimp head powder as carbon/nitrogen source

3‧‧‧Microbial extracellular polysaccharide production curve with 0.5% squid cartilage powder as carbon/nitrogen source

4‧‧‧Microbial extracellular polysaccharide production curve with 1.0% squid cartilage powder as carbon/nitrogen source

5‧‧‧Microbial extracellular polysaccharide production curve with 1.5% squid cartilage powder as carbon/nitrogen source

6‧‧‧Microbial extracellular polysaccharide production curve when the volume of culture medium is 25 ml

7‧‧‧Microbial extracellular polysaccharide production curve when the volume of culture medium is 50 ml

8‧‧‧Microbial extracellular polysaccharide production curve when the volume of culture medium is 100 ml

9‧‧‧Microbial extracellular polysaccharide production curve when the volume of culture medium is 200 ml

10‧‧‧Microbial extracellular polysaccharide production curve when cultured at 25 °C

11‧‧‧Microbial extracellular polysaccharide production curve when cultured at 30 °C

12‧‧‧Microbial extracellular polysaccharide production curve when cultured at 37 °C

Figure 1 illustrates the effect of different carbon/nitrogen sources on the production of the microbial exopolysaccharide of the present invention.

Figure 2 illustrates the effect of different concentrations of carbon/nitrogen sources on the production of the microbial exopolysaccharide of the present invention.

Figure 3 is a graph showing the effect of different culture machines on the production method of the microbial exopolysaccharide of the present invention.

Figure 4 illustrates the effect of different temperatures on the production process of the microbial exopolysaccharide of the present invention.

Figure 5 is a diagram showing the microbial cells of the present invention with different pH values of the culture medium. The effect of the production process of exopolysaccharides.

Claims (3)

A method for producing microbial exopolysaccharide, which is a strain of Paenibacillus sp. which is deposited in the Biological Resource Preservation and Research Center No. BCRC910490, and is a single carbon in 0.5% to 1.5% squid cartilage powder/ The medium having a nitrogen source volume of less than 200 ml and having a pH between 4 and 10 is cultured at a temperature of 37 ° C for at least four days, and the culture solution is subjected to purification separation to obtain a microbial exopolysaccharide. The production method according to claim 1, wherein the purification separation comprises the following steps: recovery: heating the culture solution at 121 ° C for 20 minutes, adding two volumes of methanol for decolorization, and centrifuging (13420× g ) recovery of the precipitate, re-dissolve with a small amount of deionized water and freeze-drying; deproteinization: the freeze-dried powder obtained in the above recovery step is dissolved in distilled water until it is completely dissolved, four times the volume of absolute ethanol is added, and placed in Stir at room temperature overnight at 4 ° C, and then centrifuge (13420 × g , 15 minutes) to obtain a precipitate with a small amount of distilled water to dissolve, add 1 / 5 volume of Sewag reagent (Sevag, CHCl ₃ -BuOH, v / v=5/1) The reaction is several times and freeze-dried; hydrolysis: the freeze-dried powder obtained by the above deproteination step is dissolved back in distilled water, and 0.5 U/mL of α-amylase is added at a reaction temperature of 20 ° C and pH. The mixture was hydrolyzed under the conditions of 6.9. After hydrolysis, the water was dialyzed, and the solution outside the dialysis membrane was collected and freeze-dried to obtain a powder of the microbial exopolysaccharide. The production method according to claim 2, wherein the culture solution further contains 0.1% potassium phosphate (K ₂ HPO ₄ ) and 0.05% magnesium sulfate heptahydrate (MgSO ₄ .7H ₂ O).