CN104087628A - Method for reducing viscosity of gamma-polyglutamic acid fermentation liquid - Google Patents

Abstract

The invention discloses a method for reducing the viscosity of gamma-polyglutamic acid fermentation liquid, which includes the activation of strains, the preparation of seed liquid and the fermentation of liquid shake flasks. During the fermentation of the liquid medicine bottles, high concentration KCl, the high-concentration KCl is 0.5-30g/L; the strain is Bacillus subtilis GXA-28, the preservation number is CCTCCNO: M2012347, the preservation date is September 14, 2012, and the preservation unit: China Type Culture Collection. In this method, KCl with a mass volume concentration of 0.5-30 g/L is added to the liquid fermentation medium at the initial stage of fermentation, and the viscosity is reduced to 10-80% of the original on the basis of maintaining high molecular weight and high yield of γ-polyglutamic acid , to solve the bottleneck problem of high viscosity of the fermentation broth produced by γ-PGA fermentation, and has a good application prospect; and the cost is low, which reduces the cost for the separation and purification of products in industrial production.

Description

A method for reducing the viscosity of gamma-polyglutamic acid fermentation broth

technical field

The invention belongs to the field of microbial fermentation, and in particular relates to a method for reducing the viscosity of gamma-polyglutamic acid fermentation liquid. the

Background technique

γ-polyglutamic acid is a water-soluble biopolymer formed by combining L-glutamic acid or D-glutamic acid through γ-amide groups. It has strong water absorption, degradability, hydrolysis and many other characteristics, so it is widely used in many fields such as food, agriculture, medicine, greening and water treatment, and has great development value and application prospect. the

According to current literature reports, γ-PGA is the main component of some bacterial capsules, and its function is to protect the bacteria from the harsh external environment. Therefore, its synthesis is generally in the middle and late stages of bacterial fermentation, when a large amount of metabolic waste accumulates, and the lack of nutrients promotes the bacteria to secrete and synthesize γ-PGA for self-protection. Therefore, it can be considered that the synthesis and secretion of γ-PGA is an adaptation mechanism for bacteria to cope with the harsh external environment. At present, the hotspots of research are mostly focused on microbial fermentation production to increase yield, and there are very few related reports on how to improve the yield of separation and extraction. the

It is generally believed that the viscosity of γ-PGA fermentation broth increases with the increase of yield and molecular weight. Therefore, in the later stage of liquid fermentation, the viscosity of fermentation broth increases greatly due to the increase of γ-PGA concentration, which affects the mass transfer of oxygen, and then affects γ-PGA. The synthesis of PGA is also the biggest bottleneck problem in increasing the concentration of γ-PGA. Adjusting the pH of the fermentation broth to pH<5 or pH>8 by adding acid or alkali can significantly reduce the viscosity of the fermentation broth (wherein the viscosity at pH 3.5 is only about 1/50 of that at pH 6.5). Centrifuge and sterilize the pH3.5 fermentation broth (10000g, 10min) and concentrate by ultrafiltration (filter membrane pore size 0.45μm, average pressure 0.08Mpa) 1 time, then add 95% ethanol to extract γ-PGA, and phase with neutral pH It can reduce the energy consumption by more than 50% and the solvent by 40%, but the loss of γ-polyglutamic acid is about 10%. Adding acid or alkali can change the molecular structure of γ-PGA in the fermentation broth and reduce the molecular weight, which is unfavorable for the production of γ-polyglutamic acid with high molecular weight. the

Contents of the invention

The purpose of the present invention is to provide a method for reducing the viscosity of gamma-polyglutamic acid fermentation broth in order to overcome the deficiencies in the prior art. In the initial stage of fermentation, the method adds a mass volume concentration of 0.5-30g/L to the liquid fermentation medium. KCl, on the basis of maintaining high molecular weight and high yield of γ-polyglutamic acid, reduces the viscosity to 10-80% of the original, and solves the bottleneck problem of high viscosity of the fermentation broth produced by γ-PGA fermentation, which has a good application prospect ; And the cost is low, which reduces the cost for the separation and purification of products in industrial production. the

In order to achieve the above object, the present invention is achieved through the following technical solutions:

A method for reducing the viscosity of gamma-polyglutamic acid fermentation liquid, comprising activation of strains, preparation of seed liquid and liquid shake flask fermentation, characterized in that: in the liquid medicine bottle fermentation, adding high Concentration KCl, the high concentration KCl is 0.5-30g/L; the strain is Bacillus subtilis (Bacillus subtilis) GXA-28, the preservation number is CCTCC NO: M2012347, and the preservation date is September 14, 2012. Unit: China Center for Type Culture Collection. the

The high concentration KCl mentioned above is 5-30g/L. the

The above liquid fermentation medium components also include glucose 30-50g/L, sodium glutamate 20-40g/L, yeast extract 2.5-4.0g/L, KH ₂ PO ₄ 0.5-1g/L, MgSO ₄ 0.1- 0.15g/L, pH6.5-7.5, prepared with distilled water.

The fermentation conditions of the above-mentioned liquid shake flask fermentation are as follows: the inoculum amount of the seed liquid is 1-6% by volume into the sterilized liquid fermentation medium, and the shake flask is cultivated at 40-50°C for 20-30h. the

The activation of the above-mentioned strains is to connect Bacillus subtilis (Bacillus subtilis) GXA-28 on a solid slant medium, cultivate it at 40-50°C for 8-16h, and store it for a short time at 2-8°C; the solid slant The composition of the medium is: glucose 8~12g/L, yeast extract 3~6g/L, sodium glutamate 3~6g/L, MgSO ₄ 7H ₂ O 0.1~0.2g/L, KH ₂ PO ₄ 0.3 ～0.5g/L, agar 10～15g/L, pH 6.5～7.5, prepared with distilled water.

The seed solution mentioned above is prepared by inserting the 1.0cm ² bacterial lawn on the inclined surface into a 250ml triangular flask containing 30ml liquid seed medium, the shaker speed is 160-250rpm, and the shaking flask is cultivated at 42°C-50°C for 16h-18h The composition of the liquid seed medium concentration is: glucose 10-50g/l, yeast extract 2-10g/l, sodium glutamate 5-20g/l, MgSO ₄ ·7H ₂ O 0.1-0.5g/l, KH ₂ PO ₄ 0.5~2g/l, pH 6.5~7.5, prepared with distilled water.

Compared with prior art, the beneficial effect of the present invention is:

1. The present invention uses Bacillus subtilis (Bacillus subtilis) GXA-28 as the starting strain to ferment and produce γ-PGA under high-concentration KCl conditions. On the one hand, K ⁺ increases the osmotic pressure of the substrate, and on the other hand, K ⁺ is γ-PGA The promoting ion of synthetase therefore provides a favorable environment for the synthesis of γ-PGA. As a result, on the basis of maintaining or even increasing the yield, the molecular weight is increased to 3000-4000kDa, and the viscosity is reduced to 10-80%. The technical problem that the increase of the concentration of γ-PGA leads to the increase of the viscosity of the fermented liquid has a good application prospect.

2. The cost of adding KCl in the present invention is low, which can reduce the cost for the separation and purification of products in industrial production. the

Description of drawings

Fig. 1 is the molecular weight electrophoresis figure of gamma-polyglutamic acid,

From left to right on the figure are the γ-polyglutamic acid standard product, the γ-polyglutamic acid control group of Example 1-3, and the γ-polyglutamic acid obtained after adding KCl concentrations of 0.5, 1, 5, 10, 15, and 20 g/L. - polyglutamic acid. the

The sequence listing is the 16S rDNA nucleotide sequence information of bacterial strain Bacillus subtilis (Bacillus subtilis) GXA-28, CCTCC M2012347. the

Detailed ways

The present invention will be further described in detail below in conjunction with the examples, but the embodiments of the present invention are not limited to the scope indicated by the examples. the

The Bacillus subtilis (Bacillus subtilis) GXA-28 character characteristic that the present invention utilizes is:

(1) Morphological characteristics of bacteria:

Strain CCTCC NO: M2012347 was cultured on a solid slant medium at 50°C for 16 hours and observed under an electron microscope. The bacteria were rod-shaped, with a size of 0.7-0.9×2.0-3.0 μm, movable, and Gram staining was positive. Strain CCTCC NO: M2012347 was cultured on a solid slant medium at 50°C for 30 hours and observed by spore staining, and obvious spores could be seen. the

(2) Colony morphological characteristics:

Strain CCTCC NO: M2012347 After being cultured on a solid separation medium plate at 50°C for 24 hours, on a plate added with glutamic acid, the colony is round, the surface is dendritic, the edge secretes sticky substances, and the diameter of the colony reaches 1.5-2.0cm ; On plates without glutamate, the colonies were dry, flat, with a depressed center and irregular edges. the

Strain CCTCC NO: M2012347 is cultured in liquid separation medium, and a bacterial film can be formed on the surface of the liquid, and the culture medium is slightly turbid. the

(3) The physiological and biochemical properties of CCTCC NO: M2012347 are shown in Table 3 below. the

Table 1

Note: "+" in the list means good growth or positive; "-" means no growth or negative. the

(4) CCTCC NO: 16S rDNA sequence analysis of M2012347

The 16S rDNA of the strain was analyzed using the universal amplification primers 1492r (5'-GGY TAC CTT GTT ACG ACT T-3', Y=T or C) and 27f (5'-AGA GTT TGA TCC TGG CTC AG-3'). Amplified and sequenced, the measured sequence length was 1365bp. Submit the obtained sequence to the GenBank database, obtain the sequence number GenBank ID: JN815234, and perform Blast comparison analysis with the gene sequence provided by GenBank to construct a phylogenetic tree. The results showed that the homology between CCTCC NO: M2012347 and Bacillus subtilis was 99%. The target strain was determined to be Bacillus subtilis by analyzing the morphological characteristics of the thallus and colony, physiological and biochemical characteristics and 16S rDNA sequence, and named it Bacillus subtilis (Bacillus subtilis) GXA-28. Preservation, its classification is named Bacillus subtilis (Bacillus subtilis) GXA-28, the preservation number is CCTCC NO: M2012347, the preservation date is September 14, 2012, the preservation unit: China Center for Type Culture Collection, preservation address: Hubei Province Wuhan University, Luojia Mountain, Wuchang, Wuhan. the

Example 1:

(1) Activation of strains

Bacillus subtilis (Bacillus subtilis) GXA-28 was inoculated on the solid slant medium, cultured at 40°C for 16h, and stored at 4°C for short-term; the composition of the solid slant medium was: glucose 8g/L, yeast extract 3g/L, Sodium glutamate 3g/L, MgSO ₄ ·7H ₂ O 0.1g/L, KH ₂ PO ₄ 0.3g/L, agar 10g/L, pH 6.5, distilled water.

(2) Preparation of seed solution

Put the 1.0cm ² bacterial lawn on the slope into a 250ml Erlenmeyer flask with 30ml liquid seed medium, shake the shaker at 160rpm, and culture it at 42°C for 16 hours; the concentration of the liquid seed medium is composed of: glucose 10g/l, yeast Paste 2g/l, sodium glutamate 5g/l, MgSO ₄ ·7H ₂ O 0.1g/l, KH ₂ PO ₄ 0.5g/l, pH 6.5, and distilled water.

(3) Liquid shake bottle fermentation

The seed solution was added to the sterilized liquid fermentation medium with an inoculation amount of 1% by volume, and cultured in a shaker flask at 40° C. for 20 h at a rotational speed of 160 r/min. Components of fermentation medium: glucose 30g/L, sodium glutamate 20g/L, yeast extract 2.5g/L, KH ₂ PO ₄ 0.5g/L, MgSO ₄ 0.1g/L, KCl 5g/L, pH6.5, The rest is distilled water.

The conditions of the control group and the experimental group were the same, in which KCl was added to the experimental group, and KCl was not added to the control group. the

(4) The detection results of γ-PGA are shown in Table 1 and Figure 1. the

Table 1

group test group control group Yield (g/L) 18.51 16.36 Molecular weight (Da) 2.1*10 ⁶ 2.0*10 ⁶

(5) The detection of the viscosity of the fermentation broth is shown in Table 2. the

Table 2

group test group control group Viscosity (mPa/s) 2368 4289 reduction rate 44.8% the

From Table 1 and Table 2, it can be concluded that when the concentration of γ-PGA is increased by the method of the present invention, the viscosity of the fermentation broth will not be increased, but the viscosity will be greatly reduced, while high molecular weight γ-PGA can be obtained. the

Example 2:

The difference between embodiment 2 and embodiment 1 is that KCl becomes 10g/L in the composition of fermentation medium. the

The conditions of the control group and the experimental group were the same, in which KCl was added to the experimental group, and KCl was not added to the control group. the

(4) The detection results of γ-PGA are shown in Table 3 and Figure 1. the

table 3

group test group control group Yield (g/L) 20.41 16.36 Molecular weight (Da) 3.0*10 ⁶ 2.0*10 ⁶

5) The detection of the viscosity of the fermentation broth is shown in Table 4. the

Table 4

group test group control group Viscosity (mPa/s) 932 4289 reduction rate 78.3% the

It can be concluded from Table 3 and Table 4 that when the concentration of γ-PGA is increased by the method of the present invention, the viscosity of the fermentation broth will not be increased, but the viscosity will be greatly reduced, while high molecular weight γ-PGA can be obtained. the

Example 3:

The difference between embodiment 3 and embodiment 1 is that KCl becomes 15g/L in the composition of fermentation medium. the

The conditions of the control group and the experimental group were the same, in which KCl was added to the experimental group, and KCl was not added to the control group. the

(4) The detection results of γ-PGA are shown in Table 5 and Figure 1. the

table 5

group test group control group Yield 17.55 16.36 molecular weight 4.0*10 ⁶ 2.0*10 ⁶

(5) The detection of the viscosity of the fermentation broth is shown in Table 6. the

Table 6

group test group control group Viscosity (mPa/s) 640 4289 reduction rate 85.1% the

It can be concluded from Table 5 and Table 6 that when the concentration of γ-PGA is increased by the method of the present invention, the viscosity of the fermentation broth will not be increased, but the viscosity will be greatly reduced, while high molecular weight γ-PGA can be obtained. the

Example 4:

(1) Activation of strains

Bacillus subtilis (Bacillus subtilis) GXA-28 was inoculated on the solid slant medium, cultured at 45°C for 10h, and stored at 6°C for short-term; the composition of the solid slant medium was: glucose 10g/L, yeast extract 4g/L, Sodium glutamate 6g/L, MgSO ₄ ·7H ₂ O 0.2g/L, KH ₂ PO ₄ 0.4g/L, agar 12g/L, pH 7.0, distilled water.

(2) Preparation of seed solution

Put the 1.0cm ² bacterial lawn on the slope into a 250ml Erlenmeyer flask containing 30ml liquid seed medium, shake the shaker at 200rpm, and culture the flask at 45°C for 17h; the concentration of the liquid seed medium consists of: glucose 30g/l, yeast Paste 8g/l, sodium glutamate 10g/l, MgSO ₄ ·7H ₂ O 0.3g/l, KH ₂ PO ₄ 1g/l, pH 7.0, prepared with distilled water.

(3) Liquid shake bottle fermentation

The inoculum amount of the seed liquid was 4% by volume into the sterilized liquid fermentation medium, and the shake flask was cultured at 45° C. for 25 hours, with a rotation speed of 160 r/min. The composition of the fermentation medium: glucose 40g/L, sodium glutamate 30g/L, yeast extract 3.0g/L, KH ₂ PO ₄ 0.8g/L, MgSO ₄ 0.15g/L, KCl 0.5g/L, pH7. 0, and the rest were distilled water.

The conditions of the control group and the experimental group were the same, in which KCl was added to the experimental group, and KCl was not added to the control group. the

(4) The detection results of γ-PGA are shown in Table 7. the

Table 7

group test group control group Yield 18.13 17.11 molecular weight 2.0*10 ⁶ 1.5*10 ⁶

(5) The detection of the viscosity of the fermentation broth is shown in Table 8. the

Table 8

group test group control group Viscosity (mPa/s) 1005 4255 reduction rate 76.4% the

It can be concluded from Table 7 and Table 8 that when the concentration of γ-PGA is increased by the method of the present invention, the viscosity of the fermentation broth will not be increased, but the viscosity will be greatly reduced, and high molecular weight γ-PGA can be obtained at the same time. the

Embodiment 5:

(1) Activation of strains

Bacillus subtilis (Bacillus subtilis) GXA-28 was inoculated on a solid slant medium, cultured at 50°C for 8h, and stored at 8°C for a short period of time; the composition of the solid slant medium was: glucose 12g/L, yeast extract 6g/L, Sodium glutamate 6g/L, MgSO ₄ ·7H ₂ O 0.2g/L, KH ₂ PO ₄ 0.5g/L, agar 15g/L, pH 7.5, distilled water.

(2) Preparation of seed solution

Put the 1.0cm ² bacterial lawn on the slope into a 250ml Erlenmeyer flask containing 30ml liquid seed medium, shake the shaker at 250rpm, and culture the flask at 50°C for 18h; the concentration of the liquid seed medium is composed of: glucose 50g/l, yeast Paste 10g/l, sodium glutamate 20g/l, MgSO ₄ ·7H ₂ O 0.5g/l, KH ₂ PO ₄ 2g/l, pH 7.5, prepared with distilled water.

(3) Liquid shake bottle fermentation

The seed liquid is added into the sterilized liquid fermentation medium according to the inoculum amount of 1-6% by volume, and the shake flask is cultivated at 50° C. for 30 h, and the rotation speed is 160 r/min. The composition of the fermentation medium: glucose 50g/L, sodium glutamate 40g/L, yeast extract 4.0g/L, KH ₂ PO ₄ 1g/L, MgSO ₄ 0.15g/L, KCl 30g/L, pH7.5, the rest for distilled water.

The conditions of the control group and the experimental group were the same, in which KCl was added to the experimental group, and KCl was not added to the control group. the

(4) The detection results of γ-PGA are shown in Table 9. the

Table 9

group test group control group Yield 20.41 17.58 molecular weight 4.2*10 ⁶ 2.5*10 ⁶

(5) The detection of the viscosity of the fermentation broth is shown in Table 10. the

Table 10

group test group control group Viscosity (mPa/s) 932 4380 reduction rate 78.7% the

It can be concluded from Table 9 and Table 10 that when the concentration of γ-PGA is increased by the method of the present invention, the viscosity of the fermentation broth will not be increased, but the viscosity will be greatly reduced, and high molecular weight γ-PGA can be obtained at the same time. the

Claims (6)

1. one kind is reduced the method for gamma-polyglutamic acid-fermentation broth viscosity, comprise activation, seed liquor preparation and the liquid shaking bottle fermentation of bacterial classification, it is characterized in that: in described liquid medicine bottle fermentation, add high density KCl to liquid fermentation medium, described high density KCl is 0.5-30g/L; Described bacterial classification is subtilis (Bacillus subtilis) GXA-28, and deposit number is CCTCC NO:M 2012347, and preservation date is on September 14th, 2012, depositary institution: Chinese Typical Representative culture collection center.

2. the method for reduction gamma-polyglutamic acid-fermentation broth viscosity according to claim 1, is characterized in that: described high density KCl is 5-30g/L.

3. the method for reduction gamma-polyglutamic acid-fermentation broth viscosity according to claim 1 and 2, is characterized in that: described liquid fermentation medium composition also comprises glucose 30-50g/L, Sodium Glutamate 20-40 g/L, yeast extract paste 2.5-4.0 g/L, KH ₂pO ₄0.5-1 g/L, MgSO ₄0.1-0.15 g/L, pH 6.5-7.5, distilled water preparation.

4. the method for reduction gamma-polyglutamic acid-fermentation broth viscosity according to claim 3, it is characterized in that: the fermentation condition of described liquid shaking bottle fermentation is: the seed liquor by volume inoculum size of 1-6% accesses in sterilized liquid fermentation medium, at 40-50 DEG C of shake-flask culture 20-30h.

5. the method for reduction gamma-polyglutamic acid-fermentation broth viscosity according to claim 3, it is characterized in that: the activation of described bacterial classification, that subtilis (Bacillus subtilis) GXA-28 is connected on solid slant culture base, cultivate 8-16h, make short term storage in 2 ~ 8 DEG C for 40 ~ 50 DEG C; Consisting of of described solid slant culture base: glucose 8 ~ 12g/L, yeast extract paste 3 ~ 6g/L, Sodium Glutamate 3 ~ 6g/L, MgSO ₄7H ₂o 0.1 ~ 0.2g/L, KH ₂pO ₄0.3 ~ 0.5g/L, agar 10 ~ 15g/L, pH value 6.5 ~ 7.5, distilled water preparation.

6. the method for reduction gamma-polyglutamic acid-fermentation broth viscosity according to claim 5, is characterized in that: described seed liquor preparation is by 1.0cm on inclined-plane ²lawn access be equipped with in the 250ml triangular flask of 30ml liquid seed culture medium, shaking speed 160 ~ 250rpm, 42 DEG C of-50 DEG C of shake-flask culture 16h-18h; Described liquid seed culture medium concentration consists of: glucose 10-30g/L, Sodium Glutamate 5-10 g/L, yeast extract paste 5-8 g/L, KH ₂pO ₄0.5-1 g/L, MgSO ₄0.1-0.15 g/L, pH 6.5-7.5.