CN111876364A - Bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid - Google Patents
Bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis for producing gamma-polyglutamic acid, which is classified and namedBacillus SubtilisThe culture medium has been preserved in China center for type culture Collection with the preservation number of CCTCC M2016605 and the preservation date of 2016, 11 months and 1 day. The invention also discloses a method for regulating and controlling the molecular weight of the fermentation product gamma-polyglutamic acid by using the bacillus subtilis. Compared with the prior art, the invention takes the bacillus subtilis XK as the starting strain, and can adjust the molecular weight of the gamma-polyglutamic acid by adjusting the concentrations of manganese sulfate and magnesium sulfate in a fermentation culture medium and additionally adding sugar alcohol calcium to meet the requirement of the molecular weight of the polyglutamic acid in the subdivision fields of fertilizer, cosmetics, sewage treatment and the likeAnd (4) requiring.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bacillus subtilis for producing gamma-polyglutamic acid and a method for regulating and controlling the molecular weight of a fermentation product gamma-polyglutamic acid.
Background
Gamma-polyglutamic acid is an anionic high polymer formed by connecting D-glutamic acid monomer and L-glutamic acid monomer through an amido bond formed by alpha-amino and gamma-carboxyl. Because of its water solubility, biodegradability, edibility, and non-toxicity to humans and the environment, gamma-polyglutamic acid and its derivatives have been widely used in various fields in recent years, as flocculants in water treatment, ingredients in cosmetics and medicines, and as fertilizer additives, etc.
At present, the commercial gamma-polyglutamic acid is mainly produced by submerged liquid fermentation of microorganisms, and the commonly used production strain bacillus subtilis generates the gamma-polyglutamic acid extracellularly by taking monosodium glutamate as a main substrate. In general, gamma-polyglutamic acid produced by Bacillus subtilis has an average molecular weight of 105～8×106The larger the molecular weight between Da and the larger the rheological property, the more difficult the modification by chemical agents, and the limited the application of gamma-polyglutamic acid. The molecular weight of the gamma-polyglutamic acid is required to be different in different industry fields, the molecular weight of the gamma-polyglutamic acid is required to be more than 1000 kDa by a flocculating agent in sewage treatment, more anions on the gamma-polyglutamic acid are used for attracting colloidal particles, and larger micelles are formed through adhesion and crosslinking so as to achieve the aim of flocculation; the molecular weight of gamma-polyglutamic acid is required to be about 700 kDa in the cosmetic industry, and the gamma-polyglutamic acid is used as a humectant in a product to reduce the loss rate of moisture on the surface of skin; some products such as fertilizer additives require that the molecular weight of the gamma-polyglutamic acid is below 80 kDa, and the gamma-polyglutamic acid is applied by being matched with common fertilizers, so that the utilization rate of the fertilizers is higher while a protective film is formed at the roots of plants.
The acid/base hydrolysis method is commonly used in industrial production to obtain low molecular weight gamma-polyglutamic acid. When the influence of pH on the structure of polyglutamic acid is studied, it is found that gamma-polyglutamic acid is a low molecular mass fragment at a low pH value, and after the acidic solution is neutralized to neutral pH, the fragment-like gamma-polyglutamic acid cannot return to a long-chain regular structure in a neutral state. Chinese patent CN 105296558A discloses a method for producing low molecular weight gamma-polyglutamic acid by adjusting and optimizing fermentation process, wherein in different fermentation stages, different culture temperatures, aeration flow rates and stirring speeds are controlled to obtain low molecular weight (35-260 kDa) gamma-polyglutamic acid fermentation liquor. Chinese patent CN 105385717A discloses an application of iron ions in Bacillus subtilis fermentation to produce low molecular weight gamma-polyglutamic acid, so that the content of gamma-polyglutamic acid higher than 245kD in the total gamma-polyglutamic acid is reduced to 27.38% -4.01% from the original 66.16%, and the content of gamma-polyglutamic acid lower than 100kD in the total gamma-polyglutamic acid is increased to 33.13% -81.03% from the original 4.59%.
At present, the method for obtaining the low molecular weight gamma-polyglutamic acid is not complicated in subsequent process and has higher loss rate of the gamma-polyglutamic acid, but is low in proportion of the low molecular weight gamma-polyglutamic acid in the total polyglutamic acid. Therefore, there is a need for a method of obtaining gamma-polyglutamic acid having various molecular weights by adjusting the process during fermentation, and also having a high yield of low molecular weight gamma-polyglutamic acid.
Disclosure of Invention
The invention aims to provide the bacillus subtilis for producing the gamma-polyglutamic acid, so that the gamma-polyglutamic acid with different molecular weights can be obtained by simple regulation and control, and the yield of the gamma-polyglutamic acid with low molecular weight is required to be higher.
The invention also aims to solve the technical problem of providing a method for regulating and controlling the molecular weight of the fermentation product gamma-polyglutamic acid by using the bacillus subtilis.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
bacillus subtilis for producing gamma-polyglutamic acid, which is classified and named as bacillus subtilis (Bacillus subtilis)Bacillus Subtilis) The strain number is XK, the strain number is preserved in China center for type culture Collection, the address is Wuhan university, Wuhan City, China, the postal code is 430072, and the preservation number is CCTCC NO: m2016605 with a preservation date of 2016 year and 11 monthsDay 1.
Most of the 16S rDNA sequence of the strain XK is 1420bp, which is shown in a nucleotide or amino acid sequence table. The sequences tested were compared from the correlations in the GenBank database to construct phylogenetic trees based on the full 16S rDNA sequence. The results show that: the homology of strain XK and Bacillus subtilis (Bacillus Subtilis) is 99%. The bacillus subtilis can be classified and positioned by combining the morphology, the cytochemical composition and the phylogenetic research result of 16S rDNA full-series analysis.
The application of the bacillus subtilis for producing the gamma-polyglutamic acid in producing the gamma-polyglutamic acid.
The application method comprises the following steps: inoculating the bacillus subtilis producing the gamma-polyglutamic acid into a seed culture medium for culture to obtain a seed solution, then inoculating the seed solution into a fermentation culture medium for aerobic culture, and fermenting to obtain the gamma-polyglutamic acid.
The method for producing the gamma-polyglutamic acid by fermenting the bacillus subtilis XK for producing the gamma-polyglutamic acid comprises the following steps:
(1) preparing a seed solution: inoculating bacillus subtilis XK into a seed culture medium, introducing oxygen into a shaking table at the rotating speed of 120-200 rpm, and culturing at 28-36 ℃ for 10-18 h until OD660 is greater than 5.0 to obtain a seed solution;
(2) fermentation culture: inoculating the seed solution obtained in the step (1) into a fermentation culture medium in an inoculation amount of 2-10% v/v, wherein the liquid loading amount of a fermentation culture medium of a 7.5L fermentation tank is 4L, the aeration ratio is controlled to be 1.0-1.5 VVM, the initial pH is 6.5-7.2, and the seed solution is cultured at 28-36 ℃ for 24-72 hours (preferably 36-72 hours) to obtain a fermentation liquid;
(3) diluting the fermentation liquor obtained in the step (2) (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography.
Wherein, the formula of the seed culture medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0.05-35 g/L of other inorganic salts, water as solvent and 6.5-7.2 of pH value.
Wherein the formula of the fermentation medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, water as a solvent, and 6.5-7.2 of pH.
Wherein, in the seed culture medium or the fermentation culture medium,
the carbon source is any one or combination of more of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses, preferably the carbon source is glucose and/or citric acid, and further preferably citric acid;
the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed cake powder, urea, (NH)4)2SO4、NH4Cl and NH4NO3Preferably the nitrogen source is bean cake powder, yeast extract and (NH)4)2SO4Any one or a combination of several of them, and more preferably bean cake powder.
The other inorganic salt is any one or combination of a plurality of phosphate, dihydric phosphate and hydrochloride, preferably dihydric phosphate, and more preferably potassium dihydrogen phosphate.
Wherein the content of the first and second substances,
when the concentration of manganese sulfate is more than or equal to 0g/L and less than 0.1 g/L and the concentration of magnesium sulfate is more than or equal to 1g/L and less than or equal to 2g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is less than 100 kDa, and the optimal yield reaches 46.5 g/L; the gamma-polyglutamic acid with the molecular weight is matched with an agricultural fertilizer for use, so that the damage of drought stress to plants is effectively relieved, the fresh weight, high chlorophyll content and relative water content of the rape can be maintained for a long time, and the yield increasing effect is obvious.
When the concentration of manganese sulfate is more than or equal to 0.1 g/L and less than or equal to 0.5g/L and the concentration of magnesium sulfate is more than or equal to 0.5g/L and less than or equal to 1g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than or equal to 100 kDa and less than or equal to 700 kDa, and the optimal yield reaches 43.8 g/L; the gamma-polyglutamic acid with the molecular weight can be applied to cosmetics and has better moisturizing performance.
When the concentration of manganese sulfate is more than or equal to 0.5g/L and less than or equal to 1g/L and the concentration of magnesium sulfate is more than or equal to 0.05 g/L and less than 0.5g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than 700 kDa, and the optimal yield reaches 39.5 g/L; the gamma-polyglutamic acid with the molecular weight is used as a flocculating agent in sewage treatment, and has good effect. In sewage treatment, the commonly used flocculating agent comprises inorganic flocculating agent (ferric salt and aluminum salt) and organic polymer flocculating agent (polyacrylamide), which have certain toxic action on the environment and human body, and the high molecular weight gamma-PGA has stronger adsorption and binding capacity on heavy metal and polyvalent cation, has biodegradability and nontoxicity, and is an environment-friendly microbial flocculating agent.
In another preferable scheme of the invention, in the aerobic fermentation process, after the OD value of the fermentation liquor rises to 10, sugar alcohol calcium is additionally added, and the addition amount is 1-3 g/L.
A method for increasing the molecular weight of gamma-polyglutamic acid as fermentation product comprises adding Bacillus subtilis (Bacillus subtilis)Bacillus Subtilis) CCTCC NO: m2016605 is firstly inoculated to a seed culture medium for culture to obtain a seed solution, and then the seed solution is inoculated to a fermentation culture medium for aerobic culture, and gamma-polyglutamic acid is obtained by fermentation; the formula of the seed culture medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0.05-35 g/L of other inorganic salts, water as a solvent and 6.5-7.2 of pH value; the formula of the fermentation medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, water as a solvent, and 6.5-7.2 of pH value; during seed culture, the culture temperature is 28-36 ℃, and the culture time is 10-18 h; during fermentation culture, the culture temperature is 28-36 ℃, the aeration ratio is controlled to be 1.0-1.5 VVM, and the culture time is 24-72 h; in the aerobic fermentation process, after the OD value of the fermentation liquor reaches 10, sugar alcohol calcium is additionally added, and the addition amount is 1-3 g/L.
Wherein the carbon source is any one or combination of more of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses; the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed cake powder, urea, (NH)4)2SO4、NH4Cl and NH4NO3Any one or a combination of several of them; the other inorganic salt is any one or combination of more of phosphate, dihydric phosphate and hydrochloride.
The inventors have discovered that the addition of calcium alditol during fermentation can increase the molecular weight of polyglutamic acid, and presumably calcium alditol acts to regulate the activity of polyglutamic acid synthase.
The sugar alcohol calcium of the present invention can be purchased from the market. The sugar alcohol calcium is generally used as a liquid fertilizer, and the sugar alcohol calcium used in the invention has a calcium content of 200g/L or more (organic chelated calcium) and a natural compound sugar alcohol content of 185g/L or more. The amount of calcium alditol added is calculated as the weight of the liquid.
The molecular weights referred to herein are weight average molecular weights.
The detection method of the OD value comprises the following steps: adding pure water or tap water to a 25mL volumetric flask for 1g of polyglutamic acid fermentation broth, uniformly mixing, adjusting to zero by using pure water or tap water as a blank control, and measuring the absorbance of the polyglutamic acid fermentation broth diluted by 25 times at the wavelength of 660nm, wherein the actual OD value = absorbance 25.
Has the advantages that:
compared with the prior art, the method takes the bacillus subtilis XK as an initial strain, obtains the gamma-polyglutamic acid with three different molecular weight regions of less than 100 kDa, 100-700 kDa and more than 700 kDa by adjusting the concentrations of manganese sulfate and magnesium sulfate in a fermentation medium, and the yield is more than 35 g/L. In addition, the molecular weight of the polyglutamic acid can be remarkably improved by additionally adding sugar alcohol calcium in the fermentation process. According to the invention, the molecular weight of the gamma-polyglutamic acid can be adjusted by adjusting the concentrations of manganese sulfate and magnesium sulfate in the fermentation medium and additionally adding sugar alcohol calcium so as to meet the requirements on the molecular weight of the polyglutamic acid in the subdivision fields of fertilizers, cosmetics, sewage treatment and the like. Meanwhile, the method is simple in process, suitable for industrial popularization, and good in market prospect and economic benefit.
Drawings
FIG. 1 is a graph showing the OD values of the system in the aerobic fermentation process in example 4.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1:
the method for producing the gamma-polyglutamic acid by fermenting the bacillus subtilis XK for producing the gamma-polyglutamic acid comprises the following specific steps:
(1) preparing a seed solution: inoculating bacillus subtilis to a seed culture medium containing sodium glutamate (45 g/L), citric acid (50 g/L), bean cake powder (40 g/L), potassium dihydrogen phosphate (1 g/L) and water, culturing at pH (6.5) and shaking table rotation speed of 120-200 rpm at 28-36 ℃ for 10-18 h to OD660Greater than 5.0;
(2) fermentation culture: inoculating the seed solution obtained in the step (1) into a fermentation medium containing sodium glutamate (45 g/L), citric acid (50 g/L), bean cake powder (40 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.05 g/L) and magnesium sulfate (1.2 g/L) in an inoculation amount of 3% v/v, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.0 vvm, the initial pH is 6.5, and the fermentation medium is cultured at 28 ℃ for 48 hours;
(3) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 50 kDa, and the yield of gamma-polyglutamic acid is 46.5 g/L.
Example 2:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 4% v/v, wherein the fermentation medium contains sodium glutamate (65 g/L), citric acid (34 g/L), bean cake powder (20 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.09 g/L) and magnesium sulfate (1.8 g/L), the liquid loading amount of a fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 6.9, and the fermentation medium is cultured at 32 ℃ for 52 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 89 kDa, and the yield of gamma-polyglutamic acid is 42.7 g/L.
Example 3:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 3% v/v, wherein the fermentation medium contains sodium glutamate (51 g/L), citric acid (29 g/L), bean cake powder (37 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.02 g/L) and magnesium sulfate (1.1 g/L), the liquid loading amount of a fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.2 vvm, the initial pH is 6.5, and the fermentation medium is cultured at 34 ℃ for 36 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 38 kDa and a yield of 36.8 g/L.
Example 4:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 2% v/v, wherein the fermentation medium contains sodium glutamate (45 g/L), citric acid (50 g/L), bean cake powder (40 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.2 g/L) and magnesium sulfate (0.8 g/L), the liquid loading amount of a fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.3vvm, the initial pH is 7.1, and the fermentation medium is cultured at 30 ℃ for 48 hours;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 560 kDa and a yield of 43.8 g/L.
Example 5:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed liquid into a fermentation medium with the inoculation amount of 2%, wherein the fermentation medium contains sodium glutamate (95 g/L), citric acid (89 g/L), bean cake powder (50 g/L), potassium dihydrogen phosphate (0.5 g/L), manganese sulfate (0.5 g/L) and magnesium sulfate (1.0 g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.3vvm, the initial pH is 7.0, and the fermentation medium is cultured at 30 ℃ for 52 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 340 kDa, and the yield of gamma-polyglutamic acid is 40.9 g/L.
Example 6:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 8% v/v, wherein the fermentation medium contains sodium glutamate (39 g/L), citric acid (30 g/L), bean cake powder (50 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.1 g/L) and magnesium sulfate (0.5 g/L), the liquid loading amount of a fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.0 vvm, the initial pH is 7.1, and the fermentation medium is cultured at 30 ℃ for 72 hours;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 190 kDa, and the yield of gamma-polyglutamic acid is 35.2 g/L.
Example 7:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (45 g/L), citric acid (50 g/L), bean cake powder (40 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.6 g/L) and magnesium sulfate (0.2 g/L) at an inoculation amount of 5% v/v, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 7.2, and the fermentation medium is cultured at 33 ℃ for 46 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 1190 kDa, and the yield of gamma-polyglutamic acid is 39.5 g/L.
Example 8:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (83 g/L), citric acid (51 g/L), bean cake powder (39 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (1.0 g/L) and magnesium sulfate (0.05 g/L) at an inoculation amount of 2% v/v, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.1 vvm, the initial pH is 7.1, and the fermentation medium is cultured at 35 ℃ for 68 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 960 kDa, and the yield of gamma-polyglutamic acid is 38.1 g/L.
Example 9:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (49 g/L), citric acid (59 g/L), bean cake powder (43 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate (0.5 g/L) and magnesium sulfate (0.1 g/L) in an inoculation amount of 5%, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 7.2, and the fermentation is carried out at 33 ℃ for 46 h;
(2) diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The molecular weight of the produced gamma-polyglutamic acid is about 780 kDa, and the yield of the gamma-polyglutamic acid is 35.4 g/L.
Example 10: utilization of gamma-polyglutamic acid in fertilizer
The experiment is set as 3 treatments with potted brassica chinensis as an experimental object, wherein the treatment 1 is not treated as a blank control, the treatment 2 is applied with a common fertilizer, and the treatment 3 is applied with a common fertilizer containing 0.2% of gamma-polyglutamic acid fermentation liquor, wherein the added gamma-polyglutamic acid fermentation liquor is the gamma-polyglutamic acid with the molecular weight of 50 kDa obtained in the example 1.
TABLE 1 Effect of different fertilizers on growth characteristics and yield of potted Brassica chinensis
The growth of the small green vegetables is obviously influenced by applying the common fertilizer, the leaf number and the particle weight of the small green vegetables can be increased, and the effect of the fertilizer can be exerted by matching the gamma-polyglutamic acid with the fertilizer, so that the yield increasing effect of the small green vegetables is more obvious.
Example 11: gamma-polyglutamic acid used as moisturizer in cosmetics
The gamma-polyglutamic acid used in this example was the gamma-polyglutamic acid having a molecular weight of 560 kDa obtained in example 4. The fermentation broth of example 4 was subjected to plate-and-frame filtration, alcohol precipitation, and vacuum drying to obtain gamma-polyglutamic acid powder, which was then compared with the water retentions of hyaluronic acid and collagen.
Anhydrous cosmetic formulation according to patent CN 105411975 a: 1-6% of gamma-polyglutamic acid, 11-15% of dispersing agent, 29-32% of stabilizing agent and 50-58% of oily matrix. Specifically, the gamma-polyglutamic acid group cosmetic uses 4% of the gamma-polyglutamic acid with the molecular weight of 560 kDa obtained in example 4 as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily substrate; the specific cosmetic for the hyaluronic acid group uses 4% of hyaluronic acid as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily matrix; the specific collagen group cosmetic uses 4% of collagen as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily matrix. The three groups of cosmetics were equally applied to 3 pieces of skin of the same size on the arms of the experimenters, respectively, and the moisture content was measured using a skin moisture tester SK-08, and the ratio of the measured moisture content to the moisture content of the skin was P% as shown in table 2:
TABLE 2 Effect of three groups of cosmetics on skin moisturizing Properties
Note: values are expressed as mean. + -. standard deviation
As can be seen from Table 2, the cosmetic composition containing 560 kDa gamma-polyglutamic acid as humectant has the best moisturizing effect on skin, which indicates that the molecular weight gamma-polyglutamic acid has the potential to replace hyaluronic acid and collagen in cosmetic aspect.
Example 12: gamma-polyglutamic acid is used as flocculant in sewage treatment
The gamma-polyglutamic acid used in this example was the 1190 kDa molecular weight gamma-polyglutamic acid obtained in example 7.
495mL of distilled water and 5mL of the gamma-polyglutamic acid fermentation broth obtained in example 7 were added to a 1L beaker, and 2.0g of calcium hydroxide was precisely weighed, poured into the beaker, adjusted to pH 7.4 while stirring for 5 minutes, and left to stand for 10 minutes. A liquid layer of half the height of the liquid was sucked up with a plastic dropper, the absorbance at 570nm was measured, the flocculation activity of the flocculant was determined with a calcium hydroxide suspension without addition of a γ -polyglutamic acid solution as a control, and the flocculation rate calculated from the measured absorbance was 64.5% in terms of the flocculation rate. The specific calculation method is shown in the reference (the university of Zhejiang university of Industrial science, vol.36, 2008, p.647-650).
Example 13:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation culture medium with the inoculation amount of 2% v/v, wherein the culture medium contains a fermentation culture medium containing sodium glutamate (45 g/L), citric acid (50 g/L), bean cake powder (40 g/L), potassium dihydrogen phosphate (1 g/L), manganese sulfate and magnesium sulfate, and culturing for 48h at 30 ℃ by shaking fermentation, introducing air into a shaking table with the initial pH of 7.1 and the rotation speed of 160 rpm;
the contents of manganese sulfate and magnesium sulfate in the fermentation medium are shown in Table 1. Each set of experiments was done in parallel for 3 and the results averaged.
(2) The fermentation liquor is diluted (the concentration is below 1 g/L), and then the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor are detected by high performance liquid chromatography, and the results are shown in table 3.
TABLE 3 correlation of the amount of salt used with the molecular weight of polyglutamic acid
Example 14
In example 4, after aerobic fermentation is carried out for 30h and the OD value reaches 10, sugar alcohol calcium 2.0 g/L is added, and fermentation is continued for 48h, thus finishing the fermentation.
Diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 781 kDa and a yield of 44.2 g/L.
Example 15
The OD value of the whole aerobic fermentation process system in example 4 is determined, and the specific value is shown in figure 1. As can be seen from FIG. 1, the OD value of the fermentation system gradually increased to 16 to 18 peaks and then decreased.
Example 16
The sugar alcohol calcium used in the examples below was purchased from Mol chemical group, Germany. The sugar alcohol calcium is added in an amount calculated by a liquid mixture containing the sugar alcohol calcium, and the sugar alcohol calcium is added after being sterilized.
2.0 g/L sugar alcohol calcium was added at different OD values in the aerobic fermentation in example 4, the fermentation was terminated after 48 hours, and the molecular weight of polyglutamic acid was measured, and the specific values are shown in Table 4. It can be seen that when the OD value reaches 10, the molecular weight of polyglutamic acid can be improved to the maximum extent by adding sugar alcohol calcium.
TABLE 4 fermentation results of polyglutamic acid after addition of sugar alcohol calcium at different OD values
Example 17
On the basis of example 4, after aerobic fermentation is carried out for 30 hours and the OD value reaches 10, sugar alcohol calcium with different concentrations is added, fermentation is continued for 48 hours, the fermentation is finished, and the molecular weight and the yield of the polyglutamic acid are detected, which is shown in Table 5.
TABLE 5 Effect of different concentrations of calcium alditol on fermentation results of polyglutamic acid
Example 18
In addition to example 1, after aerobic fermentation was carried out for 30 hours and the OD reached 10, sugar alcohol calcium 2.0 g/L was added and fermentation was continued for 48 hours, ending the fermentation.
Diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 85kDa, and the yield of gamma-polyglutamic acid is 47.1 g/L.
Example 19
In example 7, after aerobic fermentation was carried out for 30 hours and the OD reached 10, sugar alcohol calcium was added at 2.0 g/L, and fermentation was continued for 46 hours, thereby terminating the fermentation.
Diluting the fermentation liquor (the concentration is below 1 g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 1403 kDa, and the yield of the gamma-polyglutamic acid is 39.9 g/L.
Example 20
On the basis of example 4, when aerobic fermentation is carried out for 30 hours and the OD value reaches 10, calcium chloride is added, the addition amount is 0.4g/L calculated by calcium ions, the fermentation is continued for 48 hours, the fermentation is ended, the molecular weight and the yield of the polyglutamic acid are detected, the molecular weight of the produced gamma-polyglutamic acid is about 581kDa, and the yield of the gamma-polyglutamic acid is 44.0 g/L. This indicates that the increase in the molecular weight of polyglutamic acid of the present invention is not simple because of the addition of calcium ions, and it is presumed that sugar alcohol and chelated calcium synergistically affect the synthesis of polyglutamic acid.
Sequence listing
<110> Nanjing Xuan Kai Biotech Co., Ltd
<120> bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid
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tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240
ccgacctgagagggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420
gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200
caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380
cccgaagtcg gtgaggtaac cttttaggag ccagccgccg 1420
Claims (9)
1. Bacillus subtilis for producing gamma-polyglutamic acid, which is classified and named as bacillus subtilis (Bacillus subtilis)Bacillus Subtilis) The culture medium is preserved in China center for type culture Collection with the preservation number of CCTCC NO: m2016605, preservation date 2016, 11/1/11/2016.
2. Method for improving molecular weight of fermentation product gamma-polyglutamic acidThe method is characterized in that Bacillus subtilis (B) is addedBacillus Subtilis) CCTCC NO: inoculating the seed solution of M2016605 into a fermentation culture medium for aerobic culture, and fermenting to obtain gamma-polyglutamic acid; the formula of the fermentation medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, water as a solvent, and 6.5-7.2 of pH value; during fermentation culture, the culture temperature is 28-36 ℃, the aeration ratio is controlled to be 1.0-1.5 VVM, and the culture time is 24-72 h; in the aerobic fermentation process, after the OD value of the fermentation liquor reaches 10, sugar alcohol calcium is additionally added, and the addition amount is 1-3 g/L.
3. The method according to claim 2, wherein the carbon source is any one or a combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses; the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed cake powder, urea, (NH)4)2SO4、NH4Cl and NH4NO3Any one or a combination of several of them; the other inorganic salt is any one or combination of more of phosphate, dihydric phosphate and hydrochloride.
4. Use of the Bacillus subtilis for producing gamma-polyglutamic acid according to claim 1 for producing gamma-polyglutamic acid.
5. The application of claim 4, wherein the application method comprises: inoculating bacillus subtilis for producing gamma-polyglutamic acid into a seed culture medium for culture to obtain a seed solution, inoculating the seed solution into a fermentation culture medium for aerobic culture, and fermenting to obtain gamma-polyglutamic acid; the formula of the seed culture medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0.05-35 g/L of other inorganic salts, water as a solvent and 6.5-7.2 of pH value; the formula of the fermentation medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, water as a solvent, and 6.5-7.2 of pH value.
6. The use of claim 5, wherein the carbon source is any one or a combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses; the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed cake powder, urea, (NH)4)2SO4、NH4Cl and NH4NO3Any one or a combination of several of them; the other inorganic salt is any one or combination of more of phosphate, dihydric phosphate and hydrochloride.
7. The use according to claim 5, wherein the fermentation medium is formulated as a fermentation medium,
when the concentration of manganese sulfate is more than or equal to 0g/L and less than 0.1 g/L and the concentration of magnesium sulfate is more than or equal to 1g/L and less than or equal to 2g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is less than 100 kDa;
when the concentration of manganese sulfate is more than or equal to 0.1 g/L and less than or equal to 0.5g/L and the concentration of magnesium sulfate is more than or equal to 0.5g/L and less than or equal to 1g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than or equal to 100 kDa and less than or equal to 700 kDa;
when the concentration of manganese sulfate is more than or equal to 0.5g/L and less than or equal to 1g/L and the concentration of magnesium sulfate is more than or equal to 0.05 g/L and less than 0.5g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than 700 kDa.
8. The use of claim 5, wherein the seed culture temperature is 28-36 ℃ and the culture time is 10-18 h; during fermentation culture, the culture temperature is 28-36 ℃, the aeration ratio is controlled to be 1.0-1.5 VVM, and the culture time is 24-72 h.
9. The use of any one of claims 4 to 8, wherein in the aerobic fermentation process, after the OD value of the fermentation liquid reaches 10, sugar alcohol calcium is additionally added, and the addition amount is 1-3 g/L.
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