阅读说明：本技术 一种γ-聚谷氨酸钠溶液制备方法 (Preparation method of gamma-polyglutamic acid sodium solution ) 是由 敖德平 于 2019-10-30 设计创作，主要内容包括：本发明公开了一种γ-聚谷氨酸钠溶液制备方法,包括：取地衣芽胞杆菌原始菌株接种于LB固体培养基上,制备活化后的单菌落；将活化的单菌落接种于种子培养基中,摇床转速220rpm培养至对数生长期,得到聚谷氨酸钠种子液；按照发酵培养基体积计,将种子液以1.5％的接种量接种于发酵培养基中培养24-66.5h；在发酵至0-24h时,开始补加流加硝酸钠溶液至补加的硝酸钠的总浓度至10g/L；所述发酵培养基的组分为：蔗糖25g/L,酵母膏5g/L,谷氨酸钠40g/L,装液量70mL/250mL锥形瓶,起始pH6.5,菌种在37℃、120r/min培养24h后,于培养基中添加5％NaCl,继续培养24h,得到发酵液γ-PGA,产物水解后,初步确定产物为γ-聚谷氨酸钠,γ-聚谷氨酸钠的含量达到101.2克/L；将反应容器进行遮光处理,得到γ-聚谷氨酸钠含量为10％的溶液。(The invention discloses a preparation method of a gamma-polyglutamic acid sodium solution, which comprises the following steps: inoculating an original strain of bacillus licheniformis on an LB solid culture medium to prepare an activated single colony; inoculating the activated single colony in a seed culture medium, and culturing at a table concentrator rotating speed of 220rpm to a logarithmic phase to obtain a poly-sodium glutamate seed solution; inoculating the seed liquid into a fermentation culture medium according to the volume of the fermentation culture medium by the inoculation amount of 1.5 percent for culturing for 24-66.5 h; when the fermentation time is 0-24h, beginning to supplement and flow the sodium nitrate solution until the total concentration of the supplemented sodium nitrate reaches 10 g/L; the fermentation medium comprises the following components: 25g/L of sucrose, 5g/L of yeast extract, 40g/L of sodium glutamate, 70mL/250mL of liquid loading amount in a conical flask, the initial pH value is 6.5, 5% of NaCl is added into a culture medium after a strain is cultured for 24 hours at 37 ℃ at 120r/min, the culture medium is continuously cultured for 24 hours to obtain fermentation liquor gamma-PGA, and after the product is hydrolyzed, the product is preliminarily determined to be gamma-polyglutamic acid sodium, and the content of the gamma-polyglutamic acid sodium reaches 101.2 g/L; and shading the reaction container to obtain a solution with the content of the gamma-polyglutamic acid sodium being 10%.)

1. A preparation method of a gamma-polyglutamic acid sodium solution is characterized by comprising the following steps:

inoculating an original strain of bacillus licheniformis on an LB solid culture medium, culturing at 37 ℃ for 12-18h, and preparing an activated single colony;

inoculating the activated single colony in a seed culture medium, and culturing at 37 ℃ and a shaking table rotating speed of 220rpm to a logarithmic phase to obtain a poly-sodium glutamate seed solution;

according to the volume of the fermentation culture medium, inoculating the seed liquid into the fermentation culture medium in an inoculation amount of 1.5 percent for culturing for 24-66.5h, wherein the fermentation temperature is 28-40 ℃, the ventilation volume is 0.5-1.5vvm, and the stirring speed is 300-700 rpm; when the fermentation time is 0-24h, beginning to supplement and flow the sodium nitrate solution until the total concentration of the supplemented sodium nitrate reaches 10 g/L;

the fermentation medium comprises the following components: 25g/L of sucrose, 5g/L of yeast extract, 40g/L of sodium glutamate, 70mL/250mL of liquid loading amount in a conical flask, the initial pH value of the conical flask is 6.5, 5% of NaCl is added into a culture medium after a strain is cultured for 24 hours at 37 ℃ at 120r/min, the culture is continued for 24 hours to obtain fermentation liquid gamma-PGA, and after the product is hydrolyzed, the product is preliminarily determined to be gamma-polyglutamic acid sodium through liquid chromatography inspection, and the content of the gamma-polyglutamic acid sodium reaches 101.2 g/L;

and shading the reaction container to obtain a solution with the content of the gamma-polyglutamic acid sodium being 10%.

Technical Field

The invention relates to the technical field of production of gamma-polyglutamic acid, in particular to a preparation method of a gamma-polyglutamic acid sodium solution.

Background

Gamma-polyglutamic acid (gamma-PGA), also called natto gum and polyglutamic acid, is a water-soluble, biodegradable biological polymer prepared by using a microbial fermentation method. Gamma-polyglutamic acid is a viscous substance and is found for the first time in fermented soybeans of "natto". The gamma-PGA polyglutamic acid is a special anionic natural polymer, and is a homo-polyamide (homo-polyamide) gamma-PGA formed by amide bonding (amidinkage) between alpha-amino (alpha-ami-no) and gamma-carboxyl (gamma-caboxyyl), the molecular weight of the homo-polyamide (homo-polyamide) gamma-PGA is different from 5 ten thousand to 2 million daltons, and the moisturizing and water locking effects of the gamma-PGA polyglutamic acid are 500 times of those of hyaluronic acid.

Due to the excellent film forming property of the gamma-PGA, the skin care product added with the gamma-PGA can form a layer of breathable biological protection film on the surface of skin, can resist external invasion and ultraviolet rays while locking skin moisture, and shows excellent effects in anti-haze and sunscreen products. The gamma-PGA can increase the content of natural moisturizing factors in the skin stratum corneum, promote the growth of fibroblasts in the deep layer of the skin, balance the pH value of the skin, restore the elasticity of the skin and smooth fine wrinkles of the skin caused by water shortage. Comparison of the effects of polyglutamic acid, hyaluronic acid, and collagen on skin.

Disclosure of Invention

Based on the above, the invention aims to provide a preparation method of a gamma-polyglutamic acid sodium solution.

The preparation method of the gamma-polyglutamic acid sodium solution comprises the following steps:

inoculating an original strain of bacillus licheniformis on an LB solid culture medium, culturing at 37 ℃ for 12-18h, and preparing an activated single colony;

inoculating the activated single colony in a seed culture medium, and culturing at 37 ℃ and a shaking table rotating speed of 220rpm to a logarithmic phase to obtain a poly-sodium glutamate seed solution;

according to the volume of the fermentation culture medium, inoculating the seed liquid into the fermentation culture medium in an inoculation amount of 1.5 percent for culturing for 24-66.5h, wherein the fermentation temperature is 28-40 ℃, the ventilation volume is 0.5-1.5vvm, and the stirring speed is 300-700 rpm; when the fermentation time is 0-24h, beginning to supplement and flow the sodium nitrate solution until the total concentration of the supplemented sodium nitrate reaches 10 g/L;

the fermentation medium comprises the following components: 25g/L of sucrose, 5g/L of yeast extract, 40g/L of sodium glutamate, 70mL/250mL of liquid loading amount in a conical flask, the initial pH value of the conical flask is 6.5, 5% of NaCl is added into a culture medium after a strain is cultured for 24 hours at 37 ℃ at 120r/min, the culture is continued for 24 hours to obtain fermentation liquid gamma-PGA, and after the product is hydrolyzed, the product is preliminarily determined to be gamma-polyglutamic acid sodium through liquid chromatography inspection, and the content of the gamma-polyglutamic acid sodium reaches 101.2 g/L;

and shading the reaction container to obtain a solution with the content of the gamma-polyglutamic acid sodium being 10%.

Compared with the prior art, the preparation method of the gamma-polyglutamic acid sodium solution has the beneficial effects that:

(1) according to the preparation method of the gamma-polyglutamic acid sodium solution, sodium nitrate is not added into a culture medium at the initial fermentation stage, and the sodium nitrate solution is supplemented by adopting a proper fed-batch strategy in the fermentation process, so that the rapid accumulation of nitrite in the fermentation process is avoided, the normal glycerol metabolism of bacillus licheniformis is ensured, and the synthesis of the polyglutamic acid sodium is promoted.

(2) The preparation method of the gamma-polyglutamic acid sodium solution uses sodium nitrate to partially replace glutamic acid, thereby reducing the fermentation cost.

(3) The preparation method of the gamma-polyglutamic acid sodium solution supplements the sodium nitrate solution, controls the accumulation of nitrite in the fermentation liquor, promotes the bacillus licheniformis to utilize glycerol to produce the polyglutamic acid sodium, and provides a new solution strategy for the fermentation problem that the microorganism metabolizes the nitrate to produce nitrite.

For a better understanding and practice, the invention is described in detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a graph comparing the moisturizing ability of a solution of sodium gamma-polyglutamate.

Fig. 2 is a graph comparing skin moisture content using a sodium gamma-polyglutamate solution, propylene glycol and hyaluronic acid.

FIG. 3 is a graph comparing the effects of gamma-polyglutamic acid, HA, collagen on promoting NMF absorption in skin.

Detailed Description

The implementations described in the exemplary embodiments below are not intended to represent all implementations consistent with the present disclosure. Rather, they are merely examples of methods consistent with certain aspects of the present disclosure, as detailed in the appended claims.

The terminology used in the present disclosure is for the purpose of describing particular embodiments only and is not intended to be limiting of the disclosure. As used in this disclosure and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any and all possible combinations of one or more of the associated listed items.

The preparation method of the gamma-polyglutamic acid sodium solution comprises the following steps:

inoculating an original strain of bacillus licheniformis on an LB solid culture medium, culturing at 37 ℃ for 12-18h, and preparing an activated single colony;

inoculating the activated single colony in a seed culture medium, and culturing at 37 ℃ and a shaking table rotating speed of 220rpm to a logarithmic phase to obtain a poly-sodium glutamate seed solution;

according to the volume of the fermentation culture medium, inoculating the seed liquid into the fermentation culture medium in an inoculation amount of 1.5 percent for culturing for 24-66.5h, wherein the fermentation temperature is 28-40 ℃, the ventilation volume is 0.5-1.5vvm, and the stirring speed is 300-700 rpm; when the fermentation time is 0-24h, beginning to supplement and flow the sodium nitrate solution until the total concentration of the supplemented sodium nitrate reaches 10 g/L;

the fermentation medium comprises the following components: 25g/L of sucrose, 5g/L of yeast extract, 40g/L of sodium glutamate, 70mL/250mL of liquid loading amount in a conical flask, the initial pH value of the conical flask is 6.5, 5% of NaCl is added into a culture medium after a strain is cultured for 24 hours at 37 ℃ at 120r/min, the culture is continued for 24 hours to obtain fermentation liquid gamma-PGA, and after the product is hydrolyzed, the product is preliminarily determined to be gamma-polyglutamic acid sodium through liquid chromatography inspection, and the content of the gamma-polyglutamic acid sodium reaches 101.2 g/L;

and shading the reaction container to obtain a solution with the content of the gamma-polyglutamic acid sodium being 10%.

Compared with the prior art, the preparation method of the gamma-polyglutamic acid sodium solution has the beneficial effects that:

(1) according to the preparation method of the gamma-polyglutamic acid sodium solution, sodium nitrate is not added into a culture medium at the initial fermentation stage, and the sodium nitrate solution is supplemented by adopting a proper fed-batch strategy in the fermentation process, so that the rapid accumulation of nitrite in the fermentation process is avoided, the normal glycerol metabolism of bacillus licheniformis is ensured, and the synthesis of the polyglutamic acid sodium is promoted.

(2) The preparation method of the gamma-polyglutamic acid sodium solution uses sodium nitrate to partially replace glutamic acid, thereby reducing the fermentation cost.

(3) The preparation method of the gamma-polyglutamic acid sodium solution supplements the sodium nitrate solution, controls the accumulation of nitrite in the fermentation liquor, promotes the bacillus licheniformis to utilize glycerol to produce the polyglutamic acid sodium, and provides a new solution strategy for the fermentation problem that the microorganism metabolizes the nitrate to produce nitrite.

The advantages of the gamma-polyglutamic acid sodium solution are as follows:

1. compared with powder gamma-polyglutamic acid, the powder is easier to prevent corrosion.

2. No ion, good compatibility and no influence on the system.

3. Has the advantages of high price and high cost performance.

1. The application effect of the gamma-polyglutamic acid sodium solution in the skin care product.

The gamma-polyglutamic acid sodium solution can play a good role in moisturizing the epidermis layer and the dermis layer of the skin.

Please refer to fig. 1 and fig. 2. FIG. 1 is a graph comparing the moisturizing ability of a solution of sodium gamma-polyglutamate. Fig. 2 is a graph comparing skin moisture content using a sodium gamma-polyglutamate solution, propylene glycol and hyaluronic acid.

As shown in the comparison in FIG. 1, the moisturizing ability of the skin care product added with the gamma-polyglutamic acid sodium solution is obviously better than that of the skin care product without the gamma-polyglutamic acid sodium solution. As can be seen from FIG. 2, the water content of the skin after the application of the gamma-polyglutamic acid sodium solution is obviously better than that of propylene glycol and hyaluronic acid, and the moisturizing time is long.

Referring to fig. 3, fig. 3 is a graph comparing the effect of gamma-polyglutamic acid, HA, and collagen on promoting absorption of NMF in skin.

Gamma-polyglutamic acid is the only known effective component capable of inducing the self-increase of the natural moisturizing factor NMF, and can increase the natural moisturizing factor NMF in the skin by about 130 percent. The purpose of supplementing water from inside to outside is achieved by promoting the growth of fibroblasts in keratinocytes and increasing the content of natural moisturizing factors for growth.

The free alpha-COOH, a-CO group and a-NH-group of each unit body of the gamma-polyglutamic acid can adsorb positively charged nutrient components to create an excellent embedding and slow-release system, so that the active components in the cosmetics can slowly play the functions.

In addition, due to the excellent film forming property of the gamma-polyglutamic acid sodium solution, the skin care product added with the gamma-polyglutamic acid sodium solution can form a layer of breathable biological protection film on the surface of skin, can resist external invasion and ultraviolet rays while locking skin moisture, and shows excellent effects in anti-haze and sunscreen products. The gamma-polyglutamic acid sodium solution can increase the content of natural moisturizing factors in the horny layer of the skin, promote the growth of fibroblasts in the deep layer of the skin, balance the pH value of the skin, restore the elasticity of the skin and smooth fine wrinkles generated by water shortage of the skin.

2. The application effect of the gamma-polyglutamic acid sodium solution in oral care products.

When the gamma-polyglutamic acid sodium solution is added into an oral care product, the foam fineness can be increased, a protective film is formed on the surfaces of the oral cavity and the teeth, the tooth enamel and the oral mucosa are repaired, and the damage of the grinding agent to the teeth is reduced; meanwhile, the film formed by the gamma-polyglutamic acid can help teeth resist the invasion of external pollutants and germs and inhibit the formation of decayed teeth.

3. The application effect of the gamma-polyglutamic acid sodium solution in daily chemical washing products.

The gamma-polyglutamic acid sodium solution can be added into a washing agent and a softener of clothes to bring soft hand feeling to the fabrics, and meanwhile, because the gamma-polyglutamic acid sodium solution has extremely strong water absorption and moisture retention capacity, the gamma-polyglutamic acid sodium solution can be attached to fabric fibers to form a film to block pollution and adsorb sweat effectively, so that the clothes are more fresh and comfortable to wear, and the occurrence of rash and eczema caused by fabric stimulation is an essential component in finishing agents of babies and functional clothes.

4. The application effect of the gamma-polyglutamic acid sodium solution in the color cosmetic product.

Due to the superior water absorption and moisture retention, film forming property and biodegradability of the gamma-sodium polyglutamate solution, the characteristics of the product can be obviously improved when the gamma-sodium polyglutamate solution is added into a color cosmetic product in a proper amount, and the color cosmetic product with the innovative effects of green, nature and safety can be created. For example, the lipstick and the base powder product added with the gamma-polyglutamic acid sodium solution can form a protective film on the surface of the skin, obviously improve the dryness and crack of the lips and the dry state of the skin, prevent environmental pollution and bring moist and natural makeup.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.