CN114457136A - Fermentation liquid based on rose fermentation recombinant collagen and application thereof - Google Patents

Abstract

The invention discloses a fermentation liquid based on rose fermented recombinant collagen, comprising the following components in mass percentage: rose pollen 10%-30%, BSM medium 70%-90%. The invention also discloses the application of the fermentation liquid, including the application in the fermentation production of collagen, the application in the fermentation production of a fermentation product with the ability to promote cell proliferation and growth, and the fermentation production of a fermentation product with the functions of repairing skin damage and anti-aging applications in etc.

Description

A kind of fermentation broth based on rose fermented recombinant collagen and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a fermentation liquid based on rose fermented recombinant collagen and its application.

Background technique

Fermentation is a process in which microorganisms produce microorganisms themselves, or direct metabolites or secondary metabolites by the life activities of microorganisms under aerobic or anaerobic conditions. Fermentation originated from the process of food spoilage, and later humans used food to ferment various flavored foods. Modern fermentation technology has developed into an engineering discipline and independent industry. The technology of directional fermentation with natural plants or food is constantly developing. For example, the enzyme food fermentation technology is a technology of compound fermentation of fruits and vegetables such as lactic acid bacteria and yeast to form liquid or solid food rich in lipase, amylase, protease, lactic acid, acetic acid and other metabolites. Plants can usually provide specific nutrients for bacterial growth during fermentation, such as inorganic salts, vitamins, and can also induce microorganisms to produce special metabolites.

As a natural plant raw material, rose has been used in beauty and skin care since ancient times. In addition to volatile oil, rose essential oil, rose also contains many non-volatile chemical components, mainly flavonoids. These secondary metabolites have many This kind of physiological activity is often used in cosmetics, food industry and medicine. Rose petals are rich in anthocyanins, flavonoids, volatile oils, polyphenols, as well as a variety of vitamins, amino acids, trace elements, alkaloids, etc., and have high edible and medicinal value. Roses have a long history in my country, and are the same flower as medicine and food in my country. Their flowers have been widely used in food, wine making, tea making, beauty, cosmetics and traditional Chinese medicine. Products such as rose enzyme fermentation and rose honey brewing have been widely recognized by the market.

Pichia pastoris as an engineering strain can be genetically modified to produce specific metabolites, and recombinant human collagen has been widely obtained in Pichia pastoris engineering bacteria. However, the fermentation of the existing engineering bacteria usually uses expensive nutrients such as yeast powder and peptone, and the fermentation broth after production has a strong smell and is difficult to use directly. Usually, it is removed after fermentation, causing waste and environmental pollution.

The organic components in roses provide the nutrients needed for the growth of recombinant humanized collagen engineering strains to achieve biological fermentation, and the results will provide important application value for technological innovation in the fields of beauty, food or medicine. In the present invention, we name the fermentation broth product as "rose collagen fermentation broth".

SUMMARY OF THE INVENTION

Aiming at the above bottleneck problem, the present invention uses natural plant raw materials as the nutrient components of the culture medium to provide nutrients for the strain during the fermentation process, and can also produce some special metabolites. Maximize benefits and be green. Specifically: by adding the rose flower extract into the culture medium system of Pichia pastoris fermentation, the rose flower is innovatively used as one of the nutritional components for the fermentation of recombinant collagen engineering bacteria, and the components in the infusion liquid delay the engineering bacteria. The aging not only helps the growth of yeast engineering bacteria, but also obtains higher collagen expression. And the fermentation product shows excellent anti-oxidation, cell proliferation and growth promotion effect, has a good regulating effect on various skin anti-aging genes, and the fermentation product has a positive promoting effect on the overall anti-aging of the skin. Therefore, the first object of the present invention is to provide a fermentation broth based on rose fermented recombinant collagen. The second object of the present invention is to provide the application of the fermentation broth.

To achieve the above object, the present invention adopts the following technical solutions:

As the first aspect of the present invention, a fermentation broth based on rose fermented recombinant collagen comprises the following components by mass percentage: rose pollen 10%-30%, BSM medium 70%-90%.

According to the present invention, the above-mentioned fermentation broth based on rose fermented recombinant collagen is composed of the following components by mass percentage: 20% of rose pollen and 80% of BSM medium.

As a second aspect of the present invention, an application of the above-mentioned fermentation broth based on rose fermented recombinant collagen in fermentation production of collagen.

As a third aspect of the present invention, the above-mentioned fermentation broth based on rose fermented recombinant collagen is used in the fermentation production of a fermentation product capable of promoting cell proliferation and growth.

As the fourth aspect of the present invention, the above-mentioned fermentation broth based on rose fermented recombinant collagen is used in the fermentation production of a fermented product with skin damage repair and anti-aging properties.

As a fifth aspect of the present invention, an application of the above-mentioned fermentation broth based on rose fermented recombinant collagen in the fermentation production of a fermented product that promotes the expression of moisturizing genes.

According to the present invention, the moisturizing genes include the genes AQP-3, CD44, and HAS1.

Beneficial effects of the present invention:

1. The fermentation broth does not need further purification, and the fermentation product can be directly applied to the skin surface, which is economical and environmentally friendly, and has significant effects on skin damage repair and anti-aging;

2. The fermentation broth of the present invention is used for fermentation, and its fermentation product has the effect of promoting cell proliferation and growth;

3. The fermentation broth of the present invention can be used to increase the expression of collagen.

Description of drawings

Figure 1 shows the expression of collagen in rose fermented culture medium containing different concentrations of rose pollen for 30 hours.

Fig. 2 is the expression level of collagen in the medium containing 20% rose pollen.

FIG. 3 is the expression level of induced collagen in the fermenter of Example 3. FIG.

FIG. 4 is the density of yeast in the fermenter of Example 3. FIG.

Figure 5 shows the effect of rose collagen on cell proliferation. in,

*p<0.05; **P<0.01; ***P<0.001; ****P<0.0001;

Note: * indicates that the sample has a significant difference compared with the blank treatment group under the action of this concentration.

Figures 6-8 are the effects of rose collagen on the expression of AQP-3 gene, CD44 gene and HAS-1 gene, respectively. in,

*p<0.05; **P<0.01; ***P<0.001;

Note: * indicates that the sample has a significant difference compared with the blank treatment group under the action of this concentration.

Detailed ways

The present invention will be further explained below in conjunction with specific embodiments. The following examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the following examples are usually carried out in accordance with conventional conditions or conditions provided by manufacturers.

The recombinant human type III collagen Pichia pastoris engineering strain (GS115RHC04) of the following examples can be purchased from Merjian Biotechnology Co., Ltd.

The preparation of embodiment 1 rose fermented culture liquid

Roses are selected from Pingyin, Shandong, where the roses are grown, and the dried roses are crushed into powder with a crusher. According to the following components, it was prepared into a rose-based fermentation broth (Rose Based Mineral-Salt Medium, RMSM). The formula of rose fermentation broth is shown in Table 1, in mass percentage. When in use, the nutrients in the above formula are mixed and sterilized, but the metal inorganic salts need to be sterilized separately and then mixed aseptically at room temperature.

The formula of table 1 rose flower fermentation broth

Example 2 Shake flask fermentation of rose collagen

Pick the engineered strain of recombinant human type III collagen Pichia pastoris, inoculate it into 25ml BMGY medium, 250ml shake flask, shake at 30°C, 250rpm to _OD600 =4, centrifuge at 3000g at room temperature for 5min, collect the cells, remove the supernatant, The cells were resuspended to OD ₆₀₀ = 1.0 with different concentrations of RMSM medium in Table 1, and expression was induced to obtain a culture. The above culture was added to a 1L shake flask, covered with two layers of sterile gauze, and put into a shaker to continue growing. Every 24 hours, methanol was added to a final concentration of 0.5% to continue the induction. At various time points, 1 ml of medium was taken into a 1.5 ml centrifuge tube. These samples were used to analyze expression levels and to determine the optimal time to harvest cells after induction. Centrifuge at room temperature with a horizontal centrifuge at maximum speed for 2-3 min. For secretory expression, transfer the supernatant to a separate tube and store the supernatant and cell pellet at -80°C until assay begins. Coomassie blue staining was used, and supernatant protein expression was analyzed by SDS-PAGE. After analysis, recombinant human collagen can be expressed normally in rose-based medium, and the expression level increases with the increase of the proportion of rose pollen, but the expression level begins to decline after more than 30%, as shown in Figure 1. It may be caused by the unbalanced microbial nutrition caused by the unbalanced proportion of the remaining inorganic salts in BSM. Therefore, we selected a medium with 20% rose pollen content for cultivation, and the expression level continued to increase with the increase of time. After 30 hours of fermentation, SDS-PAGE electrophoresis staining showed that the yield reached about 2 g/L, as shown in Figure 2 .

Subsequent experiments used rose fermented broth with 20% rose pollen content.

Example 3 Large-scale fermentor fermentation of collagen

The cryopreserved recombinant human type III collagen Pichia strain was inoculated into the sterilized and cooled YPD medium, placed in a shaker for activation and culture for 28 to 30 hours, so that the OD ₆₀₀ value was between 3 and 5. between. The activated strain was inoculated into a 10L glass fermenter for expansion and culture for 6-8h. The amplified seed liquid was transferred to a stainless steel fermenter containing 150L RMSM medium at a volume ratio of 1:10 (seed liquid:fermented liquid) for 16-18 hours, and the control experiment was transferred to BSM inorganic salt medium . In the fermenter, the medium is supplemented by fed-feed mode, and the culture is continued for 3-4 hours, so that the wet weight of the bacteria reaches 150-180 g/L. Methanol was gradually added, and the methanol concentration was maintained at 0.5%, and the cells were induced and cultured for 21-24 h. The induced fermentation broth was centrifuged at high speed to separate the bacterial cells and the fermentation supernatant, and the fermentation supernatant was collected. The SDS-PAGE electrophoresis showed that the production of collagen in the fermenter was significantly increased, which was mainly due to the increase of bacterial density. The expression reached 4 g/L in RMSM and about 3 g/L in BSM medium. Rose-based medium significantly increased collagen expression. The results are shown in Figure 3. The cell density OD600 was measured with a spectrophotometer, and the results are shown in Figure 4. The results showed that the growth rate of RMSM was lower than that of BSM during the growth of bacteria, but at the end of fermentation, the density of bacteria in the two mediums was similar. Therefore, RMSM inhibited the rapid growth of bacteria and slowed down the aging of bacteria.

Example 4 Promotion of Cell Proliferation

The rose collagen fermentation broth obtained in Example 3 was freeze-dried into powder in a freeze dryer, and the powder was quantified to prepare solutions of different concentrations. 3T3 cells (purchased from the laboratory of University of Science and Technology of China) in good growth state were taken and cultured in 96-well plates for 17 hours, then replaced with serum-free basal medium and cultured for 7 hours. OD450nm was detected by CCK8 method, and the effect of the tested sample on the proliferation of 3T3 cells was obtained. See Figure 5.

The results showed that rose collagen fermentation broth could significantly promote cell proliferation, reaching the maximum value at 0.08 mg/mL, and the effect on cell proliferation was comparable to that of 10% biological FBS. It shows that in addition to collagen, the fermentation product of rose collagen may also produce a variety of substances that promote cell growth.

The effect of embodiment 5 on the expression of cellular moisturizing related genes

The rose collagen fermentation broth obtained in Example 3 was freeze-dried into powder in a freeze dryer, and the powder was quantified to prepare solutions of different concentrations with complete medium. The hacat cells were plated in 6-well plates and cultured for 24 hours, and then replaced with samples to be tested with different concentrations. Total RNA was extracted with TRIzon at 24 hours, and the RNA purity and concentration were detected by NanoDrop. Then cDNA was synthesized and amplified by PCR, and the target gene band was confirmed by agarose gel electrophoresis analysis. Finally, the cDNA was quantitatively determined by Real-Time PCR, and the effect of rose collagen on the expression of moisturizing-related genes in HACAT cells was obtained.

In this example, three genes, aquaporin-3 (AQP-3), cell surface glycoprotein (CD44) and hyaluronan synthase 1 (HAS 1), were determined, and the expressions of these three genes were all positively correlated with skin moisturizing function. Correlation, its expression reflects the effect on skin moisturizing function to a certain extent. When the sample concentration is 0.05mg/ml, the expression levels of the moisturizing-related genes AQP-3, CD44, and HAS1 in hacat cells are 94%, 104%, and 221% of the blank group, respectively; The expression levels of cell moisturizing related genes AQP-3, CD44, and HAS1 were 120%, 125% and 320% of the blank group, respectively, and the results showed a promoting effect. The results are shown in Figures 6, 7 and 8.

The above are only examples of the embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the technical principles of the present invention, several improvements and modifications can be made. These improvements and Variations should also be considered within the scope of protection of the present invention.

Claims (7)

1. A fermentation broth based on rose fermented recombinant collagen, characterized in that it comprises the following components by mass percentage: rose pollen 10%-30%, BSM medium 70%-90%. 2 . The fermentation broth of claim 1 , wherein the fermentation broth is composed of the following components by mass percentage: 20% of rose pollen and 80% of BSM medium. 3 . 3. the application of the fermentation broth based on rose fermented recombinant collagen as claimed in claim 1 or 2 in the fermentation production of collagen. 4. The application of the fermentation broth based on rose fermented recombinant collagen as claimed in claim 1 or 2 in the fermentative production of a fermented product that promotes cell proliferation and growth. 5 . The application of a fermentation broth based on rose fermented recombinant collagen as claimed in claim 1 or 2 in fermentation production of a fermented product with skin damage repair and anti-aging. 6 . 6 . The application of a fermentation broth based on rose fermented recombinant collagen as claimed in claim 1 or 2 in the fermentation production of a fermented product that promotes the expression of moisturizing genes. 7 . 7. The use of claim 6, wherein the moisturizing genes include genes AQP-3, CD44, and HAS1.

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