A fermentation liquid based on rose fermentation recombinant collagen and its application

Abstract

The present invention discloses a fermentation broth based on rose fermentation recombinant collagen, comprising the following components in percentage by mass: 10%-30% rose pollen and 70%-90% BSM culture medium. The present invention also discloses the application of the fermentation broth, including the application in the fermentation production of collagen, the application in the fermentation production of a fermentation product having the function of promoting cell proliferation and growth, the application in the fermentation production of a fermentation product having the function of repairing skin damage and anti-aging, etc.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a fermentation liquid based on rose fermentation recombinant collagen and an application thereof. Background Art

[0002] Fermentation is the process of preparing microbial cells, direct metabolites or secondary metabolites through the life activities of microorganisms under aerobic or anaerobic conditions. Fermentation originated from the food spoilage process, and later humans used food to ferment various flavored foods. Modern fermentation technology has developed into an engineering discipline and an independent industry. Directed fermentation technology based on natural plants or food is constantly developing. For example, enzyme food fermentation technology is a technology that uses lactic acid bacteria, yeast and other composite fermentation of fruits and vegetables to form liquid or solid foods rich in metabolites such as lipase, amylase, protease, lactic acid, acetic acid, etc. Plants can usually provide specific nutrients for bacterial growth during the fermentation process, such as inorganic salts and vitamins, and can also induce microorganisms to produce special metabolites.

[0003] Rose, as a natural plant raw material, has been used for beauty and skin care since ancient times. In addition to volatile oils, namely rose essential oils, roses also contain many non-volatile chemical components, mainly flavonoids. These secondary metabolites have a variety of physiological activities and are 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 a flower that is both medicinal and edible in my country. Its fresh flowers have been widely used in food, wine brewing, tea making, beauty, cosmetics and traditional Chinese medicine. Products such as rose enzyme fermentation and rose honey winemaking have been widely recognized by the market.

[0004] Pichia pastoris is an engineered strain that can produce specific metabolites after genetic modification. Recombinant human collagen has been widely obtained in Pichia pastoris engineered bacteria. However, the fermentation of existing engineered bacteria usually uses expensive nutrients such as yeast powder and peptone, and the fermentation liquid has a strong odor after production, which is difficult to use directly. It is usually removed after fermentation, causing waste and environmental pollution.

[0005] The organic components in roses provide the nutrients required for the growth of recombinant humanized collagen engineering strains to achieve biological fermentation. This achievement 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 "rose collagen fermentation broth". Summary of the invention

[0006] The present invention aims at the above bottleneck problems, 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 certain special metabolites. The culture solution after fermentation can be used as the final product, realizing maximum economic benefits and being green and environmentally friendly. Specifically, by adding rose extract to the culture medium system of Pichia yeast fermentation, rose is innovatively used as one of the nutrient components for the fermentation of recombinant collagen engineering bacteria, and the components in the immersion liquid delay the aging of the engineering bacteria, which not only helps the growth of yeast engineering bacteria, but also obtains a higher collagen expression level. And the fermentation product shows excellent antioxidant and cell proliferation and growth promoting effects, has a good regulatory effect on a variety of 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 fermentation recombinant collagen. The second object of the present invention is to provide the application of the fermentation broth.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] As a first aspect of the present invention, a fermentation broth based on rose fermentation recombinant collagen comprises the following components in percentage by mass: 10%-30% rose pollen and 70%-90% BSM culture medium.

[0009] According to the present invention, the fermentation broth based on rose fermentation recombinant collagen is composed of the following components in percentage by mass: 20% rose pollen and 80% BSM culture medium.

[0010] As a second aspect of the present invention, a use of the above-mentioned fermentation broth based on rose fermentation recombinant collagen in the fermentation production of collagen.

[0011] As a third aspect of the present invention, a fermentation broth based on the above-mentioned rose fermentation recombinant collagen is used in the fermentation production of a fermentation product having the function of promoting cell proliferation and growth.

[0012] As a fourth aspect of the present invention, a fermentation broth based on the above-mentioned rose fermentation recombinant collagen is used in the fermentation production of a fermentation product having the effects of repairing skin damage and anti-aging.

[0013] As a fifth aspect of the present invention, a fermentation broth based on the above-mentioned rose fermentation recombinant collagen is used in the fermentation production of a fermentation product having the function of promoting the expression of moisturizing genes.

[0014] According to the present invention, the moisturizing genes include genes AQP-3, CD44, and HAS1.

[0015] Beneficial effects of the present invention:

[0016] 1. The fermentation liquid does not need to be further purified, 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;

[0017] 2. The fermentation product of the fermentation broth of the present invention has the effect of promoting cell proliferation and growth;

[0018] 3. The fermentation broth of the present invention can be used to increase the expression level of collagen. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The collagen expression level in rose fermentation culture medium containing different concentrations of rose pollen after 30 hours.

[0020] Figure 2 is the expression level of collagen in the culture medium containing 20% ​​rose pollen.

[0021] Figure 3 It is the expression level of induced collagen in the fermentation tank of Example 3.

[0022] Figure 4 is the density of yeast in the fermentation tank of Example 3.

[0023] Figure 5 The effect of rose collagen on cell proliferation.

[0024] *p<0.05; **P<0.01; ***P<0.001; ****P<0.0001;

[0025] Note: * indicates that the sample has significant difference compared with the blank treatment group at this concentration.

[0026] Figure 6-Figure 8 These are the effects of rose collagen on the expression of AQP-3 gene, CD44 gene and HAS-1 gene.

[0027] *p<0.05; **P<0.01; ***P<0.001;

[0028] Note: * indicates that the sample has significant difference compared with the blank treatment group at this concentration. DETAILED DESCRIPTION

[0029] The present invention is further explained below in conjunction with specific examples. The following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions provided by the manufacturer.

[0030] The recombinant human type III collagen Pichia pastoris engineered strain (GS115RHC04) in the following examples can be purchased from Meierjian Biotechnology Co., Ltd.

[0031] Example 1 Preparation of Rose Fermentation Broth

[0032] Roses are selected from the rose planting area of ​​Pingyin, Shandong Province, and the dried roses are crushed into powder with a crusher. Rose-based fermentation medium (Rose Based Mineral-Salt Medium, RMSM) is prepared according to the following components. The formula of the rose fermentation medium is shown in Table 1, in mass percentage. When used, the nutrients in the above formula are mixed and sterilized, but the metal inorganic salts need to be sterilized separately and then aseptically mixed at room temperature.

[0033] Table 1 The formula of rose fermentation culture fluid

[0034]

[0035] Example 2 Shake flask fermentation of rose collagen

[0036] Pick the recombinant human type III collagen Pichia yeast engineering strain and inoculate it into 25 ml BMGY medium. Shake in a 250 ml shake flask at 30°C and 250 rpm until the OD 600 = 4, centrifuged at 3000 g for 5 min at room temperature, collected the cells, removed the supernatant, and resuspended the cells in RMSM medium of different concentrations in Table 1 to an OD of 600 =1.0, induce expression and obtain culture. Add the above culture to a 1L shake flask, cover with two layers of sterile gauze, and place in a shaker to continue growing. Every 24 hours, add methanol to a final concentration of 0.5% to continue induction. At multiple time points, take 1ml of culture medium to a 1.5ml centrifuge tube. These samples are used to analyze expression levels and determine the optimal time to collect cells after induction. Centrifuge at room temperature at maximum speed in a horizontal centrifuge for 2-3 minutes. During secretory expression, transfer the supernatant to a separate tube, and store the supernatant and cell pellet at -80 degrees Celsius until the start of detection. Use Coomassie Brilliant Blue staining and SDS-PAGE to analyze supernatant protein expression. After analysis, recombinant human collagen can be expressed normally in rose-based culture medium, and the expression level increases with the increase in the proportion of rose pollen, but the expression level begins to decrease after exceeding 30%, as shown. Figure 1 The reason may be that the imbalance of other inorganic salt components in BSM caused the imbalance of microbial nutrition. Therefore, we selected a medium with 20% rose pollen content for cultivation and the expression level increased with time. After 30 hours of fermentation, SDS-PAGE electrophoresis staining showed that its yield reached about 2 g / L. Figure 2 .

[0037] Subsequent experiments used rose fermentation culture fluid with 20% rose pollen content.

[0038] Example 3 Large-scale fermentation of collagen in a fermenter

[0039] The frozen recombinant human type III collagen Pichia pastoris engineered strain was inoculated into the sterilized and cooled YPD medium and placed in a shaker for activation culture for 28 to 30 hours to make its OD 600 The value is between 3 and 5. The activated strain was inoculated into a 10L glass fermenter for amplification and culture for 6 to 8 hours. The amplified seed liquid was transferred to a stainless steel fermenter containing 150L RMSM culture medium at a volume ratio of 1:10 (seed liquid: fermentation liquid) and cultured for 16 to 18 hours. The control experiment was transferred to BSM inorganic salt culture medium. The culture medium was added to the fermenter by flow addition, and the culture was continued for 3 to 4 hours to make the wet weight of the bacteria reach 150 to 180 g / L. Methanol was gradually added and the methanol concentration was maintained at 0.5% for 21 to 24 hours of induction culture. The fermentation broth after induction was centrifuged at high speed to separate the bacteria and the fermentation supernatant, and the fermentation supernatant was collected. SDS-PAGE electrophoresis showed that the production of collagen in the fermenter was significantly increased, which was mainly due to the increase in bacterial density. The expression level reached 4 grams / liter in RMSM and about 3 grams / liter in BSM culture medium. Rose-based culture medium significantly increased the expression of collagen. Results are shown in Figure 3 . The bacterial density OD600 was measured by spectrophotometer. The results are shown in Figure 4 The results showed that the growth rate of RMSM was lower than that of BSM during the bacterial growth process, but at the end of the fermentation, the bacterial density of the two culture media was similar, so RMSM inhibited the rapid growth of the bacteria and slowed down the aging of the bacteria.

[0040] Example 4 Promoting cell proliferation

[0041] The rose collagen fermentation liquid obtained in Example 3 was freeze-dried into powder in a freeze dryer, and the powder was quantified and then prepared into solutions of different concentrations. 3T3 cells with good growth status (purchased from the laboratory of the University of Science and Technology of China) were plated on a 96-well plate and cultured for 17 hours. After replacing the serum-free basal medium and culturing for 7 hours, the old medium was discarded and rose collagen solutions of different concentrations were added. After 48 hours, the OD450nm was detected by CCK8 method to obtain the effect of the tested samples on the proliferation of 3T3 cells. Figure 5 .

[0042] The results showed that rose collagen fermentation broth can significantly promote cell proliferation, reaching a maximum value at 0.08 mg / mL, and its effect on cell proliferation is equivalent to that of 10% biological FBS. This shows that in addition to collagen, rose collagen fermentation products may also produce a variety of substances that promote cell growth.

[0043] Example 5 Effect on the Expression of Cell Moisture-Related Genes

[0044] The rose collagen fermentation liquid obtained in Example 3 was freeze-dried into powder in a freeze dryer, and the powder was quantified and prepared into solutions of different concentrations with complete culture medium. After culturing HACAT cells on a 6-well plate for 24 hours, the samples to be tested were replaced with samples containing different concentrations. Total RNA was extracted with TRIzon after 24 hours, and RNA purity and concentration were tested using NanoDrop. Subsequently, cDNA was synthesized and PCR amplified, and the target gene band was confirmed by agarose gel electrophoresis analysis. Finally, cDNA was used for Real-Time PCR quantitative determination to obtain the effect of rose collagen on the expression of genes related to moisture retention in HACAT cells.

[0045] This example measures three genes, aquaporin-3 (AQP-3), cell surface glycoprotein (CD44) and hyaluronan synthase 1 (HAS 1). The expression of these three genes is positively correlated with the skin's moisturizing function, and their expression levels reflect the effect on the skin's moisturizing function to a certain extent. When the sample concentration was 0.05 mg / ml, the expression levels of the hacat cell moisturizing-related genes AQP-3, CD44, and HAS1 were 94%, 104% and 221% of the blank group, respectively; when the sample concentration was 0.5 mg / ml, the expression levels of the hacat 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. See the results. Figure 6 , Figure 7 and Figure 8 .

[0046] The above description is only an example of the implementation mode of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An application of a fermentation culture broth based on rose fermentation recombinant collagen in the fermentation production of collagen, characterized in that: The fermentation culture solution based on rose fermentation recombinant collagen comprises the following components in percentage by mass: 10%-30% rose pollen, 70%-90% BSM culture medium, The application comprises the following steps: picking a recombinant human type III collagen Pichia yeast engineering strain, inoculating it into a BMGY medium, and culturing it in a shake flask until OD 600 =4, centrifuge at room temperature, collect cells, remove supernatant, resuspend cells with the fermentation culture medium, induce expression, and obtain culture; then, add the culture into a shake flask, put it into a shaker to continue growing, and add methanol to a final concentration of 0.5% every 24 hours to continue inducing collagen expression; Alternatively, the application comprises the following steps: inoculating the recombinant human type III collagen Pichia pastoris engineered strain into YPD medium, placing it in a shaker for activation and culturing until 0D 600 The value is between 3 and 5; then, the activated strain is inoculated into a 10L glass fermenter for amplification culture; then, the amplified seed solution is transferred to the stainless steel fermenter of the fermentation culture solution at a volume ratio of 1:10 for culture; the culture medium is added to the fermenter by a flow addition method, and the culture is continued for 3 to 4 hours until the wet weight of the bacteria reaches 150 to 180 g / L; then, methanol is gradually added, and the methanol concentration is maintained at 0.5%, and the induction culture is carried out for 21 to 24 hours; then, the fermentation solution after induction is centrifuged at high speed to separate the bacteria and the fermentation supernatant, and the fermentation supernatant is collected; The recombinant human type III collagen Pichia pastoris engineered strain was purchased from Meierjian Biotechnology Co., Ltd., model GS115RHC04.

2. The use according to claim 1, characterized in that The fermentation culture solution is composed of the following components in percentage by mass: 20% rose pollen and 80% BSM culture medium.

3. An application of a fermentation culture broth based on rose fermented recombinant collagen in the fermentation production of a fermentation product having the function of promoting cell proliferation and growth, characterized in that: The fermentation culture solution based on rose fermentation recombinant collagen comprises the following components in percentage by mass: 10%-30% rose pollen, 70%-90% BSM culture medium, The application comprises the following steps: inoculating the recombinant human type III collagen Pichia pastoris engineered strain into YPD medium, placing it in a shaker for activation and culturing until 0D 600 The value is between 3 and 5; then, the activated strain is inoculated into a 10L glass fermenter for amplification culture; then, the amplified seed liquid is transferred to the stainless steel fermenter of the fermentation culture liquid at a volume ratio of 1:10 for culture; the culture medium is added into the fermenter by a flow addition method, and the culture is continued for 3 to 4 hours, so that the wet weight of the bacteria reaches 150 to 180 g / L; then, methanol is gradually added, and the methanol concentration is maintained at 0.5%, and the induction culture is carried out for 21 to 24 hours; then, the fermented liquid after the induction is centrifuged at high speed to separate the bacteria and the fermentation supernatant, and the fermentation supernatant, i.e., the fermentation product, is collected; The recombinant human type III collagen Pichia pastoris engineered strain was purchased from Meierjian Biotechnology Co., Ltd., model GS115RHC04.

4. The use according to claim 3, characterized in that The fermentation culture solution is composed of the following components in percentage by mass: 20% rose pollen and 80% BSM culture medium.

5. An application of a fermentation culture broth based on rose fermented recombinant collagen in the fermentation production of a fermentation product having the function of promoting the expression of moisturizing genes, characterized in that: The fermentation culture solution based on rose fermentation recombinant collagen comprises the following components in percentage by mass: 10%-30% rose pollen, 70%-90% BSM culture medium, The application comprises the following steps: inoculating the recombinant human type III collagen Pichia pastoris engineered strain into YPD medium, placing it in a shaker for activation and culturing until 0D 600 The value is between 3 and 5; then, the activated strain is inoculated into a 10L glass fermenter for amplification culture; then, the amplified seed liquid is transferred to the stainless steel fermenter of the fermentation culture liquid at a volume ratio of 1:10 for culture; the culture medium is added into the fermenter by a flow addition method, and the culture is continued for 3 to 4 hours, so that the wet weight of the bacteria reaches 150 to 180 g / L; then, methanol is gradually added, and the methanol concentration is maintained at 0.5%, and the induction culture is carried out for 21 to 24 hours; then, the fermented liquid after the induction is centrifuged at high speed to separate the bacteria and the fermentation supernatant, and the fermentation supernatant, i.e., the fermentation product, is collected; The recombinant human type III collagen Pichia pastoris engineering strain was purchased from Meierjian Biotechnology Co., Ltd., model GS115RHC04; The moisturizing genes include genes AQP-3 and HAS1.

6. The use according to claim 5, characterized in that The fermentation culture solution is composed of the following components in percentage by mass: 20% rose pollen and 80% BSM culture medium.

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