CN113046394B

CN113046394B - Preparation process of tectorial membrane yeast fermentation filtrate

Info

Publication number: CN113046394B
Application number: CN202110283272.7A
Authority: CN
Inventors: 杜文玉; 刘一琳; 郑春阳
Original assignee: Robustnique Corp ltd
Current assignee: Robustnique Corp ltd
Priority date: 2021-03-17
Filing date: 2021-03-17
Publication date: 2022-08-12
Anticipated expiration: 2041-03-17
Also published as: CN113046394A

Abstract

A preparation process of a tectorial membrane yeast fermentation filtrate comprises two steps, firstly, preparing seeds and a fermentation culture medium; secondly, a standing micro-aerobic culture mode is adopted in the culture process, and finally, a tectorial membrane yeast fermentation filtrate is obtained in a filtration mode. The method is characterized in that micro-aerobic fermentation is adopted in the fermentation, and compared with aerobic fermentation, the method can stimulate tectorial membrane yeast to generate rich secondary metabolites; meanwhile, the static culture not only simplifies the fermentation process and the operation flow, but also creates a micro-aerobic fermentation environment, is beneficial to the fermentation of the coated yeast, and the method is suitable for general biological laboratories, and has the advantages of simple operation, low cost and large yield.

Description

Preparation process of tectorial membrane yeast fermentation filtrate

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a preparation process of a tectorial membrane yeast fermentation product.

Background

The skin has obvious sensitivity to physical or chemical stimulation, and the self-repairing capability of the skin barrier is damaged while the inflammatory reaction is triggered by exogenous stimulation (such as ultraviolet irradiation, chemical substances and the like), so that melanin deposition is often induced, and the keratinization and skin aging are accelerated. At present, a large number of reports show that certain probiotics not only can positively influence the composition of intestinal flora, but also can regulate the immune system at local and systemic levels, thereby improving immune defense mechanisms or down regulating immune functions.

Tectorial membrane yeast is an aroma-producing yeast, and has richer secondary metabolism compared with saccharomyces cerevisiae. In the fermentation process, the tectorial membrane yeast can not only consume nutrient substances in a culture medium and generate micromolecular organic acid, vitamin, oligosaccharide and the like, but also secrete protease into fermentation filtrate and decompose macromolecular protein into micromolecular amino acid, polypeptide and the like, and the micromolecular substances are mostly remained in the fermentation filtrate except that a small part of the micromolecular substances can be absorbed and utilized by the yeast. The tectorial membrane yeast fermentation product is a light yellow transparent liquid which is rich in vitamins, amino acids, mineral substances and organic acids, can promote the expression of the filaggrin and can prevent the reduction of the filaggrin mediated by serine protein tyrosine. In addition, the tectorial membrane yeast fermentation filtrate can also increase the expression of caspase-14 in epidermal keratinocytes in a 3D model, and the expression of caspase-14 is involved in the repair of damaged skin barriers. Therefore, the natural mixed component of the fermentation filtrate of the tectorial membrane yeast can obviously improve the metabolic process of the skin epidermal layer, inhibit the generation of free radicals, protect cells from being damaged, improve the anti-aging capability of the skin and ensure that the skin is bright, tender and smooth. These effects have been demonstrated in a number of in vitro experiments and are widely used in skin care products.

In the existing reports, aerobic fermentation conditions are adopted in patents (CN 112353714A and CN109355216A), the operation steps are too complicated, about 8-10 steps are required, the equipment requirement is high, fermentation is required to be carried out in a fermentation tank, the implementation of a common laboratory is difficult, and the production cost is high. The tectorial membrane yeast is facultative anaerobic bacteria, can be massively propagated and rapidly grown under aerobic condition, is more beneficial to the growth and accumulation of thalli, but is not beneficial to the generation and accumulation of secondary metabolites; under the anaerobic condition or the micro-aerobic condition, the tectorial membrane yeast can generate more abundant secondary metabolites in order to resist the external adverse environment, so as to protect the tectorial membrane yeast against the damage of the external environment. Therefore, it is highly desirable to develop a process that is suitable for laboratory culture, simple in process, and capable of producing abundant metabolites.

Disclosure of Invention

The invention aims to provide a preparation process of a tectorial membrane yeast fermentation filtrate, which simplifies the process flow, is suitable for small-scale fermentation culture in a laboratory and can produce abundant secondary metabolites. In order to solve the technical problems, the specific technical scheme of the invention is as follows:

a preparation process of a tectorial membrane yeast fermentation filtrate comprises the following steps:

step 1: preparation of culture Medium

1) YPD medium for seed culture

YPD medium: 10 g of yeast extract, 20 g of glucose, 20 g of peptone, dissolved in 1000 ml of deionized water, autoclaved at 115 ℃ for 30 min, and added to the solid medium with 2% agar.

2) Tectorial membrane yeast fermentation culture medium for fermentation expression

The tectorial membrane yeast fermentation culture medium is divided into the part I and the part II, and the preparation and the sterilization are respectively carried out. Part I and part II were measured as 1: 1, fully mixing to obtain the required culture medium, and uniformly mixing to obtain the culture medium with the pH value of 4-6.

Step 2: expression by fermentation

(1) Activating strains:

inoculating the frozen bacteria of tectorial membrane yeast into a sterile culture plate of YPD medium, and activating at 28 deg.C for 48-60 h.

(2) Preparing a seed solution:

picking fresh colonies from the plate, suspending the colonies into sterile water, uniformly mixing the colonies, transferring the colonies into 100ml of a tectorial membrane yeast fermentation medium, and culturing the colonies for 24 hours at the temperature of 28 ℃ and the rpm of 200 in a shaking table.

(3) Fermentation culture:

inoculating the seed solution into a 0.05-5.0L shake flask containing a fermentation medium, and controlling the concentration of inoculated strain at OD ₆₀₀ And (4) the liquid filling amount is about 40% -70%, the bottle cap is used for sealing the bottle mouth, the bottle is placed in an incubator at 28 ℃, and the incubator is kept still for micro-aerobic fermentation for 60-96 hours in total.

(4) Preparing a fermentation liquid:

after fermentation, centrifuging the culture solution at 14000 rpm for 5min, and collecting supernatant; carrying out suction filtration treatment on the supernatant by using 2 sheets of medium-speed filter paper, and then weighing the fermentation filtrate after suction filtration; adding a preservative according to a certain proportion by mass, and uniformly stirring and dispersing; and (3) carrying out suction filtration on the supernatant liquid added with the preservative, and sequentially passing through 0.45 mu m and 0.2 mu m filter membranes to obtain the final tectorial membrane yeast fermentation filtrate capable of being aseptically stored.

Further, the preservative is 0.5-1.5% of PE9010 and 2-3% of butanediol.

Further, the concentration of the inoculated strain is controlled at OD ₆₀₀ =0.3-0.6。

Further, the standing micro-aerobic fermentation mode is standing fermentation in the whole culture process.

Furthermore, the standing micro-aerobic fermentation mode is that the micro-aerobic fermentation is performed by shaking once every 24 hours or 36 hours, and the rest time is the standing fermentation.

The invention has the following advantages:

(1) micro-aerobic fermentation is adopted in fermentation, and compared with aerobic fermentation, the micro-aerobic fermentation can stimulate the tectorial membrane yeast to generate rich secondary metabolites;

(2) the shake flask is sealed, and the standing culture simplifies the fermentation process and the operation flow, creates a micro-aerobic fermentation environment, is beneficial to the fermentation of the coated yeast, and the method is suitable for general biological laboratories, and has the advantages of simple operation, low cost and large yield.

Drawings

FIG. 1 is a process flow diagram of the present invention.

Detailed Description

The present invention is further illustrated by the following specific examples, which are, however, not intended to limit the scope of the invention.

The components used in the examples of the present invention were all derived from commercially available products.

Glucose and skimmed milk powder were purchased from Biotechnology engineering (Shanghai) Inc., and yeast extract was purchased from Obo Star Biotech, Beijing. Glucose is used as a carbon source of the culture medium, the yeast extract and the skim milk powder are used as a nitrogen source of the culture medium, and the mixed nitrogen source is more beneficial to the growth of the tectorial membrane yeast.

Example 1

(1) inoculating the membrane-covered yeast frozen strains into an aseptic culture plate of an YPD culture medium, activating for 48 hours at the temperature of 28 ℃, adding 50% glycerol to a final concentration of 25%, and performing frozen preservation for later use;

(2) picking fresh bacterial colonies from a flat plate, re-suspending the bacterial colonies into 15mL of sterile water, uniformly mixing the bacterial colonies, transferring the bacterial colonies into 100mL of film-covered yeast fermentation culture medium, inoculating 2mL of seed liquid into each bottle of 100mL of seed liquid, and culturing the bacterial colonies for 24 hours at the temperature of 28 ℃ and the rpm in a shaking table to obtain fermentation seed liquid;

(3) after the seed liquid culture is finished, the OD of the seeds is respectively tested by an ultraviolet spectrophotometer ₆₀₀ =5.21, transfer fresh seed liquid to 2L fermentation medium in 5L shake flask, make OD after transfer ₆₀₀ =0.21, the transferred shake flask is sealed by a bottle cap and then placed in an incubator at 28 ℃, and the whole process is kept still for fermentation for 60 hours in total;

(4) and after fermentation is finished, centrifuging the culture solution by using a 250mL centrifuge bottle, centrifuging the culture solution at the normal temperature of 14000 rpm for 5min, collecting supernatant of fermentation filtrate, filtering the centrifuged supernatant by using 2 pieces of medium-speed filter paper by using a suction filtration method, weighing about 2010 g of the fermentation filtrate, adding 901020.7 g of PE and 41.4 g of butanediol, uniformly mixing the two, adding the mixture into the fermentation filtrate, uniformly stirring and dispersing the mixture, carrying out suction filtration on the supernatant added with the preservative, and sequentially filtering the supernatant through 0.45 mu m and 0.2 mu m filter membranes to obtain the filtrate of the fermentation product of the membrane-covered yeast.

The resulting filtrate had a protein content of 1.2 mg/mL, a reducing sugar content of 8.1mg/mL, a dry matter content of 0.7%, and a total of 2L.

Example 2

(1) Inoculating the membrane-covered yeast frozen bacteria into an aseptic culture plate of an YPD culture medium, activating for 48 hours at the temperature of 28 ℃, adding 50% of glycerol to a final concentration of 25%, and preserving for later use by freezing;

(2) picking fresh colonies from the plate, resuspending the fresh colonies into 15mL of sterile water, uniformly mixing the fresh colonies, transferring the mixture into 100mL of film-covered yeast fermentation culture medium, inoculating 2mL of 100mL of seed solution into each bottle, and culturing the mixture for 24 hours at 28 ℃ and 200 rpm in a shaking table to obtain fermentation seed solution;

(3) after the seed liquid culture is finished, the OD of the seeds is respectively tested by an ultraviolet spectrophotometer ₆₀₀ = 6.0, transfer fresh seed liquid to 2L fermentation medium in 5L shake flask, make OD after transfer ₆₀₀ =0.45, the transferred shake flask is sealed by a bottle cap and then placed in an incubator at 28 ℃, shaking is carried out once every 36 hours, and the shake flask is kept stand for fermentation in the rest time, and the continuous culture lasts for 72 hours in total;

(4) and after fermentation is finished, centrifuging the culture solution by using a 250mL centrifuge bottle, centrifuging the culture solution at the normal temperature of 14000 rpm for 5min, collecting supernatant of fermentation filtrate in the previous step, filtering the centrifuged supernatant by using 2 pieces of medium-speed filter paper by using a suction filtration method, weighing about 2120 g of the fermentation filtrate, adding 21.2 g of PE9010 and 63.6 g of butanediol, uniformly mixing the supernatant and the filtrate, adding the mixture into the fermentation filtrate, uniformly stirring and dispersing the mixture, carrying out suction filtration on the supernatant added with the preservative, and sequentially filtering the mixture through 0.45 mu m and 0.2 mu m filter membranes to obtain the filtrate of the fermentation product of the membrane-coated yeast.

The resulting filtrate had a protein content of 1.4 mg/mL, a reducing sugar content of 8.0 mg/mL, and a dry matter content of 0.7% for a total of 2L.

Example 3

(1) Inoculating the membrane-covered yeast frozen bacteria into an aseptic culture plate of an YPD culture medium, activating for 60 h at 28 ℃, adding 50% of glycerol to a final concentration of 25%, and performing freeze preservation for later use;

(3) after the seed liquid culture is finished, the OD of the seeds is respectively tested by an ultraviolet spectrophotometer ₆₀₀ =7.3, transfer fresh seed liquid to 2L fresh liquid culture medium in 5L shake flask, make OD after transfer ₆₀₀ =0.95, the transferred shake flask is sealed by a bottle cap and then placed in an incubator at 28 ℃, shaking is carried out once every 24 hours, and the shake flask is kept stand for fermentation in the rest time, and the continuous culture lasts 96 hours in total;

(4) and after fermentation, centrifuging the culture solution by using a 250mL centrifuge bottle, centrifuging the culture solution for 5min at the normal temperature of 14000 rpm, collecting supernatant of fermentation filtrate in the previous step, filtering the centrifuged supernatant by using 2 pieces of medium-speed filter paper by using a suction filtration method, weighing about 2025 g of the fermentation filtrate, adding PE 901030.4 g and butanediol 60.7 g into the fermentation filtrate, uniformly mixing the two, adding the mixture into the fermentation filtrate, uniformly stirring and dispersing the mixture, carrying out suction filtration on the supernatant added with the preservative, and sequentially filtering the mixture through 0.45 mu m and 0.2 mu m filter membranes to obtain the filtrate of the fermentation product of the coated yeast.

The resulting filtrate had a protein content of 1.3mg/mL, a reducing sugar content of 8.3 mg/mL, and a dry matter content of 0.9% for a total of 2L.

A DNS method: namely, the dinitrosalicylic acid method utilizes dinitrosalicylic acid (DNS) and reducing sugar to carry out oxidation-reduction reaction under alkaline condition to generate 3-amino-5-nitro salicylic acid, the product is brownish red under boiling condition, has absorption at 540nm, and has proportional relation between color depth and reducing sugar content within a certain concentration range.

BCA method: peptide bond structure and Cu in protein molecule under alkaline environment ²⁺ Complexing and adding Cu ²⁺ Reduction to Cu ¹⁺ BCA is specific for Cu ¹⁺ The combination forms a stable purple blue complex, the absorption is at 562nm, and the color depth is in proportion to the protein content in a certain concentration range.

And (3) drying: adding 1ml of product into an evaporation dish, drying at 105 ℃ for 1h, taking out, cooling to room temperature, and weighing the empty dish, the dish loaded sample before drying and the dish loaded sample after drying in sequence in the process. Then the dry matter content = (weight after oven drying of dish sample-empty dish weight)/(weight before oven drying of dish sample-empty dish weight).

The reducing sugar content in the product is determined by using a DNS method, the protein content is determined by using a BCA method, the dry matter content is determined by using a drying method, and specific parameters are shown in a table 2 compared with the product obtained by using an aerobic fermentation mode.

The experimental results show that, compared with the competitive products, in the examples, the content of protein is increased by 30%, the content of dry matter is increased by 27.8%, and the content of reducing sugar is slightly increased, but not obviously, only by 1.6%. Except that all indexes of the product are improved, in the whole fermentation process and production process, the method of sealing and standing culture of the shake flask is adopted, so that the fermentation process and the operation process are simplified, a micro-aerobic fermentation environment is created, the fermentation of the film-covered yeast is facilitated, and the method is suitable for general biological laboratories, is simple to operate, low in cost and high in yield.

It is to be understood that the present invention has been described with reference to certain embodiments, and that various changes in the features and embodiments, or equivalent substitutions may be made therein by those skilled in the art without departing from the spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to the particular embodiment disclosed, but that the invention will include all embodiments falling within the scope of the appended claims.

Claims

1. A preparation process of a tectorial membrane yeast fermentation filtrate comprises the following steps:

step 1: preparation of culture Medium

Preparing an YPD culture medium and a culture medium for fermentation of tectorial membrane yeast;

step 2: fermentation culture

(1) Activating strains:

inoculating the membrane-covered yeast frozen bacteria into an aseptic culture plate of an YPD solid culture medium for activation;

(2) preparing a seed solution:

picking fresh colonies from a solid plate, re-suspending the colonies into sterile water, uniformly mixing the colonies, transferring the colonies into a film-covered yeast fermentation culture medium, and culturing the colonies for 24 hours at 28 ℃ and 200 rpm in a shaking table;

(3) fermentation culture:

inoculating the seed solution into a fermentation culture medium of tectorial membrane yeast for standing micro-aerobic fermentation culture, and controlling the concentration of the inoculated strain at OD ₆₀₀ =0.2-1.0, the liquid loading is about 40% -70%, and the sealed culture is placed in an incubator at 28 ℃; the micro-aerobic fermentation mode of standing is to shake once every 24 hours or 36 hours, and to perform standing culture in the rest time;

(4) preparing a fermentation liquid:

and after fermentation, centrifuging the culture solution to collect supernatant, performing suction filtration on the supernatant by using 2 pieces of medium-speed filter paper, weighing fermentation filtrate, adding a certain proportion of preservative according to mass percent, uniformly stirring and dispersing, performing suction filtration on the supernatant added with the preservative, and sequentially passing through 0.45 mu m and 0.2 mu m filter membranes to obtain final membrane-covered yeast fermentation filtrate capable of being stored in a sterile manner.

2. The process for preparing fermentation filtrate of tectorial membrane yeast as claimed in claim 1, wherein the preservative is 0.5-1.5% PE9010 and 2-3% butanediol.

3. The process for preparing fermentation filtrate of tectorial membrane yeast as claimed in claim 1, wherein the concentration of inoculated strain is controlled to OD ₆₀₀ =0.3-0.6。