阅读说明：本技术 一种植物甾醇酯发酵物的制备方法和应用 (Preparation method and application of phytosterol ester fermentation product ) 是由 管世敏 王前程 荣绍丰 黄煜玲 蔡宝国 李茜茜 于 2021-08-03 设计创作，主要内容包括：本发明公开了一种植物甾醇酯发酵物的制备方法和应用。本发明主要采用益生菌对植物甾醇酯进行生物发酵,获得抗炎活性显著提升的植物甾醇酯发酵物。小鼠耳肿抗炎活性试验显示,本发明制备的植物甾醇酯发酵物,与未发酵的植物甾醇酯相比,具有更强的的抗炎活性,同时具有修复、保湿、淡化皱纹等功效。(The invention discloses a preparation method and application of a phytosterol ester fermentation product. The invention mainly adopts probiotics to carry out biological fermentation on phytosterol ester, and the phytosterol ester fermentation product with obviously improved anti-inflammatory activity is obtained. An anti-inflammatory activity test of the mouse ear swelling shows that the phytosterol ester fermentation product prepared by the invention has stronger anti-inflammatory activity and has the effects of repairing, moisturizing, fading wrinkles and the like compared with the non-fermented phytosterol ester.)

1. A preparation method of a phytosterol ester fermentation product is characterized in that the phytosterol ester fermentation product with anti-inflammatory activity is prepared by taking phytosterol ester as a substrate and fermenting the phytosterol ester by yeast, and the preparation method specifically comprises the following steps:

step 1: respectively weighing and mixing the phytosterol ester, tween, span and glycerol, and heating and stirring to obtain a phytosterol ester mixed system;

step 2: adding the phytosterol ester mixed system into a culture medium, continuously stirring, and then cooling to obtain a conversion system;

and step 3: adding saccharomycete powder into the conversion system under aseptic operation, and fermenting and culturing;

and 4, step 4: cooling to room temperature after inactivation, and then homogenizing to obtain the phytosterol ester fermentation product.

2. The method for preparing the phytosterol ester fermentation product according to claim 1, wherein the mixing mass ratio of span, tween, phytosterol ester and glycerol in the step 1 is 1-9: 5-14: 9-47: 47-68; the span is span-40, and the Tween is Tween-20.

3. The method for preparing a phytosterol ester fermentation product according to claim 1, wherein the heating and stirring conditions in the step 1 are as follows: the temperature is 40-90 ℃, the stirring speed is 200-500 rpm, and the stirring time is 1-3 h.

4. The method of preparing a phytosterol ester fermentation product according to claim 1, wherein the formula of the culture medium in the step 2 is as follows: 1.0-3.0 wt% of glucose, 0.2-0.7 wt% of soybean peptone, 0.1-0.4 wt% of dipotassium hydrogen phosphate, 0.1-0.3 wt% of magnesium chloride and the balance of water, wherein the pH value is 5.0-6.5; the medium was sterilized at 121 ℃ for 15min before use.

5. The method for preparing a phytosterol ester fermentation product according to claim 1, wherein the mass ratio of the phytosterol ester mixed system to the culture medium in the step 2 is 1: 50-1: 5.

6. The method for preparing phytosterol ester fermentation product according to claim 1, wherein the stirring time in the step 2 is 1-2 h, and the temperature reduction is that the temperature is reduced to 30-40 ℃.

7. The method for preparing phytosterol ester fermentation product according to claim 1, wherein the mass of the saccharomycete powder added in the step 3 is 0.1-0.5% of the mass of the transformation system; the conditions of the fermentation culture are as follows: standing and fermenting for 6-36 h at the temperature of 35-40 ℃.

8. The method of preparing a phytosterol ester fermentation product according to claim 1, wherein the conditions for inactivation in step 4 are as follows: inactivating for 2-20 min at 80-100 ℃; the rotation speed of the homogenization treatment is 15000-30000 rpm, and the time is 10-20 min.

9. Use of a phytosterol ester fermentation product prepared by the method for preparing a phytosterol ester fermentation product according to any one of claims 1-8 in the preparation of cosmetics.

10. The use according to claim 9, wherein said cosmetic products comprise skin lotions and creams with anti-inflammatory rejuvenating properties.

Technical Field

The invention relates to a preparation method and application of a phytosterol fermentation product, and belongs to the technical field of cosmetics.

Background

The phytosterol ester is derived from the esterification reaction of phytosterol and oleic acid, has the same function as the phytosterol in function, and is superior to the phytosterol in physical property and solubility. Phytosterol esters have excellent anti-inflammatory, skin tissue repair and antioxidant effects, and thus are widely used as functional substances in cosmetics.

At present, the research on the phytosterol ester is only limited in the application aspect, the research on the biological modification of the phytosterol ester is very few, and the research and the report on the modification of the phytosterol ester by adopting a biotechnology means are not found.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: how to biologically modify phytosterol esters to improve their anti-inflammatory activity.

In order to solve the technical problems, the invention provides a preparation method of a phytosterol ester fermentation product, which uses phytosterol ester as a substrate to prepare the phytosterol ester fermentation product with anti-inflammatory activity by yeast fermentation, and specifically comprises the following steps:

step 1: respectively weighing and mixing the phytosterol ester, tween, span and glycerol, and heating and stirring to obtain a phytosterol ester mixed system;

step 2: adding the phytosterol ester mixed system into a culture medium, continuously stirring, and then cooling to obtain a conversion system;

and step 3: adding saccharomycete powder into the conversion system under aseptic operation, and fermenting and culturing;

and 4, step 4: cooling to room temperature after inactivation, and then homogenizing to obtain the phytosterol ester fermentation product.

Preferably, the mixing mass ratio of span, tween, phytosterol ester and glycerol in the step 1 is 1-9: 5-14: 9-47: 47-68; the span is span-40, and the Tween is Tween-20.

Preferably, the heating and stirring conditions in the step 1 are as follows: the temperature is 40-90 ℃, the stirring speed is 200-500 rpm, and the stirring time is 1-3 h.

Preferably, the formula of the culture medium in the step 2 is as follows: 1.0-3.0 wt% of glucose, 0.2-0.7 wt% of soybean peptone, 0.1-0.4 wt% of dipotassium hydrogen phosphate, 0.1-0.3 wt% of magnesium chloride and the balance of water, wherein the pH value is 5.0-6.5; the medium was sterilized at 121 ℃ for 15min before use.

Preferably, the mass ratio of the phytosterol ester mixed system to the culture medium in the step 2 is 1: 50-1: 5.

Preferably, the stirring time in the step 2 is 1-2 hours, and the temperature is reduced to 30-40 ℃.

Preferably, the mass of the saccharomycete powder added in the step 3 is 0.1-0.5% of the mass of the transformation system; the conditions of the fermentation culture are as follows: standing and fermenting for 6-36 h at the temperature of 35-40 ℃.

Preferably, the conditions for inactivation in step 4 are: inactivating for 2-20 min at 80-100 ℃; the rotation speed of the homogenization treatment is 15000-30000 rpm, and the time is 10-20 min.

The invention also provides application of the phytosterol ester fermentation product prepared by the preparation method of the phytosterol ester fermentation product in preparation of cosmetics.

Preferably, the cosmetic comprises skin care emulsions and creams having anti-inflammatory rejuvenating properties.

Compared with the prior art, the invention has the beneficial effects that:

1. the anti-inflammatory activity of the phytosterol ester fermentation product prepared by yeast fermentation is obviously improved compared with that of the non-fermented phytosterol ester;

2. the preparation process and the production process related by the invention are green production in the whole process, no wastewater or waste residue pollution is caused, and all raw materials used for preparation enter a final product;

3. the phytosterol ester fermentation product prepared by the method does not need complicated and complicated purification steps, can be directly used in cosmetics, and has huge application space and market in the cosmetic industry.

Drawings

Figure 1 shows a comparison of the components before and after fermentation of phytosterol esters.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

The detection method related by the invention comprises the following steps:

(1) phytosterol ester content determination-gas chromatography

The method for detecting the content of phytosterol ester in a sample by adopting a gas chromatography comprises the following steps: chromatographic conditions are as follows: HP-5 capillary column, split ratio 30: 1, 1 mu L of sample feeding amount, adopting a programmed temperature rise, raising the initial temperature to 120 ℃, keeping the initial temperature for 2min, raising the initial temperature to 240 ℃ at 15 ℃/min, keeping the initial temperature for 2min, raising the initial temperature to 280 ℃ at 5 ℃/min, keeping the initial temperature for 10min, and totally consuming for 30 min.

And (3) standard curve preparation: preparing phytosterol oleate standard solutions with the concentrations of 0.1mg/mL, 0.2mg/mL, 0.4mg/mL, 0.8mg/mL, 1.6mg/mL, 3.2mg/mL and 6.4mg/mL respectively, performing the same chromatographic procedures to obtain a gas chromatogram, performing integration treatment and drawing a standard curve.

Sample treatment: adding a sample to be detected into a solvent dichloromethane, sealing a cover, performing ultrasonic treatment in an ultrasonic instrument, centrifuging to obtain a liquid to be detected, and calculating the content of the phytosterol ester according to a standard curve after gas phase detection.

(2) Animal experiment-anti-inflammatory activity experiment of mouse ear swelling model

Reference (pharmacological experimental methodology) fourth edition of the method was performed.

The first experiment method comprises the following steps:

1. preparing an inflammation-causing agent: 100% xylene.

2. The experimental or control samples were dissolved in the inflammatory agent at 1 mL/mL.

3. The right ear of the experimental mice was coated with either an inflammatory agent (control) or an inflammatory agent containing sample (experimental group) on both the front and back sides. The volume of the inflammatory agent was 0.02 mL/tube. The left ear did not do anything.

After 4.1h, the animals were sacrificed by anesthesia, and the ears were cut off and the round ears were punched out of the same portion with a punch of 9mm diameter, respectively, and weighed.

5. The swelling degree is obtained by subtracting the left ear piece weight from the right ear piece weight of each mouse, and the swelling degrees of the sample group, the control group and the positive group are subjected to statistical treatment, and the swelling inhibition rate (%) is determined.

II, test materials:

1. test animals: ICR mice (male), body weight: 22g to 25g, 50, and 5 groups.

2. Clobetasol propionate cream (10 g: 2 mg): jiangsu Yuansheng pharmaceutical industry 19010701# (positive).

3. Xylene: national reagents company AR20161810# (modeling reagent).

Thirdly, test conditions:

1. laboratory temperature: 20-24 ℃;

2. relative humidity: 60-70%.

Example 1

A method for preparing phytosterol fermentation product comprises the following steps:

1) preparation of a culture medium: the components are calculated according to mass fraction, specifically 1.0 percent of glucose, 0.2 percent of soybean peptone, 0.1 percent of dipotassium hydrogen phosphate and 0.1 percent of magnesium chloride, the rest is complemented with water, the pH is adjusted to 6.5, the mixture is sterilized for 15min at 121 ℃, and the mixture is cooled to 40 ℃ after the sterilization;

2) preparing a phytosterol ester mixed system: mixing the components according to the mass ratio of span-40, tween-20, phytosterol ester and glycerol of 9:14:9:68, and fully stirring for 1h at the temperature of 40 ℃ and the stirring speed of 200 rpm;

3) preparation of a transformation system: adding the phytosterol ester mixed system obtained in the step 2) into the culture medium obtained in the step 1), wherein the mass ratio of the phytosterol ester mixed system to the culture medium is 1:50, continuously stirring for 1h, and cooling to 30 ℃;

4) fermentation: adding 0.1% of saccharomycete powder into the conversion system obtained in the step 3) according to the mass fraction under the aseptic operation, and standing and fermenting for 6 hours at the culture temperature of 35 ℃;

5) inactivation: inactivating the fermentation liquor obtained in the step 4) at 80 ℃ for 2min, and then cooling to room temperature;

6) homogenizing: homogenizing the inactivated fermentation liquor obtained in the step 5), wherein the homogenizing rotating speed is 15000rpm, and the homogenizing time is 10min, so as to obtain the final product phytosterol ester fermentation product.

Example 2

1) Preparation of a culture medium: the components are prepared according to mass fraction, specifically 2.5% of glucose, 0.5% of soybean peptone, 0.3% of dipotassium hydrogen phosphate and 0.2% of magnesium chloride, the rest is complemented with water, the pH is adjusted to 5.5, the mixture is sterilized at 121 ℃ for 15min, and the mixture is cooled to 60 ℃ after the sterilization;

2) preparing a phytosterol ester mixed system: mixing the components according to the mass ratio of span-40, tween-20, phytosterol ester and glycerol of 5:9:26:60, and fully stirring for 2 hours at the temperature of 60 ℃ and the stirring speed of 350 rpm;

3) preparation of a transformation system: adding the phytosterol ester mixed system obtained in the step 2) into the culture medium obtained in the step 1), wherein the mass ratio of the phytosterol ester mixed system to the culture medium is 1:9, continuously stirring for 1h, and cooling to 37 ℃;

4) fermentation: adding 0.3 percent of saccharomycete powder into the conversion system obtained in the step 3) according to the mass fraction under the aseptic operation, and standing and fermenting for 24 hours at the culture temperature of 37 ℃;

5) inactivation: inactivating the fermentation liquor obtained in the step 4) at 90 ℃ for 10min, and then cooling to room temperature;

6) homogenizing: homogenizing the inactivated fermentation liquor obtained in the step 5), wherein the homogenizing rotating speed is 20000rpm, and the homogenizing time is 15min, so as to obtain the final product phytosterol ester fermentation product.

Example 3

1) Preparation of a culture medium: the components are prepared according to mass fraction, specifically 3.0% of glucose, 0.7% of soybean peptone, 0.4% of dipotassium hydrogen phosphate and 0.3% of magnesium chloride, the rest is complemented with water, the pH is adjusted to 6.5, the mixture is sterilized at 121 ℃ for 15min, and the mixture is cooled to 90 ℃ after the sterilization;

2) preparing a phytosterol ester mixed system: mixing the components according to the mass ratio of span-40 to tween-20 to the phytosterol ester to the glycerol of 1:5:47:47, and fully stirring for 3 hours at the temperature of 90 ℃ and the stirring speed of 500 rpm;

3) preparation of a transformation system: adding the phytosterol ester mixed system obtained in the step 2) into the culture medium obtained in the step 1), wherein the mass ratio of the phytosterol ester mixed system to the culture medium is 1:5, continuously stirring for 2h, and cooling to 40 ℃;

4) fermentation: adding 0.3 percent of saccharomycete powder into the conversion system obtained in the step 3) according to the mass fraction under the aseptic operation, and standing and fermenting for 36 hours at the culture temperature of 40 ℃;

5) inactivation: inactivating the fermentation liquor obtained in the step 4) at 100 ℃ for 20min, and then cooling to room temperature;

6) homogenizing: homogenizing the inactivated fermentation liquor obtained in the step 5), wherein the homogenizing rotating speed is 30000rpm, and the homogenizing time is 20min, so as to obtain a final product, namely a phytosterol ester fermentation product;

comparative example 1

1) Preparation of a culture medium: the components are prepared according to mass fraction, specifically 2.5% of glucose, 0.5% of soybean peptone, 0.3% of dipotassium hydrogen phosphate and 0.2% of magnesium chloride, the rest is complemented with water, the pH is adjusted to 5.5, the mixture is sterilized at 121 ℃ for 15min, and the mixture is cooled to 60 ℃ after the sterilization is finished;

2) preparing a phytosterol ester mixed system: mixing the components according to the mass ratio of span-40 to tween-20 to the phytosterol ester to the glycerol of 5:9:26:60, and fully stirring for 2 hours at the temperature of 60 ℃ and the stirring speed of 350 rpm;

3) preparation of a transformation system: adding the phytosterol ester mixed system obtained in the step 2) into the culture medium obtained in the step 1), wherein the mass ratio of the phytosterol ester mixed system to the culture medium is 1:9, continuously stirring for 1h, and cooling to 37 ℃;

4) inoculation and inactivation: adding 0.3% of saccharomycete powder into the conversion system obtained in the step 3) according to the mass fraction under the aseptic operation, inactivating at 90 ℃ for 10min, and cooling to room temperature;

5) homogenizing: homogenizing the inactivated solution obtained in the step 4), wherein the homogenizing speed is 20000rpm, and the homogenizing time is 15min, so as to obtain a mixture of unfermented phytosterol and culture medium;

example 4

The phytosterol ester fermentation sample liquid prepared in the example 2 is prepared into the skin care emulsion with the anti-inflammatory repair function, and the specific components are shown in the table 1.

TABLE 1 skin care lotion composition (content by mass fraction)

The preparation method of the skin care emulsion comprises the following steps:

homogenizing A-phase phytosterol oleate fermented product with a homogenizer for 10min, slowly adding B-phase into A, and stirring at 400r/min for 30 min; adding phase C card wave into distilled water of 60 deg.C to dissolve completely, mixing the above two mixed systems, stirring at 400r/min for 60min, stirring, and cooling to room temperature to obtain skin care emulsion.

Example 5

The phytosterol ester fermentation sample liquid prepared in example 2 is taken to prepare the skin cream with the anti-inflammatory and repairing functions, and the specific components are shown in table 2.

TABLE 2 skin cream ingredients (content by mass fraction)

The preparation method of the skin cream comprises the following steps:

homogenizing A phase phytosterol ester fermentation product with homogenizer for 10min, slowly adding B phase into A, and stirring at 400r/min for 30 min; adding phase C card wave into distilled water of 60 deg.C to dissolve completely, mixing the above two mixed systems, stirring at 400r/min for 60min, stirring, and cooling to room temperature to obtain skin cream.

Comparative example 2

A skin cream formulated from the unfermented phytosterol ester of comparative example 1 in combination with the medium, the specific ingredients of which are shown in table 3.

TABLE 3 skin cream compositions (content by mass fraction)

The preparation method of the skin cream comprises the following steps:

homogenizing A-phase phytosterol oleate biological fermentation liquid for 10min by using a homogenizer, slowly adding B-phase into A, and stirring for 30min at a speed of 400 r/min; adding phase C card wave into distilled water of 60 deg.C to dissolve completely, mixing the above two mixed systems, stirring at 400r/min for 60min, stirring, and cooling to room temperature to obtain skin cream.

The result of the detection

(1) Gas phase detection results

The phytosterol ester fermentation product obtained in example 2 and the phytosterol ester and culture medium mixture obtained in comparative example 1 were subjected to gas chromatography, and the specific results are shown in table 4:

TABLE 4 comparison of conversion before and after fermentation of phytosterol esters

As can be seen from the results of table 4 and fig. 1, the components of phytosterol ester are significantly changed after fermentation by yeast.

(2) Results of animal experiments

The skin care emulsions or creams obtained in examples 4 to 5 and comparative example 2 were subjected to anti-inflammatory activity test in mouse ear swelling model, and the results are shown in table 5:

TABLE 5 Effect of swelling of auricles in mice Experimental results

Example 4 the sample solution has certain effect of inhibiting mouse auricle swelling compared with the model group (50.1 percent, P is less than 0.01); example 5 the sample solution has certain effect of inhibiting mouse auricle swelling compared with the model group (54.2 percent, P is less than 0.01); compared with the model group, the control group has certain effect of inhibiting the auricle swelling of the mice (26.4 percent, and P is less than 0.05). Namely, examples 4 to 5 and comparative example 2 all had inhibitory effects on the swelling of mouse pinna, and the inhibition rates were 50.1%, 54.2% and 26.4% respectively, as compared with the model group. The results show that the anti-inflammatory activity of the phytosterol ester fermentation product is increased by about 1 time compared with that of the unfermented phytosterol ester, and is close to that of the positive control drug clobetasol propionate.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.