CN115837060B - Application of millet ferment in preparation of medicine for preventing and treating eczema and cream - Google Patents

Abstract

The invention provides application of millet ferment in preparing a medicament for preventing and treating eczema, and a cream containing the millet ferment for preventing and treating eczema. The millet ferment can effectively relieve the degree of epidermal hypertrophy, keratinization and dermal edema caused by chronic eczema, improve the infiltration of eudermatitis cells, and the moisturizing component in the cream can make the millet ferment liquid cream have mild efficacy, small skin irritation and no obvious side effect, is beneficial to maintaining the skin barrier function, promotes the recovery and regulation of skin microcirculation of skin due to the skin damage such as scabbing, chapping, lichenification and the like caused by repeated itching and scratching, improves skin metabolism and eliminates skin nutrition loss. The invention provides a novel cream preparation for preventing and treating chronic eczema, and is expected to provide a novel scheme for clinical treatment.

Description

Application of millet ferment in preparation of medicine for preventing and treating eczema and cream

Field of the art

The invention belongs to the technical field of medicines, and relates to application of millet ferment in preparation of a medicament for preventing and treating eczema, and a cream containing the millet ferment for preventing and treating eczema.

(II) background art

Eczema is a common inflammatory skin condition of the epidermis and superficial dermis. The skin is characterized by pachylosis, redness, edema, itching and dry hair, which can be accompanied by foaming, cracking, crusting, flaking, bleeding or bleeding, and the course of the disease is irregular, and the itching is severe in repeated attacks. The pathogenesis of eczema is complex, and the etiology of the eczema is divided into two types: an internal cause and an external cause. Internal causes such as gastrointestinal dysfunction, mental stress, overfatigue, nerve dysfunction, endocrine disturbance, infection focus in vivo, etc. Eczema is caused by external factors such as cold, heat, sunlight, wind, scratching, friction, animal fur, chemical substances and the like.

Chronic eczema often results from repeated attacks of acute or subacute eczema without healing. Has the problems of high recurrence and difficult radical cure.

At present, the treatment of chronic eczema is divided into western medicines and traditional Chinese medicines. Wherein the western medicine is mainly glucocorticoid, antibiotic, immunosuppressant, antihistamine, etc. Western medicines have quick response to eczema, but have the defects of incomplete treatment, easy recurrence, large toxic and side effects after long-term application of hormone, and low patient compliance. The traditional Chinese medicine treatment is prepared by decocting, the raw materials and the preparation method are relatively simple, the active ingredients are difficult to completely extract, and certain heat-sensitive active ingredients can be damaged, so that various itch, infection and the like caused by skin eczema of the prepared medicine can not be relieved rapidly, and the ideal effect can not be achieved. In view of the above, the development of a safe and effective medicament with little or no side effect has practical significance for treating skin chronic eczema.

(III) summary of the invention

In order to solve the problems in the prior art, the invention provides application of millet ferment in preparing a medicament for preventing and treating eczema, and a cream containing the millet ferment for preventing and treating eczema.

In order to achieve the above purpose, the technical scheme of the invention is as follows:

the preparation method of the millet fermentation broth comprises the following steps:

application of millet ferment in preparing medicament for preventing and treating eczema is provided.

The millet ferment is prepared and obtained according to the following method:

(1) Soaking: soaking millet in water at 60-80 ℃ for 10-15 min according to a feed-liquid ratio of 1:10-20;

(2) Homogenizing by colloid mill: uniformly stirring the soaked millet and the soaking solution, pouring the mixture into a colloid mill, homogenizing and crushing for 3-5 min to obtain millet pulp;

(3) Gelatinization: heating millet paste to 80-100 ℃ and keeping for 15-20min to gelatinize millet;

(4) And (3) high-temperature sterilization: subpackaging the gelatinized millet paste, sterilizing at high temperature, and taking out and cooling to room temperature for standby;

(5) Inoculating and fermenting: lactobacillus plantarum and saccharomycetes are used for mixed fermentation, activated and rejuvenated strains are centrifuged, a culture medium is washed, sterile water is used for washing for 2-3 times, a starter is prepared by the sterile water, the starter is inoculated into sterilized millet pulp, and after uniform mixing, the millet pulp is cultured for 48-60 hours at the temperature of 30-40 ℃ to obtain millet fermentation liquor;

(6) Centrifuging the obtained millet fermentation liquor, taking supernatant, and freeze-drying to obtain millet fermentation product.

In particular, the medicament is a medicament for treating skin inflammatory skin thickening.

The medicament is prepared into one of the following dosage forms: cream, gel, lotion, spray.

The invention also relates to a cream for preventing and treating eczema, which is prepared from the following raw materials in parts by mass:

preferably, the cream is prepared from the following raw materials in parts by mass:

the millet ferment is prepared and obtained according to the following method:

(1) Soaking: soaking millet in water at 60-80 ℃ for 10-15 min according to a feed-liquid ratio of 1:10-20;

(2) Homogenizing by colloid mill: uniformly stirring the soaked millet and the soaking solution, pouring the mixture into a colloid mill, homogenizing and crushing for 3-5 min to obtain millet pulp;

(3) Gelatinization: heating millet paste to 80-100 ℃ and keeping for 15-20min to gelatinize millet;

(4) And (3) high-temperature sterilization: subpackaging the gelatinized millet paste, sterilizing at high temperature, and taking out and cooling to room temperature for standby;

(5) Inoculating and fermenting: lactobacillus plantarum and saccharomycetes are used for mixed fermentation, activated and rejuvenated strains are centrifuged, a culture medium is washed, sterile water is used for washing for 2-3 times, a starter is prepared by the sterile water, the starter is inoculated into sterilized millet pulp, and after uniform mixing, the millet pulp is cultured for 48-60 hours at the temperature of 30-40 ℃ to obtain millet fermentation liquor;

(6) Centrifuging the obtained millet fermentation liquor, taking supernatant, and freeze-drying to obtain millet fermentation product.

The cream is prepared by the following steps: uniformly mixing the polyethylene glycol-7-stearate, stearyl alcohol, oleoyl polyoxyethylene glyceride and ethylparaben according to a prescription ratio to obtain a phase A; uniformly mixing the millet fermentation broth and purified water to obtain a phase B; heating the phase A and the phase B in a water bath kettle respectively until transparent and clear solution is formed; stopping heating, dropwise adding the phase A into the phase B under the magnetic stirring condition at 70-72 ℃, continuously stirring until a uniform and stable white suspension is formed, magnetically stirring and cooling to room temperature, and obtaining the emulsifiable paste.

The beneficial effects of the invention are mainly as follows:

(1) The cream provided by the invention is convenient to carry, simple in use, convenient to apply and capable of being independently finished by users.

(2) The millet fermentation broth emulsifiable paste prepared by the invention has high active ingredient content and has remarkable effect of relieving chronic eczema. The invention has the advantages of mild drug effect, small skin irritation and no obvious side effect in the use process through a mouse experiment. In addition, the cream can form a transparent film when being coated on skin, has the effects of moisturizing skin, is beneficial to maintaining the skin barrier function, promotes the recovery and regulation of skin microcirculation of skin due to skin lesions such as scabs, chaps, lichenification and the like caused by repeated itching and scratching, improves skin metabolism and eliminates skin nutrition loss.

(3) The invention has the advantages of optimized production process, simple required conditions and equipment and mass production.

(IV) description of the drawings

FIG. 1 is a HE picture and skin thickness control (magnification: 200 times) of mice of different treatment groups.

Fig. 2 is a drawing of Filaggrin immunohistochemistry and area percent (magnification: 200 times) of mice of different treatment groups.

FIG. 3 is an immunohistochemical and magnification image (magnification: 200 times) of ICAM-1 of mice in different treatment groups.

Fig. 4 is a graph of infγ concentration analysis of mice from different treatment groups.

(fifth) detailed description of the invention

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:

example 1:

1. millet ferment preparation

(1) Soaking: soaking millet in purified water at 80deg.C for 15min at a feed-liquid ratio of 1:20 to obtain full soaking of millet.

(2) Homogenizing by colloid mill: uniformly stirring the soaked millet, pouring into a colloid mill, homogenizing and crushing for 3min.

(3) Gelatinization: heating the homogenized millet paste at high temperature (85deg.C) for 20min to gelatinize the millet.

(4) And (3) high-temperature sterilization: subpackaging the gelatinized millet paste into 250mL conical flasks, sterilizing at 115 ℃ for 12min, taking out after finishing, and cooling to room temperature for standby.

(5) Inoculating and fermenting: inoculating lactobacillus plantarum (Lp 299 vL) and saccharomycetes (CICC 1921) according to the proportion of 1:1 to obtain mixed bacterial liquid, centrifuging at 4 ℃ for 10min at 5000r/min, washing off a culture medium, repeatedly washing for 2 times with sterile water, preparing a starter by using the sterile water, inoculating the starter into sterilized millet paste at the volume ratio of 5%, uniformly mixing, and culturing at the temperature of 30-40 ℃ for 60h to obtain millet fermentation liquor.

(6) Centrifuging the obtained millet fermentation broth at 3500rpm/min for 10min, collecting supernatant, and lyophilizing to obtain millet fermentation product.

2. Preparation of the cream

(1) Tefose63, stearyl alcohol, oleoyl polyoxyethylene glyceride and ethylparaben are weighed, and the weight of the tefose63, the weight of the stearyl alcohol, the weight of the oleoyl polyoxyethylene glyceride and the weight of the ethylparaben are 18.0g, 2.0g, 5.0g and 0.1g in sequence, and the tefose63, the stearyl alcohol, the oleoyl polyoxyethylene glyceride and the ethylparaben are uniformly mixed to obtain a phase A.

(2) Phase A was placed in a 200ml beaker, heated in a water bath and stirred with a glass rod until completely dissolved and slowly warmed to 72 ℃.

(3) 2g of millet ferment was weighed and 77.9g of purified water was added to give phase B.

(4) Phase B was placed in a 200ml beaker, heated in a water bath and stirred with a glass rod until completely dissolved and slowly warmed to 70 ℃.

(5) And stopping heating, quickly taking out the beaker filled with the phase A and the phase B, placing the beaker with the phase B on a magnetic stirrer, slowly dropwise adding the phase A under the stirring condition, and stirring until the phase A is uniformly dissolved.

(6) After the phase A is completely added into the phase B, magnetically stirring for 30min, and subpackaging to obtain the emulsifiable paste.

3. Animal experiment

3.1 Experimental materials

Experimental animals:

clean grade 6 week old female C57BL/6 mice were purchased from the national academy of sciences of medicine, zhejiang. Mice were randomly divided into: untreated, DNCB (dinitrochlorobenzene) treated, millet ferment cream, matrix (cream without millet ferment), compound glycyrrhizin tablet (purchased from minox origin pharmaceutical co., japan) 4 animals per group were housed in separate cages.

3.2 feed:

the animal experiment center of Zhejiang province medical academy of sciences provides the basic feed for the mice.

3.3 reagent: DNCB (dinitrochlorobenzene), millet ferment cream, millet ferment-free cream and compound glycyrrhizin. Filaggrin (filaggrin) antibody kit, human intercellular adhesion molecule 1 (ICAM-1) detection kit.

3.3 Experimental methods

Experiment on the day mice were dehaired on the abdomen with an area of approximately 2cm x 2cm, and 25 μl of 0.5% dncb solution was applied to the dehaired site for 3 consecutive days once a day.

On day 4, the back of the mice was dehaired by about 2cm x 2cm, and the dehaired area was applied with 25 μl of 0.25% dncb solution daily at morning for 14 days, 1 time every 2 days.

The corresponding reagents described above were applied in different groups every afternoon during these 14 days. (DNCB solution is applied on the same day, after 6h from it, other cream applications are performed).

The abdominal skin of the mice was collected on day 18 for subsequent index detection.

3.5 detection of skin efficacy

(1) Hematoxylin-eosin staining observed skin morphology:

the slices were soaked in xylene for 2 times, 15min each time, the dewaxed slices were dehydrated step by step and placed in an aqueous hematoxylin solution for 5min, color separated in aqueous ammonia for several seconds. Washing with running water for 15min, and dehydrating with 70% (v/v) and 90% ethanol for 10min each. Adding 0.5% alcohol eosin staining solution for staining for 2min, washing with running water, and dehydrating the stained slice with pure alcohol. The cut part is placed in xylene for transparency for 3min multiplied by 2 times, and then is sealed by neutral resin, and is observed and analyzed by microscopic photographing.

(2) Immunohistochemical detection of Filaggrin and ICAM-1 expression:

1) Skin tissue paraffin sections were routinely deparaffinized to water.

2) Washing with distilled water, and soaking in PBS for 5min.

3)3％H ₂ O ₂ Treating at room temperature for 5-10 min to inactivate endogenous enzyme and washing with distilled water for 3 times.

4) The antigen is thermally repaired, the slice is immersed in 0.01M sodium citrate buffer solution (pH6.0), the electric furnace is heated to boiling and then is powered off, and after 5-10 min intervals, the process is repeated for 1-2 times. After cooling, PBS (pH 7.2-7.6) is washed 1-2 times. And dripping the sealing liquid at room temperature for 20min. And (5) removing redundant liquid without washing.

5) And (3) dripping diluted primary antibody working solution, and incubating at 37 ℃ for one night.

6) PBS rinse, 5min 3 times.

7) Dilute solution Bio-sheep anti-mouse lgG concentrate was diluted 1:100 into working solution, and Bio-sheep anti-mouse lgG working solution was added dropwise, incubated at 37℃for 2 hours, and then strictly operated according to the procedures of the specification.

8) DAB color development.

9) And (5) fully flushing with tap water.

10 Hematoxylin counterstain, hydrochloric acid alcohol differentiation.

11 Transparent, and sealing.

12 1 slice was observed per mouse, and each slice was counted under a microscope (200 x) with different fields of view.

13 Semi-quantitative analysis of the color area using ImageJ.

(3) ELISA method for detecting INFgamma content:

1) Taking a whole blood sample of the mouse, naturally agglutinating for 30min, centrifuging for 15min at 1000x g, and taking the supernatant for detection.

2) And (3) adding samples, namely respectively arranging blank holes, standard holes and sample holes to be tested. 100uL of sample diluent is added to a blank hole, 100uL of standard sample is added to a standard hole or 100uL of sample to be detected is added to a hole of an ELISA plate, air bubbles are not needed, and the sample is added to the bottom of the hole of the ELISA plate during sample adding, so that the hole wall is not touched as much as possible. Ensuring uninterrupted sample feeding and finishing the sample feeding for 5-10 min.

3) The ELISA plate is covered with a film and incubated for 120min at 37 ℃.

4) Washing, uncovering the sealing plate film, discarding liquid, beating residual liquid in the plate by using a towel or filter paper, washing the batten for 4 times by using washing liquid (1×), and ensuring that no residual liquid exists in the plate after the last washing, so that the towel or filter paper fiber is prevented from entering the plate, and the batten cannot be dried out after being cooled at room temperature for a long time.

And (3) adding samples, namely respectively arranging blank holes, standard holes and sample holes to be tested. 100uL of sample diluent is added to a blank hole, 100uL of standard sample is added to a standard hole or 100uL of sample to be detected is added to a hole of an ELISA plate, air bubbles are not needed, and the sample is added to the bottom of the hole of the ELISA plate during sample adding, so that the hole wall is not touched as much as possible. Ensuring uninterrupted sample feeding and finishing the sample feeding for 5-10 min.

5) 100uL (1X) of detection antibody was added to each well, the membrane was covered and incubated at 37℃for 1h. Washing is the same as in step 4.

6) 100uL (1X) of detection antibody was added to each well, the membrane was covered and incubated at 37℃for 40min. Washing is the same as in step 4.

7) Color development: TMB color development liquid 100uL is added into each hole, and the color development is carried out in a dark place at 37 ℃ for 15-20min (if the color is too light, the color development time can be prolonged appropriately and does not exceed 30 min).

8) And (3) terminating: 100uL of stop solution was added to each well, at which time the blue color changed to yellow. The addition sequence of the stop solution is consistent with that of the TMB color development solution.

9) Reading: the optical density (OD value) of each well was measured at 450nm using an enzyme-labeled instrument with 630nm as a calibration wavelength. Readings were taken within 5min after the addition of the stop solution.

(6) Statistical testing

Data analysis was performed using GraphPad 8.0 software, all data were expressed as mean ± standard deviation, one-way ANOVA (One-way ANOVA) was used for mean comparison between groups, P <0.05 was statistically significant for differences, and P <0.01 was statistically significant for differences.

4. Experimental results

(1) Hematoxylin-eosin staining observed skin morphology:

see figure 1 for HE pictures, skin lesion histopathological changes, and skin thickness control for the different treatment groups of mice.

As can be seen from fig. 1, the experimental results show that the skin tissue pathology of the untreated mice has no obvious change, and the epidermis and dermis have complete structures and distinct layers. Compared with untreated group, DNCB group has the characteristics of hypertrophic epidermis, hyperkeratosis, oedema of dermis layer, infiltration of a large number of neutrophils and lymphocytes, shrinkage of hair follicle, and overall characteristic of typical chronic eczema pathology, which indicates successful modeling. The millet ferment cream group and the compound glycyrrhizin group have obviously improved epidermis hypertrophy, keratinization and dermis edema degree, and the hair follicle sebaceous glands have no obvious inflammatory cell infiltration. The cream group without millet ferment has lighter skin damage degree than the model group, but the epidermis is slightly fat, and the dermis can be infiltrated by a small amount of inflammatory cells, so the effect is not obvious. Two different parts of the epidermis of each group of mice are randomly taken, the thickness of each group of mice is measured, and the calculated thickness is shown in the dotted graph of fig. 2. Of the different treatment groups, DNCB group skin thickness (18.159.+ -. 3.12) (P < 0.001) was the thickest, being significantly higher than the other treatment groups. The untreated group had no significant statistical difference (P > 0.05) from the millet ferment cream group and the compound glycyrrhizin group. Statistical analysis was performed on DNCB group, millet ferment cream group, matrix group, compound glycyrrhizin group, and the millet ferment cream group (17.321 ±2.534) and matrix group (32.191 ± 7.034) were significant (P < 0.004). The result shows that the millet ferment as an effective component has a key effect on repairing the epidermal hypertrophy, hyperplasia and hyperkeratosis caused by eczema.

(2) Immunohistochemical detection of Filaggrin and ICAM-1 expression:

filaggrin is an important molecule in the stratum corneum of human skin that links keratin fibers. filaggrin plays an important role in maintaining normal proliferation, keratinization, and maintenance of normal skin barrier function of skin keratinocytes, thereby preventing loss of epidermal moisture and invasion of external allergens. The immunohistochemical detection of the expression level of the Filaggrin is shown in figure 2, the Filaggrin is marked as light pink, the DNCB group has the most light obvious color, and the untreated group, the millet ferment cream group and the compound glycyrrhizin group have the light pink and light pink. By analyzing the occupied area of the light pink color of the Filaggrin in different groups, the DNCB group and the millet ferment cream group have significant statistical difference (P < 0.001), the untreated group and the millet ferment cream group have no significant statistical difference (P > 0.05), and the millet ferment cream group and the matrix group have slight difference (P < 0.003). The millet ferment cream is also very important in protecting the skin cuticle and protein fiber of human body, and forms a solid physical barrier of the outermost layer of epidermis.

ICAM-1 plays an important role in immune and inflammatory responses, and FIG. 3 is an enlarged view of the expression of ICAM-1 in different groups. The DNCB group had increased ICAM-1 expression compared to the other groups, and a large number of neutrophil and lymphocyte infiltrates. ICAM-1 expression was significantly down-regulated after the millet ferment cream group was used. The ICAM-1 expression level is sequentially selected from untreated group, compound glycyrrhizin group, millet ferment cream group, matrix group and DNCB group. The results suggest that millet ferment cream can achieve the aim of treating chronic eczema by regulating the immune system and inhibiting inflammatory response mechanism.

(3) ELISA method for detecting INFgamma content:

millet broth cream was also found to reduce inflammatory response by induced release of infγ by examining peripheral blood of different groups of mice, and the results are shown in fig. 4. IFN-gamma is a main factor secreted by Th1 cells, and can reflect the degree of Th1 and Th2 immune imbalance, thereby evaluating the disease condition and the degree of skin damage. Compared with untreated groups, DNCB groups are extremely remarkable (P < 0.0008), and compound glycyrrhizin groups, millet ferment cream groups and matrix groups have no statistical difference (P > 0.05). By carrying out statistical analysis on the millet ferment cream group, the matrix group and the compound glycyrrhizin group, the significant difference (P < 0.0005) exists between the millet ferment cream group and the matrix group, and further the effective components of the millet ferment cream can be suggested to relieve inflammatory reaction.

The results show that the millet ferment cream preparation prepared by the formula has the functions of resisting pathological changes of chronic eczema and inhibiting the infiltration of eudermatitis cells.

Claims (6)

1. The application of the millet ferment in preparing the medicine for preventing and treating eczema is characterized in that the millet ferment is prepared and obtained by the following method:

(1) Soaking: soaking millet in water at 60-80 ℃ for 10-15 min according to a feed-liquid ratio of 1:10-20;

(2) Homogenizing by colloid mill: uniformly stirring the soaked millet and the soaking solution, pouring the mixture into a colloid mill, homogenizing and crushing for 3-5 min to obtain millet slurry;

(3) Gelatinization: heating millet paste to 80-100 ℃ and keeping for 15-20min to gelatinize millet;

(4) And (3) high-temperature sterilization: subpackaging the gelatinized millet paste, sterilizing at high temperature, and taking out and cooling to room temperature for standby;

(5) Inoculating and fermenting: using lactobacillus plantarum and saccharomycetes for mixed fermentation, centrifuging activated and rejuvenated strains, washing a culture medium, washing the culture medium for 2-3 times with sterile water, preparing a starter with the sterile water, inoculating the starter into sterilized millet pulp, uniformly mixing, and culturing the millet pulp at 30-40 ℃ for 48-60 hours to obtain millet fermentation liquor;

(6) Centrifuging the obtained millet fermentation liquor, taking supernatant, and freeze-drying to obtain millet fermentation product.

2. The use according to claim 1, characterized in that the medicament is a medicament for the treatment of inflammatory skin thickening of the skin.

3. The use according to claim 1, wherein the medicament is formulated as one of the following: cream, gel, lotion, spray.

4. A emulsifiable paste for preventing and treating eczema is prepared from the following raw materials in parts by mass:

1-5% of millet ferment

10-20% of polyethylene glycol-7-stearate

Stearyl alcohol 1-3%

0.05-0.2% of ethylparaben

3-8% of oleoyl polyoxyethylene glyceride

The balance being purified water;

the millet ferment is prepared and obtained according to the following method:

(1) Soaking: soaking millet in water at 60-80 ℃ for 10-15 min according to a feed-liquid ratio of 1:10-20;

(2) Homogenizing by colloid mill: uniformly stirring the soaked millet and the soaking solution, pouring the mixture into a colloid mill, homogenizing and crushing for 3-5 min to obtain millet slurry;

(3) Gelatinization: heating millet paste to 80-100 ℃ and keeping for 15-20min to gelatinize millet;

(4) And (3) high-temperature sterilization: subpackaging the gelatinized millet paste, sterilizing at high temperature, and taking out and cooling to room temperature for standby;

(5) Inoculating and fermenting: using lactobacillus plantarum and saccharomycetes for mixed fermentation, centrifuging activated and rejuvenated strains, washing a culture medium, washing the culture medium for 2-3 times with sterile water, preparing a starter with the sterile water, inoculating the starter into sterilized millet pulp, uniformly mixing, and culturing the millet pulp at 30-40 ℃ for 48-60 hours to obtain millet fermentation liquor;

(6) Centrifuging the obtained millet fermentation liquor, taking supernatant, and freeze-drying to obtain millet fermentation product.

5. The cream according to claim 4, characterized in that it is made of the following raw materials by mass:

millet ferment 2%

Polyethylene glycol-7-stearate 18%

Stearyl alcohol 2%

0.1% of ethylparaben

Oleoyl polyoxyethylene glyceride 5%

The balance being purified water.

6. The cream according to claim 4 or 5, characterized in that it is prepared by the following method: uniformly mixing the polyethylene glycol-7-stearate, stearyl alcohol, oleoyl polyoxyethylene glyceride and ethylparaben according to a prescription ratio to obtain a phase A; uniformly mixing the millet fermentation liquor and purified water to obtain a phase B; heating the phase A and the phase B in a water bath kettle respectively until transparent and clear solution is formed; stopping heating, dropwise adding the phase A into the phase B under the magnetic stirring condition at 70-72 ℃, continuously stirring until a uniform and stable white suspension is formed, magnetically stirring and cooling to room temperature, and obtaining the emulsifiable paste.