CN112494578A - Chamomile fermentation liquor antibacterial gel and preparation method thereof - Google Patents
Chamomile fermentation liquor antibacterial gel and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of plant fermentation, and particularly relates to a chamomile fermentation liquid bacteriostatic gel and a preparation method thereof. Aiming at the problems of weak sterilization capability, complicated preparation process and the like of the existing antibacterial gel, the invention provides a preparation method of a chamomile fermentation liquor antibacterial gel, which comprises the following steps: a. preparing a fermentation raw material liquid; b. activating lactobacillus rhamnosus and lactobacillus plantarum strains; c. culturing the activated two strains to obtain two seed solutions; d. preparing plant fermentation liquor; e. preparing the antibacterial gel. According to the invention, chamomile and aloe raw materials are adopted to prepare the gel, so that damaged vaginal mucosa can be effectively repaired, the angelica sinensis cortex moutan is labor-intensive and itching-relieving, the two lactic acid bacteria can balance the flora in order to balance the vaginal flora and resist the propagation of harmful bacteria, the bacteriostatic ability reaches over 90%, and the vaginal gynecological diseases can be effectively relieved.
Description
Technical Field
The invention belongs to the technical field of plant fermentation, and particularly relates to a chamomile fermentation liquid bacteriostatic gel and a preparation method thereof.
Background
The female vagina has a special physiological structure and an anatomical structure, which causes the multiple gynecopathy of the female, mainly comprising vulvar diseases, vaginal diseases, uterine diseases, fallopian tube diseases, ovarian diseases and the like. Many people lack the attention to the cognition and physical health of gynecological diseases, and certain bad living habits gradually reduce the physiological health, so that some female diseases are entangled and can not be cured for a long time, and great inconvenience is brought to normal life and work.
Dysbacteriosis of the female genital tract is the root of gynecological diseases. Under physiological conditions, the vaginal ecological balance is maintained mainly by lactobacilli, estrogen and vaginal pH. The estrogen thickens the vaginal epithelium and increases the intracellular glycogen content, the vaginal epithelial cells decompose glycogen into monosaccharide, the vaginal lactobacillus converts the monosaccharide into lactic acid, the normal acidic environment of the vagina is maintained (the pH is less than or equal to 4.5 and is mostly 3.8-4.4), and the growth of other pathogens is inhibited. When the body resistance is low, the vaginal mucosa is injured or the microenvironment in the vagina is changed, the virulence of some resident flora in the vagina, such as bacteria, fungi, spirochetes, mycoplasma and the like, is increased, so that the outbreak of gynecological diseases is caused. In the prior art, some varieties of vaginal diseases for treating gynecological diseases are antibiotic products mainly used for killing, beneficial flora for protecting human bodies is killed while inflammation is cured, harmful bacteria are more wanting to be inundated once beneficial flora is not protected, and more gynecological diseases follow.
In recent years, antibacterial gels are widely researched, and because the gel material has biological adhesiveness and good biocompatibility, the gel can slowly diffuse to mucous membranes, has long retention time, is beneficial to thoroughly killing pathogens, and is favored by patients due to the advantages of unique lubricity, cool feeling, convenience in use and the like. Therefore, the research on the gynecological external antibacterial gel with good curative effect and small side effect has very important practical significance.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: the existing antibacterial gel has the problems of weak sterilization capability, complex preparation process and the like.
The technical scheme for solving the technical problems comprises the following steps: the preparation method of the chamomile fermentation liquid bacteriostatic gel comprises the following steps:
a. preparation of fermentation feedstock
Preparing raw materials, by weight, 40-70 parts of chamomile, 0.1-0.2 part of angelica sinensis, 0.1-0.4 part of cortex moutan, 800-1000 parts of water, 0.3-0.8 part of cellulase, 0.1-0.4 part of pectinase and 60-150 parts of aloe juice; crushing chamomile and angelica sinensis cortex moutan, adding water, adding cellulase and pectinase, performing enzymolysis for 2-6 hours at 45-55 ℃, cooling to room temperature, filtering, taking supernate, adding aloe juice, mixing uniformly, and sterilizing to obtain a fermentation raw material solution;
b. activated bacterial strain
Inoculating lactobacillus rhamnosus and lactobacillus plantarum in an MRS liquid culture medium, and culturing in an incubator at 37 ℃ for 24h to obtain activated lactobacillus rhamnosus and lactobacillus plantarum strains;
c. seed culture
B, respectively putting the two activated strains in the step b into an MRS liquid culture medium, culturing at the constant temperature of 37 ℃ for 10-12 h, centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate with a PBS buffer solution to obtain two seed solutions;
d. preparation of plant fermentation broth
B, inoculating the two seed solutions obtained in the step c into the fermentation raw material solution obtained in the step a, fermenting for 1-7 days, adding chitosan, settling for 5-10 hours, filtering, and sterilizing the supernatant to obtain plant fermentation liquor;
e. preparing bacteriostatic gel
The raw materials comprise: 2-6 parts of hydroxypropyl methyl cellulose, 5-30 parts of hydroxyethyl cellulose, 10-50 parts of glycerol and 0.1-0.8 part of stachyose, 20-60 parts of the plant fermentation liquid obtained in the step d, 600 parts of water, 30-50 parts of chitosan, 10-30 parts of lactic acid and 200-600 parts of calcium chloride solution;
adding hydroxypropyl methylcellulose, hydroxyethyl cellulose, glycerol, stachyose, and plant fermentation liquid into water, dripping two drops of tea tree essential oil, and mixing; sequentially adding chitosan and lactic acid, stirring into gel, adding calcium chloride solution, and mixing to obtain antibacterial gel.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the enzyme activity of the cellulase in the step a is more than or equal to 1 wu/g.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the enzyme activity of the pectinase obtained in the step a is more than or equal to 50 wu/g.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the raw materials in the step a are as follows: 45-60 parts of chamomile, 0.1-0.15 part of angelica, 0.15-0.35 part of cortex moutan, 800-950 parts of water, 0.45-0.65 part of cellulase, 0.2-0.35 part of pectinase and 80-120 parts of aloe juice by weight; the enzymolysis temperature is 45-50 ℃, and the enzymolysis time is 3-5 h.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the lactobacillus rhamnosus strain obtained in the step b is purchased from a strain with the preservation number of ATCC7469, and the lactobacillus plantarum strain is purchased from a strain with the preservation number of ATCC 8014.
In the preparation method of the chamomile fermentation liquid antibacterial gel, the inoculation amounts of the lactobacillus rhamnosus and the lactobacillus plantarum in the step d are respectively 5-10%, and the inoculation concentrations are respectively 1.0 multiplied by 106～1.0×108CFU/mL。
In the preparation method of the chamomile fermentation liquid antibacterial gel, the addition amount of the chitosan in the step d is 2-10% of the total volume of the solution, and preferably 5-8%.
In the preparation method of the chamomile fermentation liquid antibacterial gel, the fermentation temperature in the step d is 37 ℃, the fermentation time is 2-4 days, and the settling time is 6-8 hours.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the concentration of the calcium chloride solution in the step e is 0.5%.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the raw materials in the step e comprise: the plant fermentation liquid comprises, by weight, 3-5 parts of hydroxypropyl methyl cellulose, 5-20 parts of hydroxyethyl cellulose, 20-40 parts of glycerol, 0.2-0.5 part of stachyose, 30-50 parts of plant fermentation liquid, 30-45 parts of chitosan, 15-28 parts of lactic acid and 300-500 parts of calcium chloride solution.
Furthermore, in the preparation method of the chamomile fermentation liquid antibacterial gel, in the raw materials in the step e, the viscosity of hydroxymethyl propyl cellulose is 400mPa.s, the viscosity of hydroxyethyl cellulose is 5000mPa.s, the purity of stachyose is 85%, and the deacetylation degree of chitosan is 85% -90%.
The invention also provides the chamomile fermentation liquid bacteriostatic gel prepared by the method.
The invention has the beneficial effects that:
the invention provides a preparation method of a chamomile fermentation liquid antibacterial gel, chamomile is used as a raw material, and lactobacillus rhamnosus and lactobacillus plantarum are used for fermentation to prepare the antibacterial gel, chamomile and aloe can effectively repair damaged vaginal mucosa, the addition of angelica and cortex moutan mainly has the effects of relieving itching and easing pain, two lactic acid bacteria are used for balancing vaginal flora and resisting the propagation of harmful bacteria, glycerin is mainly used as a humectant, chitosan is used as a main gel matrix, and the antibacterial gel has good adhesiveness and biocompatibility, prolongs the acting time of the gel, and better exerts curative effect. The chamomile fermentation liquid antibacterial gel disclosed by the invention is strong in antibacterial ability, safe, free of side effects, capable of relieving itching and pain, repairing damaged mucosa and effectively improving female gynecological inflammation.
Drawings
FIG. 1 is a diagram showing the bacteriostatic results of the bacteriostatic gel of the chamomile fermentation liquid prepared in the example.
Detailed Description
The invention provides a preparation method of a chamomile fermentation liquid bacteriostatic gel, which comprises the following steps:
a. preparation of fermentation feedstock
Preparing raw materials, by weight, 40-70 parts of chamomile, 0.1-0.2 part of angelica sinensis, 0.1-0.4 part of cortex moutan, 800-1000 parts of water, 0.3-0.8 part of cellulase, 0.1-0.4 part of pectinase and 60-150 parts of aloe juice; crushing chamomile and angelica sinensis cortex moutan, adding water, adding cellulase and pectinase, performing enzymolysis for 2-6 hours at 45-55 ℃, cooling to room temperature, filtering, taking supernate, adding aloe juice, mixing uniformly, and sterilizing to obtain a fermentation raw material solution;
b. activated bacterial strain
Inoculating lactobacillus rhamnosus and lactobacillus plantarum in an MRS liquid culture medium, and culturing in an incubator at 37 ℃ for 24h to obtain activated lactobacillus rhamnosus and lactobacillus plantarum strains;
c. seed culture
B, respectively putting the two activated strains in the step b into an MRS liquid culture medium, culturing at the constant temperature of 37 ℃ for 10-12 h, centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate with a PBS buffer solution to obtain two seed solutions;
d. preparation of plant fermentation broth
B, inoculating the two seed solutions obtained in the step c into the fermentation raw material solution obtained in the step a, fermenting for 1-7 days, adding chitosan, settling for 5-10 hours, filtering, and sterilizing the supernatant to obtain plant fermentation liquor;
e. preparing bacteriostatic gel
The raw materials comprise: 2-6 parts of hydroxypropyl methyl cellulose, 5-30 parts of hydroxyethyl cellulose, 10-50 parts of glycerol and 0.1-0.8 part of stachyose, 20-60 parts of the plant fermentation liquid obtained in the step d, 600 parts of water, 30-50 parts of chitosan, 10-30 parts of lactic acid and 200-600 parts of calcium chloride solution;
adding hydroxypropyl methylcellulose, hydroxyethyl cellulose, glycerol, stachyose, and plant fermentation liquid into water, dripping two drops of tea tree essential oil, and mixing; sequentially adding chitosan and lactic acid, stirring into gel, adding calcium chloride solution, and mixing to obtain antibacterial gel.
In order to better perform fermentation, in the preparation method of the chamomile fermentation liquid antibacterial gel, the enzyme activity of the cellulase in the step a is more than or equal to 1 wu/g.
In order to better perform fermentation, in the preparation method of the chamomile fermentation liquid antibacterial gel, the enzyme activity of the pectinase in the step a is more than or equal to 50 wu/g.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the raw materials in the step a are as follows: 45-60 parts of chamomile, 0.1-0.15 part of angelica, 0.15-0.35 part of cortex moutan, 800-950 parts of water, 0.45-0.65 part of cellulase, 0.2-0.35 part of pectinase and 80-120 parts of aloe juice by weight; the enzymolysis temperature is 45-50 ℃, and the enzymolysis time is 3-5 h.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the lactobacillus rhamnosus strain obtained in the step b is purchased from a strain with the preservation number of ATCC7469, and the lactobacillus plantarum strain is purchased from a strain with the preservation number of ATCC 8014. According to the invention, the lactobacillus rhamnosus and the lactobacillus plantarum are adopted to prepare the antibacterial gel, the lactobacillus rhamnosus is beneficial lactobacillus separated from human intestinal tracts and can adjust intestinal flora, and researches show that when the lactobacillus rhamnosus is used for preparing the gynecological antibacterial gel, the lactobacillus rhamnosus can also adjust vaginal flora balance, inhibit the propagation and growth of harmful bacteria and promote the flora balance. The lactobacillus plantarum is only reported in research on regulating flora in human gastrointestinal tracts, and particularly used for regulating female vaginal flora, and can inhibit reproduction of vaginal harmful flora, improve vaginal environment and prevent gynecological diseases.
In the preparation method of the chamomile fermentation liquor bacteriostatic gel, the inoculation amounts of lactobacillus rhamnosus and lactobacillus plantarum in the step d are respectively 5-10%, and the inoculation concentrations are respectively 1.0 multiplied by 10 so as to balance flora and inhibit the growth and reproduction of harmful bacteria6～1.0×108CFU/m L。
In the preparation method of the chamomile fermentation liquid antibacterial gel, the addition amount of the chitosan in the step d is 2-10% of the total volume of the solution, and preferably 5-8%. The invention adopts chitosan to prepare the gel, has good adhesiveness and biocompatibility, can prolong the acting time of the gel and better exert the curative effect.
In the preparation method of the chamomile fermentation liquid antibacterial gel, the fermentation temperature in the step d is 37 ℃, the fermentation time is 2-4 days, and the settling time is 6-8 hours.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the concentration of the calcium chloride solution in the step e is 0.5%.
In the preparation method of the chamomile fermentation liquid bacteriostatic gel, the raw materials in the step e comprise: the plant fermentation liquid comprises, by weight, 3-5 parts of hydroxypropyl methyl cellulose, 5-20 parts of hydroxyethyl cellulose, 20-40 parts of glycerol, 0.2-0.5 part of stachyose, 30-50 parts of plant fermentation liquid, 30-45 parts of chitosan, 15-28 parts of lactic acid and 300-500 parts of calcium chloride solution.
Furthermore, in the preparation method of the chamomile fermentation liquid antibacterial gel, in the raw materials in the step e, the viscosity of hydroxymethyl propyl cellulose is 400mPa.s, the viscosity of hydroxyethyl cellulose is 5000mPa.s, the purity of stachyose is 85%, and the deacetylation degree of chitosan is 85% -90%.
The invention also provides the chamomile fermentation liquid bacteriostatic gel prepared by the method.
The following examples are intended to illustrate specific embodiments of the present invention without limiting the scope of the invention to the examples.
Example 1 preparation of Chamomile fermentation broth bacteriostatic gel
The specific operation steps are as follows:
(1) taking 45 parts of chamomile, 0.1 part of angelica and 0.15 part of cortex moutan by weight, crushing, adding 800 parts of pure water, adding 0.45 part of cellulase and 0.2 part of pectinase, carrying out enzymolysis at 45 ℃ for 3 hours, cooling to room temperature, filtering to obtain supernatant, namely plant enzymolysis liquid, taking 80 parts of fresh aloe juice, uniformly mixing, and carrying out pasteurization to room temperature to obtain fermentation raw material liquid;
(2) respectively sucking Lactobacillus rhamnosus (Lactobacillus rhamnous) ATCC7469 and Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014 which are stored in a freeze-drying tube dissolved in sterile water, inoculating the Lactobacillus rhamnosus ATCC7469 and the Lactobacillus plantarum ATCC8014 into a 5 mM MRS liquid culture medium, and culturing the Lactobacillus rhamnosus and the Lactobacillus plantarum in an incubator at 37 ℃ for 24 hours to obtain activated Lactobacillus rhamnosus and Lactobacillus plantarum strains;
(3) respectively putting 2mL of the two activated strains obtained in the step (2) into fresh MRS liquid culture media, and culturing in a constant-temperature incubator at 37 ℃ to obtain a culture solution; centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate obtained by centrifugation with sterile PBS buffer solution to obtain seed solution;
(4) inoculating 5% of the seed liquid obtained in the step (3) into the fermentation raw material liquid obtained in the step (1) according to the volume percentage, fermenting for 2 days at 37 ℃, adding 5% of chitosan in volume ratio of the fermentation liquid, settling for 6 hours, filtering the fermentation liquid to obtain clarified liquid, pasteurizing the clarified liquid, and cooling to obtain plant fermentation liquid;
(5) preparing gynecological bacteriostatic gel: soaking 3 parts of hydroxypropyl methyl cellulose, 10 parts of hydroxyethyl cellulose, 20 parts of glycerol, 0.2 part of stachyose, 30 parts of plant fermentation liquor and 600 parts of pure water, adding 0.1 mu L of tea tree essential oil after fully dissolving, and uniformly mixing;
weighing 30 parts of chitosan, adding the chitosan into the solution, stirring the chitosan uniformly, adding 18 parts of lactic acid, stirring the mixture into gel, finally adding 300 parts of 0.5% calcium chloride solution, and mixing the mixture uniformly to obtain the gynecological antibacterial gel 1.
Example 2 preparation of Chamomile fermentation broth bacteriostatic gel
The specific operation steps are as follows:
(1) taking 60 parts of chamomile, 0.15 part of angelica and 0.2 part of cortex moutan by weight parts, crushing, adding 920 parts of pure water, adding 0.55 part of cellulase and 0.2 part of pectinase, carrying out enzymolysis for 3 hours at 45 ℃, cooling to room temperature, filtering to obtain supernatant, namely plant enzymolysis liquid, taking 90 parts of fresh aloe juice, uniformly mixing, and carrying out pasteurization to room temperature to obtain fermentation raw material liquid;
(2) respectively sucking Lactobacillus rhamnosus (Lactobacillus rhamnous) ATCC7469 and Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014 which are stored in a freeze-drying tube dissolved in sterile water, inoculating the Lactobacillus rhamnosus ATCC7469 and the Lactobacillus plantarum ATCC8014 into a 5 mM MRS liquid culture medium, and culturing the Lactobacillus rhamnosus and the Lactobacillus plantarum in an incubator at 37 ℃ for 24 hours to obtain activated Lactobacillus rhamnosus and Lactobacillus plantarum strains;
(3) respectively putting 2mL of the two activated strains obtained in the step (2) into fresh MRS liquid culture media, and culturing in a constant-temperature incubator at 37 ℃ to obtain a culture solution; centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate obtained by centrifugation with sterile PBS buffer solution to obtain seed solution;
(4) inoculating 10% of the seed liquid obtained in the step (3) into the fermentation raw material liquid obtained in the step (1) according to the volume percentage, fermenting for 3 days at 37 ℃, adding chitosan accounting for 5% of the volume ratio of the fermentation liquid, settling for 6 hours, filtering the fermentation liquid to obtain clear liquid, pasteurizing the clear liquid, and cooling to obtain plant fermentation liquid;
(5) preparing gynecological bacteriostatic gel: soaking 4.5 parts of hydroxypropyl methyl cellulose, 15 parts of hydroxyethyl cellulose, 20 parts of glycerol, 0.2 part of stachyose, 30 parts of plant fermentation liquor and 600 parts of pure water, adding 0.1 mu L of tea tree essential oil after fully dissolving, and uniformly mixing;
weighing 30 parts of chitosan, adding the chitosan into the solution, stirring the chitosan uniformly, adding 18 parts of lactic acid, stirring the mixture into gel, finally adding 300 parts of 0.5% calcium chloride solution, and mixing the mixture uniformly to obtain the gynecological antibacterial gel 2.
Example 3 preparation of Chamomile fermentation broth bacteriostatic gel
The specific operation steps are as follows:
(1) taking 60 parts of chamomile, 0.15 part of angelica and 0.3 part of cortex moutan by weight parts, crushing, adding 900 parts of pure water, adding 0.6 part of cellulase and 0.3 part of pectinase, carrying out enzymolysis at 45 ℃ for 4 hours, cooling to room temperature, filtering to obtain supernatant, namely plant enzymolysis liquid, taking 100 parts of fresh aloe juice, uniformly mixing, and carrying out pasteurization to room temperature to obtain fermentation raw material liquid;
(2) respectively sucking Lactobacillus rhamnosus (Lactobacillus rhamnous) ATCC7469 and Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014 which are stored in a freeze-drying tube dissolved in sterile water, inoculating the Lactobacillus rhamnosus ATCC7469 and the Lactobacillus plantarum ATCC8014 into a 5 mM MRS liquid culture medium, and culturing the Lactobacillus rhamnosus and the Lactobacillus plantarum in an incubator at 37 ℃ for 24 hours to obtain activated Lactobacillus rhamnosus and Lactobacillus plantarum strains;
(3) respectively putting 2mL of the two activated strains obtained in the step (2) into fresh MRS liquid culture media, and culturing in a constant-temperature incubator at 37 ℃ to obtain a culture solution; centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate obtained by centrifugation with sterile PBS buffer solution to obtain seed solution;
(4) inoculating 10% of the seed liquid obtained in the step (3) into the fermentation raw material liquid obtained in the step (1) according to the volume percentage, fermenting for 3 days at 37 ℃, adding chitosan accounting for 5% of the volume ratio of the fermentation liquid, settling for 7 hours, filtering the fermentation liquid to obtain clear liquid, pasteurizing the clear liquid, and cooling to obtain plant fermentation liquid;
(5) preparing gynecological bacteriostatic gel: soaking 4 parts of hydroxypropyl methyl cellulose, 20 parts of hydroxyethyl cellulose, 30 parts of glycerol, 0.2 part of stachyose, 30 parts of plant fermentation liquor and 600 parts of pure water, adding 0.2 mu L of tea tree essential oil after fully dissolving, and uniformly mixing;
weighing 40 parts of chitosan, adding the chitosan into the solution, stirring the chitosan uniformly, adding 25 parts of lactic acid, stirring the mixture into gel, finally adding 400 parts of 0.5% calcium chloride solution, and mixing the mixture uniformly to obtain the gynecological antibacterial gel 3.
Example 4 preparation of Chamomile fermentation broth bacteriostatic gel
The specific operation steps are as follows:
(1) taking 60 parts of chamomile, 0.15 part of angelica and 0.3 part of cortex moutan by weight parts, crushing, adding 900 parts of pure water, adding 0.6 part of cellulase and 0.3 part of pectinase, carrying out enzymolysis at 50 ℃ for 4 hours, cooling to room temperature, filtering to obtain supernatant, namely plant enzymolysis liquid, taking 100 parts of fresh aloe juice, uniformly mixing, and carrying out pasteurization to room temperature to obtain fermentation raw material liquid;
(2) respectively sucking Lactobacillus rhamnosus (Lactobacillus rhamnous) ATCC7469 and Lactobacillus plantarum (Lactobacillus plantarum) ATCC8014 which are stored in a freeze-drying tube dissolved in sterile water, inoculating the Lactobacillus rhamnosus ATCC7469 and the Lactobacillus plantarum ATCC8014 into a 5 mM MRS liquid culture medium, and culturing the Lactobacillus rhamnosus and the Lactobacillus plantarum in an incubator at 37 ℃ for 24 hours to obtain activated Lactobacillus rhamnosus and Lactobacillus plantarum strains;
(3) respectively putting 2mL of the two activated strains obtained in the step (2) into fresh MRS liquid culture media, and culturing in a constant-temperature incubator at 37 ℃ to obtain a culture solution; centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate obtained by centrifugation with sterile PBS buffer solution to obtain seed solution;
(4) inoculating 10% of the seed liquid obtained in the step (3) into the fermentation raw material liquid obtained in the step (1) according to the volume percentage, fermenting for 4 days at 37 ℃, adding chitosan accounting for 5% of the volume ratio of the fermentation liquid, settling for 6 hours, filtering the fermentation liquid to obtain clear liquid, pasteurizing the clear liquid, and cooling to obtain plant fermentation liquid;
(5) preparing gynecological bacteriostatic gel: soaking 4 parts of hydroxypropyl methyl cellulose, 15 parts of hydroxyethyl cellulose, 30 parts of glycerol, 0.2 part of stachyose, 40 parts of plant fermentation liquor and 600 parts of pure water, adding 0.2 mu L of tea tree essential oil after fully dissolving, and uniformly mixing;
weighing 35 parts of chitosan, adding the chitosan into the solution, stirring the chitosan uniformly, adding 25 parts of lactic acid, stirring the mixture into gel, finally adding 450 parts of 0.5% calcium chloride solution, and mixing the mixture uniformly to obtain the gynecological antibacterial gel 4.
Effect verification
And (3) testing the antibacterial effect:
the gynecological bacteriostatic gel prepared in the examples 1-4 is tested for bacteriostatic effect, and the method comprises the following steps:
the bacteriostasis test mainly adopts a bacteriostasis ring method to test whether the gel has the inhibition effect on escherichia coli (8099), staphylococcus aureus (ATCC 6538) and candida albicans (ATCC 10231).
(1) The test bacteria were washed 24h fresh slant cultures with PBS and diluted to about 5.0X 10 with PBS5CFU/mL～4.5×106CFU/mL bacterial suspension is ready for use.
(2) The prepared agar medium was sterilized and poured into culture plates, approximately 25mL per plate, in a clean bench and allowed to set until needed. 0.5mL of the bacterial liquid is added into the plate, and the bacterial liquid is uniformly coated by a coater.
(3) And vertically placing an oxford cup on the surface of the culture medium, and slightly pressurizing to ensure that the oxford cup is firmly fixed on the culture medium. Different volumes of bacteriostatic gel were added to the cups. Culturing at 37 ℃, culturing bacterial propagules for 24 hours, and observing the final result; the Candida albicans is cultured for 48h to observe the final result. The bacteriostatic effect is shown in figure 1.
As can be seen from the figure 1, the bacteriostatic gel obtained in the embodiments 1 to 4 has a good bacteriostatic effect on Escherichia coli, Staphylococcus aureus and Candida albicans, wherein the diameter of the bacteriostatic ring is more than or equal to 12mm, and the bacteriostatic requirement is met.
And (3) testing the bacteriostatic rate:
the gynecological bacteriostatic gel prepared in the examples 1-4 is tested for bacteriostatic rate, and the determination method is as follows:
(1) test strains: staphylococcus aureus (ATCC 6538), Escherichia coli (8099), Candida albicans (ATCC 10231). 0.03mol/L Phosphate Buffered Saline (PBS) (pH7.2-7.4), culture medium: the culture of Staphylococcus aureus and Escherichia coli was performed using trypticase Soytone agar medium, and the culture of Candida albicans was performed using Sabouraud agar medium.
(2) Equipment: a constant temperature water bath box, a timer, an ultra-clean workbench and the like. The carrier is 10mm × 10mm degreased white plain cloth, the degreasing method is carried out according to the sterilization technical specification (2002 edition), and the carrier is sterilized by pressure steam for standby before use.
(3) The method comprises the following operation steps: the test bacteria were washed 24h fresh slant cultures with PBS and diluted to about 5.0X 10 with PBS6CFU/mL～5.0×107CFU/mL was made into a bacterial suspension for use. Dripping 10 μ L of the bacterial suspension on a sterilization carrier by using a micropipettor, and drying at 36 +/-1 ℃ orAnd drying at room temperature for later use. Weighing the sample according to the amount of 5 g/piece, placing the sample in a sterile plate, placing the sterile plate in a water bath at the temperature of 20 +/-1 ℃ for 5min, taking the carrier of the infected bacteria by using sterile forceps, completely immersing the carrier in the sample, and immediately timing. And (3) when the interaction between the carrier and the sample is carried out for the specified time in the specification, adding the carrier into a 5.0mL PBS test tube, mixing uniformly, oscillating, washing the test bacteria, sucking 1.0mL of sample liquid respectively, measuring the number of surviving bacteria according to a viable bacteria culture counting method, and inoculating 2 plates into each tube of sample liquid. If the number of colonies growing on the plate is large, the plate can be serially diluted 10 times with PBS and then counted by viable bacteria culture. The same batch of PBS and culture medium were used as negative controls. All test samples and control samples are cultured at 36 +/-1 ℃, and bacterial propagules are cultured for 48h to observe results; the Candida albicans was cultured for 72h for observation. The test is repeated for 3 times, and the bacteriostasis rate is calculated.
(4) Calculation of the bacteriostatic Rate
In the formula:
x-bacteriostasis rate,%;
A0-the amount of recovered bacteria in the positive control group in CFU/mL;
A1recovery of bacterial load in CFU/mL in the test group.
(5) And (4) judging a result: the bacteriostasis rate is 50-90 percent, and the antibacterial effect is judged; the bacteriostasis rate is more than or equal to 90 percent, and the antibacterial effect is stronger. The test results are shown in Table 1.
TABLE 1 results of the bacteriostatic ratio test
The results in table 1 show that the carrier soaked in the gel obtained in the example has fewer viable bacteria, and even in example 4, the bacteriostatic rate reaches 100%, which indicates that the bacteriostatic gel prepared by the invention has stronger bacteriostatic action, can effectively kill pathogenic bacteria and balance vaginal bacterial colonies.
The embodiment shows that the chamomile fermentation liquid bacteriostatic gel provided by the invention has a strong bacteriostatic action, has strong affinity with a human body, can be used for inhibiting bacteria, balancing the colony environment in a vagina, repairing the damaged mucous membrane and has a good gynecological bacteriostatic action. The gel of the invention has simple preparation method, easily obtained raw materials and good practical value.
Claims (10)
1. The preparation method of the chamomile fermentation liquid bacteriostatic gel is characterized by comprising the following steps of:
a. preparation of fermentation feedstock
Preparing raw materials, by weight, 40-70 parts of chamomile, 0.1-0.2 part of angelica sinensis, 0.1-0.4 part of cortex moutan, 800-1000 parts of water, 0.3-0.8 part of cellulase, 0.1-0.4 part of pectinase and 60-150 parts of aloe juice; crushing chamomile and angelica sinensis cortex moutan, adding water, adding cellulase and pectinase, performing enzymolysis for 2-6 hours at 45-55 ℃, cooling to room temperature, filtering, taking supernate, adding aloe juice, mixing uniformly, and sterilizing to obtain a fermentation raw material solution;
b. activated bacterial strain
Inoculating lactobacillus rhamnosus and lactobacillus plantarum in an MRS liquid culture medium, and culturing in an incubator at 37 ℃ for 24h to obtain activated lactobacillus rhamnosus and lactobacillus plantarum strains;
c. seed culture
B, respectively putting the two activated strains in the step b into an MRS liquid culture medium, culturing at the constant temperature of 37 ℃ for 10-12 h, centrifuging the culture solution at 3000rpm for 10min, and dispersing the precipitate with a PBS buffer solution to obtain two seed solutions;
d. preparation of plant fermentation broth
B, inoculating the two seed solutions obtained in the step c into the fermentation raw material solution obtained in the step a, fermenting for 1-7 days, adding chitosan, settling for 5-10 hours, filtering, and sterilizing the supernatant to obtain plant fermentation liquor;
e. preparing bacteriostatic gel
The raw materials comprise: 2-6 parts of hydroxypropyl methyl cellulose, 5-30 parts of hydroxyethyl cellulose, 10-50 parts of glycerol and 0.1-0.8 part of stachyose, 20-60 parts of the plant fermentation liquid obtained in the step d, 600 parts of water, 30-50 parts of chitosan, 10-30 parts of lactic acid and 200-600 parts of calcium chloride solution;
adding hydroxypropyl methylcellulose, hydroxyethyl cellulose, glycerol, stachyose, and plant fermentation liquid into water, dripping two drops of tea tree essential oil, and mixing; sequentially adding chitosan and lactic acid, stirring into gel, adding calcium chloride solution, and mixing to obtain antibacterial gel.
2. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: the enzyme activity of the cellulase in the step a is more than or equal to 1 wu/g; the enzyme activity of the pectinase is more than or equal to 50 wu/g.
3. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: the raw materials in the step a are as follows: 45-60 parts of chamomile, 0.1-0.15 part of angelica, 0.15-0.35 part of cortex moutan, 800-950 parts of water, 0.45-0.65 part of cellulase, 0.2-0.35 part of pectinase and 80-120 parts of aloe juice by weight; the enzymolysis temperature is 45-50 ℃, and the enzymolysis time is 3-5 h.
4. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: the lactobacillus rhamnosus strain obtained in the step b is purchased from a strain with the preservation number of ATCC7469, and the lactobacillus plantarum strain is purchased from a strain with the preservation number of ATCC 8014.
5. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: d, the inoculation amount of the lactobacillus rhamnosus and the lactobacillus plantarum is 5-10% respectively, and the inoculation concentration is 1.0 multiplied by 10 respectively6～1.0×108CFU/mL。
6. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: and d, adding the chitosan in an amount of 2-10% of the total volume of the solution, preferably 5-8%.
7. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: and d, fermenting at 37 ℃ for 2-4 days, and settling for 6-8 h.
8. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: the raw materials in the step e comprise: the plant fermentation liquid comprises, by weight, 3-5 parts of hydroxypropyl methyl cellulose, 5-20 parts of hydroxyethyl cellulose, 20-40 parts of glycerol, 0.2-0.5 part of stachyose, 30-50 parts of plant fermentation liquid, 30-45 parts of chitosan, 15-28 parts of lactic acid and 300-500 parts of calcium chloride solution.
9. The preparation method of the chamomile fermentation broth bacteriostatic gel according to claim 1, which is characterized in that: in the raw materials in the step e, the viscosity of the hydroxymethyl propyl cellulose is 400mPa.s, the viscosity of the hydroxyethyl cellulose is 5000mPa.s, the purity of the stachyose is 85 percent, and the deacetylation degree of the chitosan is 85 to 90 percent.
10. The chamomile fermentation liquid bacteriostatic gel prepared by the method of any one of claims 1-9.
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