CN115957148A - Nutrient containing composite plant antibacterial peptide and microcapsule thereof - Google Patents
Nutrient containing composite plant antibacterial peptide and microcapsule thereof Download PDFInfo
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- CN115957148A CN115957148A CN202310037579.8A CN202310037579A CN115957148A CN 115957148 A CN115957148 A CN 115957148A CN 202310037579 A CN202310037579 A CN 202310037579A CN 115957148 A CN115957148 A CN 115957148A
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention provides a nutrient containing composite plant antibacterial peptide, which is prepared by fermenting a fermentation substrate containing composite plant protein by using bacillus subtilis, wherein the plant raw materials for extracting the composite plant protein comprise gastrodia elata, pseudo-ginseng, ginkgo, sweet wormwood, honeysuckle, rosemary and purslane. In the invention, the composite vegetable protein extracted from specific plant raw materials is fermented by using bacillus subtilis, on one hand, the bacillus subtilis can carry out enzymolysis on the composite vegetable protein extracted from the specific plant raw materials in the fermentation process to prepare the high-activity composite plant antibacterial peptide, the composite plant antibacterial peptide has broad-spectrum antibacterial action and has remarkable antibacterial and bacteriostatic activity on bacteria such as propionibacterium, staphylococcus aureus, malassezia and the like and dermatophytes, on the other hand, the composite plant antibacterial peptide can play a good desensitization effect on the composite vegetable protein through fermentation treatment, and the prepared composite plant antibacterial peptide has excellent mildness and biological safety.
Description
Technical Field
The invention belongs to the technical field of plant and microorganism application, and particularly relates to a nutrient containing composite plant antibacterial peptide and a microcapsule thereof.
Background
Proteins are important components of human body, such as skin, hair, nails and various organs, and the proteins play important physiological functions in maintaining normal metabolism of the body, repairing and updating body tissues, providing energy and the like. Because the plant protein has various varieties and abundant resources, the active ingredients of the plant protein have good efficacy and small side effect, the plant protein has good affinity to skin and scalp, and can improve the skin moisturizing performance, the natural beauty care product mainly based on the plant protein is more and more favored by consumers.
The vegetable protein used in the daily chemical products is generally hydrolysate of vegetable protein and derivatives of the hydrolysate. The hydrolysate of plant protein, namely plant polypeptide, can be processed by different methods such as chemical hydrolysis, enzymatic hydrolysis, microbial fermentation and the like. The relative molecular mass of the plant polypeptide is about 1000-30000 Da, has good physiological activity, is easy to be absorbed by human body, and has the functions of nutrition, film forming and moisture preservation. Therefore, the application of the compound in daily necessities and cosmetics is very wide.
The plant protein and the polypeptide thereof have great development potential as protein sources for sustainable application in the fields of food industry, daily chemical industry and the like. In the development process of plant protein resources, the quality and functional characteristics of plant protein varieties are important concerns. The antibacterial performance and the antiallergic performance are important performances of daily chemical products, but many daily chemical products at present cannot solve skin problems caused by bacteria and fungi and allergy problems caused by vegetable protein allergens, and have potential risks in the safety aspect of vegetable polypeptide raw materials and products. Therefore, the provision of plant polypeptides with antibacterial and hypoallergenic effects, which are applied to daily chemical care products, thereby inhibiting fungi on skin and other parts and ensuring the safety of the skin, becomes a new idea in the daily chemical industry.
Disclosure of Invention
The invention aims to provide a nutrient containing composite plant antibacterial peptide and a microcapsule thereof, which have excellent antibacterial and bacteriostatic activity, low sensitization and good biological safety.
According to one aspect of the invention, the nutrient containing the composite plant antibacterial peptide is prepared by fermenting a fermentation substrate containing composite plant protein by using bacillus subtilis, wherein plant raw materials for extracting the composite plant protein comprise gastrodia elata, pseudo-ginseng, ginkgo, sweet wormwood, honeysuckle, rosemary and purslane.
In the invention, the composite vegetable protein extracted from specific plant raw materials is fermented by using bacillus subtilis, on one hand, the bacillus subtilis can carry out enzymolysis on the composite vegetable protein extracted from the specific plant raw materials in the fermentation process to prepare the high-activity composite plant antibacterial peptide, the composite plant antibacterial peptide has broad-spectrum antibacterial action and has remarkable antibacterial and bacteriostatic activity on bacteria such as propionibacterium, staphylococcus aureus, malassezia and the like and dermatophytes, on the other hand, the composite plant antibacterial peptide can play a good desensitization effect on the composite vegetable protein through fermentation treatment, and the prepared composite plant antibacterial peptide has excellent mildness and biological safety.
Preferably, in the plant material, the ratio of gastrodia elata: pseudo-ginseng: and (3) ginkgo biloba: sweet wormwood herb: and (3) moxa: honeysuckle flower: rosemary: 0.5-2 parts of purslane, namely 0.5-1 part of purslane. Through reasonable setting of the plant raw material ratio, the composite plant protein extracted from the plant raw materials has excellent antibacterial and bacteriostatic properties and is mild.
Preferably, in the plant material, the ratio of gastrodia elata: pseudo-ginseng: ginkgo biloba: sweet wormwood herb: and (3) moxa: honeysuckle flower: rosemary: 1. The composite plant protein extracted from the plant raw materials with the mixture ratio has more excellent broad-spectrum antibacterial performance and mildness.
Preferably, the preparation method of the composite vegetable protein comprises the following steps:
s1, performing wall breaking treatment on the plant raw materials to obtain composite plant powder.
S2, carrying out low-temperature treatment on the composite plant powder, wherein the temperature of the low-temperature treatment is-60 ℃ to-30 ℃, and the time of the low-temperature treatment is 12-24 hours.
S3, after the low-temperature treatment is finished, adding hot water into the composite plant powder, wherein the water temperature of the hot water is 60-80 ℃, and taking the obtained mixed solution as a mixed plant solution.
S4, salting out the mixed plant solution, and collecting precipitate containing the composite plant protein.
By utilizing the preparation method of the composite vegetable protein, the plant raw material can be treated on the premise of ensuring the activity of the effective components of the plant raw material, the composite vegetable protein can be fully extracted and obtained, the extraction rate of the composite vegetable protein is ensured, and the preparation method is simple and convenient.
Preferably, the ratio of the mixed plant powder to hot water is 1g.
Preferably, in the above S3, after adding hot water and stirring, further performing an ultrasonic operation to obtain a mixed plant solution.
Preferably, the intensity of the ultrasonic operation is 200-600 w, and the ultrasonic time is 10-40 min.
Preferably, the stirring temperature is 30-50 ℃ and the stirring time is 4-6 h in the stirring and centrifuging process of the mixed plant solution and the salted-out mixed solution.
Preferably, the centrifugal rotation speed is 6000-8000 r/min and the centrifugal time is 10-20 min in the stirring and centrifugation process of the mixed plant solution and the salted-out mixed solution.
Preferably, the preparation method of the composite vegetable protein further comprises the following steps:
s5, dissolving the precipitate containing the composite vegetable protein by using a phosphate buffer solution with the pH value of 5.0-7.0, separating insoluble substances, and collecting clear liquid.
And S6, drying the clear liquid to remove the solvent, and obtaining the solid material which is the composite vegetable protein.
Preferably, in S6, the drying treatment of the clear liquid is vacuum freeze drying, wherein the temperature of the vacuum freeze drying is-60 ℃ to-40 ℃, and the time of the vacuum freeze drying is 6-8 h.
Preferably, the content of the composite vegetable protein in the fermentation substrate is 2.85-6.65% by mass percentage.
Preferably, the fermentation substrate is a composite plant protein fermentation culture medium, and the raw materials for preparing the composite plant protein fermentation culture medium comprise, by mass, 6-10 parts of composite plant protein, 0.1-0.3 part of glucose, 0.3-0.7 part of sodium chloride, 0.01-0.02 part of camphorsulfonic acid and 150-200 parts of water. The composite protein fermentation culture medium prepared by the composite vegetable protein can ensure that the bacillus subtilis as a fermentation microbial inoculum fully exerts the fermentation activity in the composite protein fermentation culture medium, thereby ensuring the conversion rate of the composite vegetable protein to the composite plant antibacterial peptide.
Preferably, the pH value of the composite plant protein fermentation medium is 6.6-6.8.
Preferably, the preparation method of the composite plant antibacterial peptide comprises the following steps: sterilizing the composite vegetable protein fermentation culture medium at 110-120 ℃ for 20-30 min, inoculating bacillus subtilis into the sterilized composite vegetable protein fermentation culture medium, and after fermentation is finished, heating the fermentation product to sterilize the fermentation product; and centrifuging the sterilized fermentation product, taking supernatant, filtering, taking filtrate, and drying. In particular cases, the fermentation product may optionally be heat sterilized in a form that provides a boiling water bath (in which the fermentation product is placed in boiling water with its vessel).
Preferably, the inoculation amount of the bacillus subtilis in the fermentation substrate is 1.6-1.8 multiplied by 10 viable count 8 cfu/mL。
Preferably, in the process of fermenting the fermentation substrate containing the composite vegetable protein by using the bacillus subtilis, the fermentation time is 18 to 20 hours, and the fermentation temperature is 30 to 40 ℃. The bacillus subtilis can keep excellent fermentation activity by fermenting under the fermentation time and the fermentation temperature, so that the bacillus subtilis can efficiently and fully carry out enzymolysis on the composite plant protein into the composite plant polypeptide in the fermentation process, the effect of improving the extraction rate of the composite plant antibacterial peptide is achieved, and meanwhile, the process of forming the composite plant antibacterial peptide by carrying out enzymolysis on the composite plant protein can be accelerated.
Preferably, after the fermentation treatment is completed, the composite vegetable protein fermentation medium is sterilized and centrifuged, and clear liquid is collected and filtered and purified by adopting a 0.45 mu m filter membrane and a 10kDa ultrafiltration membrane in sequence.
Preferably, after the fermentation treatment is completed, the method further comprises the operation of drying the fermentation product: the drying treatment is realized by adopting a spray drying mode, and the spray drying condition is 0.08-0.2 atmospheric pressure and 70-130 ℃; and/or the drying treatment is realized by adopting a vacuum freeze drying mode, wherein the drying temperature of the vacuum freeze drying is-55 ℃ to-40 ℃.
According to another aspect of the present invention, there is provided an antibacterial microcapsule comprising the complex plant antibacterial peptide-containing nutrient as described above; wherein, the raw materials for preparing the antibacterial microcapsule comprise 0.002-0.02% of composite plant antibacterial peptide, 10-30% of glycerol, 0.5-2.6% of polyglyceryl nonionic surfactant and 0.0002-0.2% of glucan by weight percentage. In the invention, a double-layer molecular structure is formed by adopting a polyglyceryl nonionic surfactant, so that the composite plant antibacterial peptide can be spontaneously wrapped in a microcapsule double-layer formed by the nonionic surfactant, thereby forming a high-stability microcapsule inclusion structure; and the microcapsule bilayer structure is similar to a bilayer phospholipid structure of skin cells, so that the composite plant antibacterial peptide can be better absorbed by the skin, and the permeability of the composite plant antibacterial peptide is obviously improved.
Preferably, the preparation method of the antibacterial microcapsule comprises the following steps: fully stirring and dissolving 0.002-0.02% of composite plant antibacterial peptide, 0.2-2% of polyglycerol-10 diisostearate, 0.3-0.6% of polyglycerol-10 laurate, 10-30% of glycerol and 0.0002-0.2% of glucan to obtain a uniform mixed solution; carrying out wrapping auxiliary treatment on the mixed solution to obtain an aqueous suspension; and removing impurities to obtain the antibacterial microcapsule.
Preferably, the wrapping-assistance treatment is selected from one of high-voltage pulsed electric field assistance, ultrasonic assistance, and free diffusion.
Preferably, the wrapping auxiliary treatment is high-voltage pulse electric field auxiliary, and the high-voltage pulse electric field technology is beneficial to improving the wrapping efficiency of the composite plant antibacterial peptide in the microcapsule.
Preferably, the technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 0.5 kV/cm-2 kV/cm, the number of pulse treatments is 10-60, and the material conductivity is 2000 mu S/cm-6000 mu S/cm.
According to another aspect of the present invention, there is provided a daily chemical product comprising the antibacterial microcapsules, wherein the daily chemical product is one of a skin care lotion, a shampoo, a body wash, a cream, a face cleanser, an essence or a lotion.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following will clearly and completely describe the technical solution of the present invention with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
A preparation method of microcapsules containing composite plant antibacterial peptides comprises the following steps:
1. preparation of composite vegetable protein
The preparation method comprises the following steps of S1, uniformly mixing and crushing gastrodia elata, pseudo-ginseng, ginkgo, sweet wormwood, honeysuckle, rosemary and purslane in a mass part ratio of 1;
s2, performing low-temperature freezing treatment on the composite plant powder obtained in the step S1, wherein the freezing treatment temperature is-45 ℃ and the freezing treatment time is 18 hours;
s3, after the low-temperature freezing treatment is finished, adding hot water with the water temperature of 68 ℃ into the composite plant powder and stirring, wherein the ratio of the mixed plant powder to the hot water is 1g;
s4, centrifuging the mixed plant solution obtained in the step S3, collecting supernatant, adding ammonium sulfate solid into the supernatant, stirring and centrifuging (the stirring temperature is 40 ℃, the stirring time is 6 hours, the centrifugal rotation speed is 8000r/min, and the centrifugal time is 15 min), and collecting precipitate containing the composite plant protein;
s5, adding a phosphate buffer solution with the pH value of 6.5 into the precipitate obtained in the step S4, dissolving the precipitate, performing ultrafiltration, and collecting clear liquid to obtain a mixed plant crude protein solution (the ultrafiltration adopts a 100KDa ultrafiltration membrane);
s6, carrying out vacuum freeze-drying treatment on the mixed crude plant protein solution, wherein the temperature of vacuum freeze-drying is-55 ℃, the time of vacuum freeze-drying is 6.5 hours, and the finally obtained solid material is the composite plant protein.
2. Preparation of composite vegetable protein fermentation culture medium
And (2) dissolving 10 parts of the composite plant protein prepared in the step (1), 0.2 part of glucose, 0.5 part of sodium chloride and 0.01 part of camphorsulfonic acid in 160 parts of distilled water, adjusting the pH to 6.6, and sterilizing at 110 ℃ for 30min to obtain the composite plant protein fermentation medium.
3. Preparation of nutrient containing composite plant antibacterial peptide
Inoculating bacillus subtilis to the composite vegetable protein fermentation culture medium prepared in the step 2 (the inoculation amount of the bacillus subtilis is 3 percent of the volume of the vegetable protein fermentation culture medium, and the viable count of the bacillus subtilis is 1.7 multiplied by 10 8 cfu/mL), fermenting at 34 ℃ for 18h, and sterilizing in a boiling water bath after the fermentation is finished; centrifuging the sterilized composite plant protein fermentation culture medium (the centrifugation speed is 8000r/min, the centrifugation time is 15 min), taking the supernatant, filtering (the filtering method is that a 0.45 mu m filter membrane is adopted for filtering, then a 10kDa ultrafiltration membrane is adopted for ultrafiltration), taking the filtrate for drying (the drying method is vacuum freeze drying at-50 ℃), and obtaining the nutrient containing the composite plant antibacterial peptide.
4. Preparation of antibacterial microcapsules
Taking 0.01% of the nutrient containing the composite plant antibacterial peptide prepared in the step 3, 0.1% of polyglycerol-10 diisostearate, 0.4% of polyglycerol-10 laurate, 25% of glycerol and 0.1% of glucan, and fully stirring and dissolving to obtain a uniform mixed solution; carrying out high-voltage pulse electric field auxiliary treatment on the mixed solution, wherein the technical parameters of the high-voltage pulse electric field are as follows: the field intensity is 1kV/cm, the number of pulse treatments is 60, the material conductivity is 4000 mu S/cm, an aqueous suspension is obtained, and the antibacterial microcapsule is obtained after impurity removal.
Example 2
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in the preparation of the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein are composed of gastrodia elata, pseudo-ginseng, ginkgo biloba, sweet wormwood, honeysuckle, rosemary and purslane, wherein the weight ratio of gastrodia elata, pseudo-ginseng, ginkgo biloba, sweet wormwood, honeysuckle, rosemary and purslane is 0.5. Except for the differences described above, the materials and process operations used in this example were strictly consistent with example 1.
Example 3
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in the preparation of the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein are composed of gastrodia elata, pseudo-ginseng, ginkgo biloba, sweet wormwood, honeysuckle, rosemary and purslane, wherein the weight ratio of gastrodia elata, pseudo-ginseng, ginkgo biloba, sweet wormwood, honeysuckle, rosemary and purslane is (1. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 4
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in the preparation process of the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein comprise gastrodia elata, pseudo-ginseng, ginkgo, sweet wormwood, honeysuckle, rosemary and purslane, wherein the weight ratio of the gastrodia elata, the pseudo-ginseng, the ginkgo, the sweet wormwood, the honeysuckle, the rosemary and the purslane is 0.3. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 5
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in the preparation process of the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein comprise gastrodia elata, pseudo-ginseng, ginkgo, sweet wormwood, honeysuckle, rosemary and purslane, wherein the weight ratio of the gastrodia elata, the pseudo-ginseng, the ginkgo, the sweet wormwood, the honeysuckle, the rosemary and the purslane is (1). Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 6
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in this example, the freezing temperature of-45 ℃ in step S2 of example 1 was replaced by-38 ℃ in the process of preparing antibacterial microcapsules. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 7
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the step S3 of example 1 is replaced by adding hot water with the water temperature of 68 ℃ to dissolve the composite plant powder, and adding water with the water temperature of 25 ℃ to dissolve the composite plant powder. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 8
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: this example replaces the fermentation at 34 c in step 3 of example 1 with a 28 c fermentation in the preparation of antimicrobial microcapsules. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Example 9
This example refers to example 1 to prepare antibacterial microcapsules, which differs from example 1 in that: this example replaces the fermentation at 34 c in step 3 of example 1 with a 42 c fermentation in the preparation of antimicrobial microcapsules. Except for the differences, the materials and process operations used in this example were strictly consistent with those of example 1.
Comparative example 1
This comparative example an antibacterial microcapsule was prepared with reference to example 1, which differs from example 1 in that: this comparative example omits the fermentation operation in step 3 of example 1 in the process of preparing antibacterial microcapsules. In addition to the differences described above, the materials used and the process operation in this comparative example remained strictly identical to example 1.
Comparative example 2
This comparative example an antibacterial microcapsule was prepared with reference to example 1, which differs from example 1 in that: this comparative example in the process of preparing the antibacterial microcapsule, bacillus subtilis was inoculated into the composite vegetable protein fermentation medium in step 3 of example 1, instead of bacillus licheniformis. In addition to the differences described above, the materials used and the process operation in this comparative example remained strictly identical to example 1.
Comparative example 3
This comparative example an antibacterial microcapsule was prepared with reference to example 1, which differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the culture medium for inoculating the bacillus subtilis is a culture medium without composite plant protein, and the material composition of the culture medium is that peptone with equal mass parts is adopted to replace the composite plant protein used for preparing the composite plant protein fermentation culture medium in the step 2 of the example 1. Except for the differences described above, the materials used and the process operation in this comparative example were strictly in accordance with example 1.
Comparative example 4
This comparative example prepares antibacterial microcapsules with reference to example 1, and differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the plant material for extracting the composite plant protein consists of only artemisia apiacea. In addition to the differences described above, the materials used and the process operation in this comparative example remained strictly identical to example 1.
Comparative example 5
This comparative example an antibacterial microcapsule was prepared with reference to example 1, which differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the plant material for extracting the composite plant protein was composed of only rosemary. In addition to the differences described above, the materials used and the process operation in this comparative example remained strictly identical to example 1.
Comparative example 6
This comparative example prepares antibacterial microcapsules with reference to example 1, and differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein comprise gastrodia elata, gingko, wormwood and rosemary, wherein the gastrodia elata: and (3) ginkgo biloba: and (3) moxa: rosemary = 1. Except for the differences described above, the materials used and the process operation in this comparative example were strictly in accordance with example 1.
Comparative example 7
This comparative example an antibacterial microcapsule was prepared with reference to example 1, which differs from example 1 in that: in the process of preparing the antibacterial microcapsule, the plant raw materials for extracting the composite plant protein comprise pseudo-ginseng, sweet wormwood, honeysuckle and purslane, wherein the pseudo-ginseng comprises the following components in parts by mass: sweet wormwood herb: honeysuckle flower: purslane = 1. In addition to the differences described above, the materials used and the process operation in this comparative example remained strictly identical to example 1.
Test example 1
1. Test objects: the microcapsules containing complex plant antibacterial peptides obtained in examples 1 to 9 and comparative examples 1 to 7.
2. The test method comprises the following steps:
(1) And (3) testing antibacterial and bacteriostatic properties: the test is carried out according to the content specified in the national standard QB/T2738-2012 evaluation method for antibacterial and bacteriostatic effects of daily chemical products. And (3) testing the bacteriostatic rate and the bacteriostatic effect of bacteria and dermatophytes such as propionibacterium, staphylococcus aureus, malassezia, pityrosporum ovale, trichophyton rubrum, microsporum canis and the like. Wherein, the bacteriostasis rate = (total number of colonies on the culture medium of the control group-total number of colonies on the culture medium of the test group)/total number of colonies on the culture medium of the control group x 100%. Evaluation standard of antibacterial effect: the bacteriostatic rate is less than 50%, the bacteriostatic effect is graded (-), and the product has no bacteriostatic action; the bacteriostasis rate is more than or equal to 50 percent, and the bacteriostasis effect is graded (+), so that the product has the bacteriostasis function; the antibacterial rate is more than or equal to 90 percent, the antibacterial effect is graded (+ +), and the product has strong antibacterial effect; the antibacterial rate is more than or equal to 99 percent, the antibacterial effect is graded (+++), and the product has strong antibacterial and bacteriostatic effects.
(2) Skin irritation test: a skin irritation test was performed using a mouse as a skin test subject. The hairs on both sides of the spinal column of the back of the mouse are cut off without damaging the epidermis, and the hair removal range is about 2cm multiplied by 2cm. 1mL of the sample to be tested was dropped on two layers of sterilized gauze (1.5 cm. Times.1.5 cm), and the gauze soaked with the sample to be tested was covered on the exposed skin area of the mouse and fixed with a transparent adhesive tape. The application time is 4h, and after continuously applying for 4h, residual test samples are washed off by warm water to mark the tested part. After 1h of removing the samples, the skin reaction of the tested part is observed, the result is interpreted, and the result is interpreted again after 24h and 48h. Skin irritation test scoring criteria: in the skin irritation test, the scoring criteria are as follows: skin does not have obvious discomfort reaction, and the skin state rating is four-star; the skin appears light erythema and is not infiltrated, and the skin state rating is four-star; it is four-star that the skin has obvious or large-area erythema and no infiltration, and the skin state rating is good; it is four-star that the skin shows obvious or large-area erythema accompanied by small-area edema; it is possible that the skin is rated for a marked or extensive erythema with marked edema, papules or blisters.
TABLE 1 results of antibacterial and bacteriostatic property test of examples 1 to 9 and comparative examples 1 to 7
TABLE 2 skin irritation test results of examples 1 to 9 and comparative examples 1 to 7
3. And (3) analyzing a test result:
the results of the antibacterial and bacteriostatic performance test and the skin irritation test are shown in tables 1 and 2. As shown in tables 1 and 2, the composite plant antibacterial peptide provided by the invention has strong killing effect on bacteria and dermatophytes, and has good mildness.
From tables 1 and 2, it can be seen that the composition of the plant materials, the ratio of the plant materials, whether fermentation is performed or not, the selection of the fermentation strain, the fermentation temperature in the process of preparing the composite plant antibacterial peptide, the low temperature treatment temperature in the process of preparing the composite plant protein and the temperature for adding hot water to dissolve the composite plant antibacterial peptide can have certain influence on the antibacterial and bacteriostatic performance and the mild type of the composite plant antibacterial peptide.
The results of the performance tests corresponding to example 1 are compared with examples 2, 3, 4, 5 and comparative examples 3, 4, 5, 6, 7. As can be seen from tables 1 and 2, under the same conditions of other components and operations for preparing the composite plant antibacterial peptide, the fermentation substrate of comparative example 3 is a conventional fermentation medium without composite plant protein, while the fermentation substrate of example 1 is a composite protein medium containing composite plant protein, and compared with the composite plant antibacterial peptide prepared in example 1, the reference product provided in comparative example 3 basically cannot show obvious antibacterial and bacteriostatic properties and shows obvious sensitization; under the condition that other components and operations for preparing the composite plant antibacterial peptide are the same, the plant raw material for preparing the antibacterial peptide in the comparative example 4 only comprises sweet wormwood, and the plant raw material for preparing the antibacterial peptide in the comparative example 5 only comprises rosemary, and the test results show that the antibacterial and bacteriostatic properties and the broad-spectrum antibacterial property of the composite plant antibacterial peptide obtained in the comparative examples 4 and 5 are obviously lower than those of the composite plant antibacterial peptide prepared from the plant raw material provided in the embodiment 1 of the invention; under the condition that other components and operations for preparing the composite plant antibacterial peptide are the same, plant raw materials for preparing the antibacterial peptide in the comparative example 6 comprise the following components in parts by mass of 1; in contrast, the plant raw materials for preparing the composite plant antibacterial peptide in examples 2 and 3 respectively comprise the following components in parts by mass of 0.5. In contrast, the plant raw materials for preparing the composite plant antibacterial peptide in examples 4 and 5 respectively comprise gastrodia elata, panax notoginseng, ginkgo biloba, sweet wormwood, honeysuckle, rosemary and purslane in a mass ratio of 0.3. Therefore, the composite plant antibacterial peptide prepared by extracting the composite plant protein according to the components and the proportion of the plant raw materials has antibacterial and bacteriostatic activity, and has low sensitization and good biological safety, so that the antibacterial microcapsule provided by the invention has excellent antibacterial performance and low sensitization.
The results of the corresponding performance tests in tables 1, 2 of example 1 and examples 8, 9 and comparative examples 1, 2 are compared. As can be seen from tables 1 and 2, under the same conditions of other components and operations for preparing the composite plant antibacterial peptide, the composite plant antibacterial peptide obtained in comparative example 1 without fermentation has lower antibacterial and bacteriostatic properties and shows more obvious allergenicity; in addition, in the comparative example 2, bacillus licheniformis is adopted to replace bacillus subtilis as a fermentation inoculant in the process of preparing the composite plant antibacterial peptide, and compared with the example 1, the composite plant antibacterial peptide prepared in the comparative example 2 has lower antibacterial and bacteriostatic performance and poorer mildness. Therefore, the composite plant antibacterial peptide obtained by fermenting the composite plant protein fermentation culture medium by using the bacillus subtilis can better extract antibacterial active ingredients in plant raw materials, and the prepared composite plant antibacterial peptide has good biological safety by fermenting and selecting proper strains to be beneficial to not generating sensitization, so that the antibacterial microcapsule provided by the invention has the characteristics of excellent antibacterial and bacteriostatic properties and high temperature and type. Under the same conditions of components and operation for preparing the composite plant antibacterial peptide, compared with the composite plant antibacterial peptide obtained at the fermentation temperature of 34 ℃, the composite plant antibacterial peptide obtained by respectively fermenting at the temperature of 28 ℃ and 42 ℃ in the embodiments 8 and 9 has lower antibacterial and bacteriostatic properties and lower mild type. Therefore, the invention can better extract and maintain the antibacterial active ingredients in the plant raw materials by reasonably setting the fermentation temperature, so that the antibacterial microcapsule has excellent antibacterial and bacteriostatic properties and low sensitization.
Comparing the corresponding performance test results of the embodiment 1 and the embodiments 6 and 7 in the tables 1 and 2, under the condition that other components and operations for preparing the composite plant antibacterial peptide are the same, the antibacterial and bacteriostatic performance and the broad-spectrum antibacterial performance of the composite plant protein obtained in the embodiment 6 at the freezing temperature of-38 ℃ are slightly lower than those of the composite plant protein obtained at the freezing temperature of-45 ℃; in example 7, the antibacterial and bacteriostatic properties and broad-spectrum antibacterial properties of the composite vegetable protein obtained by adding hot water at a water temperature of 25 ℃ in step S3 were slightly lower than those of the composite vegetable protein obtained by adding water at a water temperature of 68 ℃. Thus, compared with examples 4 and 5, example 1 performs the operations of freezing and reheating, and reasonably sets the temperature of the frozen plant material and the temperature of the plant material dissolved by adding water, which is beneficial to ensuring the activity of effective antibacterial ingredients in the plant material and fully extracting the active ingredients in the composite plant, so that the antibacterial peptide prepared from the composite plant protein has better antibacterial and bacteriostatic effects and broad-spectrum antibacterial effects.
Although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that various changes, modifications and equivalents may be made therein without departing from the spirit and scope of the invention.
Claims (10)
1. A nutrient containing composite plant antibacterial peptide, which is characterized in that: the nutrient is prepared by fermenting a fermentation substrate containing composite vegetable protein by using bacillus subtilis, wherein the plant raw materials for extracting the composite vegetable protein comprise gastrodia elata, pseudo-ginseng, gingko, sweet wormwood, honeysuckle, rosemary and purslane.
2. The complex plant antimicrobial peptide-containing nutrient according to claim 1, wherein: according to the mass ratio, in the plant raw materials, the gastrodia elata: the pseudo-ginseng: the ginkgo biloba: the sweet wormwood herb: the wormwood is: the honeysuckle flower: the rosemary is: 0.5 to 2, wherein the purslane =0.5 to 1.
3. The complex plant antimicrobial peptide-containing nutrient according to claim 1, wherein: the preparation method of the composite vegetable protein comprises the following steps:
s1, performing wall breaking treatment on the plant raw materials to obtain composite plant powder;
s2, carrying out low-temperature treatment on the composite plant powder, wherein the temperature of the low-temperature treatment is-60 ℃ to-30 ℃, and the time of the low-temperature treatment is 12-24 hours;
s3, after the low-temperature treatment is finished, adding hot water into the composite plant powder, wherein the water temperature of the hot water is 60-80 ℃, and taking the obtained mixed solution as a mixed plant solution;
s4, salting out the mixed plant solution, and collecting the precipitate containing the composite plant protein.
4. The complex plant antimicrobial peptide-containing nutrient according to claim 3, wherein: the preparation method of the composite vegetable protein also comprises the following steps:
s5, dissolving the precipitate by using a phosphate buffer solution with the pH value of 5.0-7.0, separating insoluble substances, and collecting clear liquid;
and S6, drying the clear liquid to remove the solvent, and obtaining the solid material which is the composite vegetable protein.
5. The complex plant antimicrobial peptide-containing nutrient according to any one of claims 1 to 4, wherein: the content of the composite plant protein in the fermentation substrate is 2.85-6.65% by mass percent.
6. The complex plant antimicrobial peptide-containing nutrient according to claim 5, wherein: the fermentation substrate is a composite plant protein fermentation culture medium, and the raw materials for preparing the composite plant protein fermentation culture medium comprise, by mass, 6-10 parts of the composite plant protein, 0.1-0.3 part of glucose, 0.3-0.7 part of sodium chloride, 0.01-0.02 part of camphorsulfonic acid and 150-200 parts of water.
7. The complex plant antimicrobial peptide-containing nutrient according to claim 6, wherein: the inoculation amount of the bacillus subtilis in the fermentation substrate meets the condition that the viable count is 1.6-1.8 multiplied by 10 8 cfu/mL。
8. The complex plant antimicrobial peptide-containing nutrient according to claim 1, wherein: in the fermentation treatment, the fermentation time is 18-20 hours, and the fermentation temperature is 30-40 ℃.
9. An antibacterial microcapsule, which is characterized in that: the antimicrobial microcapsules comprising the nutrient of any one of claims 1 to 8;
wherein, the raw materials for preparing the antibacterial microcapsule comprise, by mass, 0.002-0.02% of the composite plant antibacterial peptide, 10-30% of glycerol, 0.5-2.6% of polyglyceryl nonionic surfactant and 0.0002-0.2% of glucan.
10. A daily chemical product characterized by: the daily chemical product comprises the antibacterial microcapsule according to claim 9, and the daily chemical product is one of skin care lotion, shampoo, shower gel, cream, face wash, essence or lotion.
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