CN113712888A

CN113712888A - Royal jelly separated liquid fermented product, skin external preparation containing the same, and preparation method and application thereof

Info

Publication number: CN113712888A
Application number: CN202111041355.1A
Authority: CN
Inventors: 史豆豆; 邓朝庆
Original assignee: Zhejiang Chenhai Life Science Co ltd
Current assignee: Zhejiang Chenhai Life Science Co ltd
Priority date: 2021-09-06
Filing date: 2021-09-06
Publication date: 2021-11-30
Anticipated expiration: 2041-09-06
Also published as: CN113712888B

Abstract

The invention discloses a fermented product of royal jelly separating liquid, a skin external preparation containing the fermented product, a preparation method and application of the fermented product. The preparation method of the fermented product of the royal jelly separation liquid comprises the following steps: inoculating lactobacillus into the fermentation substrate, fermenting, culturing, and sterilizing; wherein the fermentation substrate comprises a royal jelly separating medium which is a material prepared by removing protein from fresh royal jelly. The royal jelly isolate fermentation product prepared by the invention has good antioxidant and anti-inflammatory effects, has the effect of promoting the growth of human skin fibroblasts, has high safety, does not irritate the skin, has simple preparation process and low preparation cost, and can be widely applied to the field of skin external preparations.

Description

Royal jelly separated liquid fermented product, skin external preparation containing the same, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of fermentation, and particularly relates to a royal jelly isolate fermentation product, a skin external preparation containing the same, a preparation method and application.

Background

The royal jelly is a milky substance secreted by worker bee after eating honey and bee pollen, and is completely secreted by bee itself. Royal jelly contains abundant proteins, for example, royal jelly contains a large amount of royal jelly main proteins, so that the royal jelly is concerned about and becomes a research hotspot because of multiple effects of promoting cell growth, resisting tumors, resisting bacteria and the like. At present, China develops and produces functional beverages by using royal jelly main protein in royal jelly. After royal jelly main protein is extracted from royal jelly, a large amount of royal jelly separation liquid can be generated. The royal jelly separating liquid is transparent green or yellow green, contains a large amount of organic acids, vitamins, amino acids, polypeptides and other rich nutrient substances and active ingredients, but is generally discarded as waste, and the utilization rate is low, so that the resource waste is caused.

At present, the active substance extraction methods commonly used in the field of skin external preparations include water extraction, organic solvent extraction, ultrasonic extraction, microwave extraction, supercritical fluid extraction, microbial fermentation, and the like. In the face of such a large number of extraction methods, how to screen a method which is more favorable for extracting active substances which can be used in the field of skin external preparations from a royal jelly separation liquid and realize the maximum utilization degree of resources still remains a technical problem faced by researchers in the field.

Therefore, there is a need in the art to develop an extraction method suitable for extracting active ingredients from a royal jelly separation liquid, which can be used in the field of skin external preparations, so as to realize the purpose of turning the royal jelly into a treasure and improve the availability of the royal jelly separation liquid.

Disclosure of Invention

The invention aims to overcome the defects that in the prior art, royal jelly separating liquid is usually discarded as waste, the utilization degree is low, the existing extraction processes are numerous, and a method which is more favorable for extracting active ingredients which can be used in the field of skin external preparations from the royal jelly separating liquid is difficult to screen, and the like, and provides a fermented product of the royal jelly separating liquid, the skin external preparations containing the fermented product, a preparation method and application thereof. The royal jelly isolate fermentation product prepared by the invention has good antioxidant and anti-inflammatory effects, has the effect of promoting the growth of human skin fibroblasts, has high safety, does not irritate the skin, has simple preparation process and low preparation cost, and can be widely applied to the field of skin external preparations.

The invention adopts the following technical scheme to solve the technical problems:

the invention provides a preparation method of a royal jelly separation liquid leavening, which comprises the following steps: inoculating lactobacillus into the fermentation substrate, fermenting, culturing, and sterilizing; the fermentation substrate comprises a royal jelly separating liquid, and the royal jelly separating liquid is a material prepared by removing protein components from fresh royal jelly.

In some embodiments, the lactic acid bacteria can include streptococcus thermophilus and/or lactobacillus.

Wherein the Lactobacillus may include any one or more of Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus bulgaricus (Lactobacillus bulgaricus) and Lactobacillus johnsonii (Lactobacillus johnsonii).

Wherein the Lactobacillus rhamnosus may include Lactobacillus rhamnosus having strain number PB-LR76, produced by Zhengzhou and Synbiotic engineering technologies, Inc.

Wherein the Lactobacillus johnsonii may include Lactobacillus johnsonii having a strain number LBJ 456 manufactured by Zhengzhou and Synbiotic engineering technologies, Inc.

The Lactobacillus bulgaricus may be Lactobacillus bulgaricus of BMZ124644 available from Ningbo boat Biotechnology Ltd.

Wherein said Streptococcus thermophilus may comprise Streptococcus thermophilus manufactured by Ningbo boat Biotechnology Ltd under product number BMZ 132094.

In a preferred embodiment, said lactic acid bacterium is a mixed strain of said Streptococcus thermophilus and said Lactobacillus bulgaricus, said Lactobacillus rhamnosus or said Lactobacillus johnsonii.

When the lactic acid bacteria are mixed strains of the streptococcus thermophilus and the lactobacillus bulgaricus, the number ratio of the streptococcus thermophilus to the lactobacillus bulgaricus can be (1-3): 1.

in some embodiments, the lactic acid isThe bacteria can be added in the form of lactobacillus liquid according to the routine in the field, and the concentration of the lactobacillus in the lactobacillus liquid can be 10⁶～10⁸CFU/mL, preferably 10⁶～10⁷CFU/mL。

The mass ratio of the lactobacillus liquid to the fermentation substrate can be conventional in the field, and is preferably (1-5): 100.

the preparation method of the lactobacillus liquid can be conventional in the field, and specifically comprises the following steps: inoculating the lactobacillus into an MRS broth culture medium with the pH value of 6.8-7.2, and performing fermentation culture at the temperature of 37-50 ℃.

In some embodiments, the fermentation substrate may further comprise a sterilization operation prior to use. The sterilization conditions and methods may be conventional in the art and may generally be autoclaving.

When the high-pressure steam sterilization method is adopted for sterilization, the sterilization temperature can be the temperature which is conventional in the operation in the field, preferably 90-100 ℃, and more preferably 95-100 ℃.

Wherein, when the high-pressure steam sterilization method is adopted for sterilization, the sterilization time can be the time of the operation routine in the field, and is preferably 20-30 min.

Wherein, when the sterilization is performed by the high pressure steam sterilization method, the sterilization pressure may be a pressure conventional in such operations in the art, and is preferably 0.09 to 0.15 MPa.

In some embodiments, the royal jelly separation liquid is preferably a small molecular water liquid of royal jelly from Apis cerana health technologies.

In some embodiments, the preparation method of the royal jelly separation liquid can comprise the following steps: extracting fresh Lac Regis Apis with water, and removing proteins by membrane separation.

In some embodiments, the royal jelly isolate may comprise 1% to 3%, for example 1.50%, by weight of the fermentation substrate.

In some embodiments, the fermentation substrate may further comprise water.

In a preferred embodiment, the fermentation substrate comprises 1-3 wt% of the royal jelly separation liquid and the balance of water.

In some embodiments, the fermentation time may be a time that is conventional in the art, preferably 10-15 hours, and more preferably 13-15 hours.

In some embodiments, the temperature of the fermentation culture may be a temperature conventional in the art, preferably 37-45 deg.C, more preferably 37-43 deg.C.

In some embodiments, the sterilization conditions and methods may be those conventional in such operations in the art, and may generally be autoclaving.

Wherein, when the high-pressure steam sterilization method is adopted for the sterilization, the sterilization pressure can be the pressure which is conventional in the operation in the field, and is preferably 0.09-0.15 MPa.

In some embodiments, the sterilization may be followed by any one or more of filtration, secondary sterilization, and mixing with a preservative.

Wherein the filtration can be performed in a filter as is conventional in the art. The pore size of the filter can be conventional in the art, and can be generally 0.4-0.6 μm, preferably 0.45 μm.

The conditions and method for secondary sterilization can be the conditions and method which are conventional in the operation in the field, and the high-pressure steam sterilization method can be generally adopted.

Wherein, when the high-pressure steam sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization can be the temperature which is conventional in the operation in the field, preferably 90-100 ℃, and more preferably 95-100 ℃.

Wherein, when the high-pressure steam sterilization method is adopted for the secondary sterilization, the time for the secondary sterilization can be the time which is conventional in the operation in the field, and is preferably 20-30 min.

Wherein, when the high pressure steam sterilization method is adopted for the secondary sterilization, the pressure of the secondary sterilization can be the pressure which is conventional in the operation in the field, and is preferably 0.09-0.15 MPa.

Wherein, in the process of mixing with the preservative, the mixing temperature can be the temperature which is conventional in the operation in the field, preferably 65-80 ℃, and more preferably 75-80 ℃.

Among them, the kind of the preservative may be conventional in the art, and preferably includes 1, 2-hexanediol and/or p-hydroxyacetophenone.

When the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone in the sterilized material can be 0.3% -0.5%, and the mass percentage of the 1, 2-hexanediol in the sterilized material can be 0.5% -1%; preferably, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.5%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 0.5%.

The invention also provides a royal jelly isolate fermentation product which is prepared by the preparation method of the royal jelly isolate fermentation product.

The invention also provides application of the royal jelly isolate fermentation product as a product, a product additive or a product substrate in preparing a skin external preparation.

In some embodiments, the fermented royal jelly isolate can be used as an antioxidant active ingredient and/or an anti-inflammatory active ingredient in the skin external preparation.

Wherein the antioxidant active ingredient is an antioxidant active ingredient having DPPH free radical scavenging ability.

Wherein the anti-inflammatory active ingredient can be an anti-inflammatory active ingredient which can inhibit the expression of KLK7 inflammatory factors and/or IL-1 inflammatory factors.

The invention also provides a skin external agent, which comprises the fermented product of the royal jelly separating liquid.

In some embodiments, the external skin preparation may include, as is conventional in the art, and is not limited to, a mask, a serum, or a toner.

In some embodiments, the mass percentage of the fermented royal jelly isolate in the external skin preparation may be 5% to 100%, preferably 50% to 100%.

In some embodiments, the skin external preparation may further include any one or more of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergy active ingredient, and an antioxidant active ingredient.

On the basis of the common knowledge in the field, the above preferred conditions can be combined randomly to obtain the preferred embodiments of the invention.

The reagents and starting materials used in the present invention are commercially available.

The positive progress effects of the invention are as follows: the royal jelly isolate fermentation product prepared by the invention has good antioxidant effect and anti-inflammatory effect, has the effect of promoting the growth of human skin fibroblasts, has high safety, does not irritate the skin, has simple preparation process and low preparation cost, and can be used as an excellent raw material of a skin external preparation or directly used as the skin external preparation.

Drawings

The disclosure may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are incorporated in and form a part of this specification, illustrate preferred embodiments of the present disclosure and, together with the detailed description, serve to explain the principles and advantages of the disclosure. Wherein:

FIG. 1 is a graph comparing the total antioxidant capacity of products prepared in examples 1-3 and comparative example 1;

FIG. 2 is a graph comparing the DPPH radical scavenging ability of the products obtained in examples 1 to 3 and comparative example 1;

FIG. 3 is a graph comparing the release levels of KLK7 inflammatory factor in HaCat cells after HaCat cells were treated with the product of example 3, blank control and model groups, respectively;

FIG. 4 is a graph comparing the IL-1 inflammatory factor release levels in HaCat cells after HaCat cells were treated with the product of example 3, blank control and model groups, respectively;

FIG. 5 is a graph showing the results of cell viability after human skin fibroblasts were treated with the products of example 3 at various concentrations.

Detailed Description

The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention. The experimental methods without specifying specific conditions in the following examples were selected according to the conventional methods and conditions, or according to the commercial instructions.

Exemplary embodiments of the present disclosure will be described hereinafter with reference to the accompanying drawings.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

In the following examples, lactic acid bacterium 1 was Lactobacillus rhamnosus (Lactobacillus rhamnosus) produced by Zhengzhou and Synbiotic engineering Co., Ltd, strain No. PB-LR 76;

in the following examples, lactic acid bacterium 2 was Lactobacillus johnsonii (Lactobacillus johnsonii) produced by Zhengzhou and Synbiotic engineering technology Co., Ltd, strain number LBJ 456;

in the following examples, lactic acid bacterium 3 was a mixed strain of Streptococcus thermophilus (Streptococcus thermophilus) with product number BMZ132094 and Lactobacillus bulgaricus (Lactobacillus bulgaricus) with product number BMZ124644, which were produced by Ningbo Ming boat Biotech Co., Ltd, and the number ratio of Streptococcus thermophilus to Lactobacillus bulgaricus was 1: 1;

in the following embodiments, the royal jelly separation liquid can be royal jelly small molecule water liquid of Apis cerana health science and technology company, and the preparation process comprises water leaching of fresh royal jelly, membrane separation for three times, and protein removal.

In the following examples, the formulation of MRS broth was: 10g of meat juice, 5g of peptone, 3g of yeast powder, 5g of D (+) -glucose, 1g of starch, 5g of NaCl, 3g of NaAc, 0.5g of L-cysteine hydrochloride and 1.0L of distilled water; the MRS broth solid culture medium is prepared by adding agar powder into an MRS broth liquid culture medium, wherein the agar powder accounts for 1% of the mass of the MRS broth liquid culture medium.

The preparation method of the lactobacillus 1 bacterial liquid in the following embodiment comprises the following steps:

(1) activation of strains: inoculating lactobacillus 1 into MRS broth solid culture medium for streak activation, and culturing in 43 deg.C incubator for 48 hr to obtain single colony;

(2) and (3) expanding culture of strains: inoculating the single colony obtained in the step (1) into 100mL of MRS broth liquid culture medium with the pH value of 7, and culturing for 48h in an incubator at 43 ℃ to obtain lactobacillus 1 bacterial liquid with the concentration of 10⁷CFU/mL。

The preparation method of the lactobacillus 2 bacterial liquid is the same as that of the lactobacillus 1 bacterial liquid, and the difference is only that the inoculated strains are respectively lactobacillus 2.

The preparation method of the lactobacillus 3 bacterial liquid is the same as that of the lactobacillus 1 bacterial liquid, and the difference is that the inoculated strains are respectively lactobacillus 3, and the culture temperature is 37 ℃.

Example 1

The preparation method of the fermentation substrate comprises the following steps: adding appropriate amount of Lac Regis Apis separating solution into water, sterilizing at 95 deg.C and 0.09MPa for 30min to obtain Lac Regis Apis separating solution fermentation substrate; the mass ratio of the royal jelly separating medium to water in the royal jelly separating medium fermentation substrate is 1: 100, respectively;

the concentration obtained by the above method is 10⁷Preparing a royal jelly separating liquid leavening by taking CFU/mL lactobacillus rhamnosus liquid and a royal jelly separating liquid leavening substrate as raw materials, and specifically comprising the following steps: inoculating 3g of lactobacillus liquid into 300g of royal jelly separation liquid fermentation substrate, and fermenting and culturing for 15h at the temperature of 43 ℃ under the condition of stirring; sterilizing the obtained material at 95 deg.C under 0.09MPa for 30 min; transferring to a filter with pore diameter of 0.45 μm for filtering after sterilization, and performing secondary sterilization at 95 deg.C under 0.09MPa for 30 min; two timesSterilizing, cooling, mixing with antiseptic at 75 deg.C, wherein the antiseptic comprises 1, 2-hexanediol and p-hydroxyacetophenone, the p-hydroxyacetophenone accounts for 0.5 wt% of the sterilized material, and the 1, 2-hexanediol accounts for 0.5 wt% of the sterilized material, and making into Lac Regis Apis isolate fermented product.

Example 2

The difference from example 1 is that only the lactic acid bacteria 1 liquid was replaced with the lactic acid bacteria 2 liquid, and the royal jelly isolate fermentation product was obtained in the same manner as in example 1 except for the other condition parameters.

Example 3

The difference from example 1 is that the same amount of lactic acid bacteria 3 liquid was used instead of lactic acid bacteria 1 liquid, the fermentation temperature was 37 ℃, and the other condition parameters were the same as example 1, to obtain a fermented product of royal jelly separation liquid.

Comparative example 1

Adding appropriate amount of Lac Regis Apis separating solution into water, sterilizing at 95 deg.C under 0.09MPa for 30min, transferring to filter with aperture of 0.45 μm, filtering, and sterilizing at 95 deg.C under 0.09MPa for 30 min; after secondary sterilization, cooling to obtain a fermentation substrate of the royal jelly separation liquid; the mass ratio of the royal jelly separating medium to water in the royal jelly separating medium fermentation substrate is 1: 100.

effect example 1 Total antioxidant Capacity

The product prepared in the comparative example 1 and the fermented product of the royal jelly separation liquid prepared in the above examples 1 to 3 were respectively diluted into a liquid to be tested with a volume percentage of 50%.

Taking the solution to be tested in the embodiment 1 as an example, the preparation method comprises the following steps: collecting 150 μ L of Lac Regis Apis separated liquid fermented product, adding PBS, and making into 300 μ L of the solution to be tested in example 1.

Firstly preparing ABTS working mother liquor by 200uL of ABTS and 200uL of oxidant solution, storing for 14 hours at room temperature in a dark place, and diluting by 40 times with PBS to obtain the ABTS working liquor.

Preparation of standard curve assay: standards were diluted in PBS and 10mM Trolox standard solutions were diluted to 0.15, 0.3, 0.6, 0.9, 1.2 and 1.5 mM.

Adding 200uL of ABTS working solution into each detection hole of a 96-hole plate, and adding 10uL of PBS solution into a blank control hole; 10uL of Trolox standard solutions with various concentrations are added into the standard curve detection holes; and adding 10uL of the solution to be detected in the embodiment into the sample detection hole, adding 10uL of the solution to be detected in the comparative example 1 into the control group, and gently mixing the solution and the control group. After incubation for 4 minutes at room temperature, A734 was determined. The total antioxidant capacity of the samples was calculated from the standard curve and the results are shown in table 1 and figure 1.

The resulting standard curve is: y-0.6923 x +0.456, R²＝0.9993。

TABLE 1 Total antioxidant Capacity

The results show that the total antioxidant capacity of the fermentation product of the royal jelly separation liquid prepared in the examples 1 to 3 is obviously higher than that of the fermentation product of the comparative example 1. The total antioxidant capacity of the fermented product of the royal jelly separation liquid prepared in examples 1 to 3 is not very different.

Effect example 2DPPH radical scavenging ability

The product obtained in comparative example 1 and the fermented products of the royal jelly separation liquid obtained in the above examples 1,2 and 3 were diluted to a test liquid having a volume percentage of 50%, respectively.

Taking the solution to be tested in the embodiment 1 as an example, the preparation method comprises the following steps: and (3) adding water into 1.5mL of the fermented product of the royal jelly separation liquid to prepare the solution to be detected in the embodiment 1 with the volume of 3 mL.

20mg of DPPH is dissolved in 250ml of absolute ethanol and stored at 0-4 ℃ in the dark.

3mL of DPPH ethanol solution was added to each tube, an equal volume of water (A1) was added to the control tube, an equal volume of example test solution (A2) was added to the sample tube, and 3mL of water and an equal volume of example test solution (A3) were added to the blank.

After reacting for 30min, the absorbance of each tube was measured at 517nm and DPPH radical clearance was calculated. The DPPH radical clearance calculation formula is as follows: DPPH radical clearance ═ [ (a1+ A3) -a2]/a1 × 100% × dilution factor, in the present effect example, the dilution factor was 2. The results are shown in Table 2 and FIG. 2.

TABLE 2DPPH radical scavenging Capacity

As can be seen from the results of Table 2 and FIG. 2, the DPPH radical scavenging ability of the fermented products of the royal jelly isolate prepared in examples 1 to 3 is significantly higher than that of comparative example 1. Wherein the DPPH radical scavenging rate (70.13%) of the fermented product of the royal jelly isolate obtained in example 3 was 2.3 times that of the product obtained in comparative example 1. The DPPH radical scavenging effect of the fermented product of the royal jelly-isolated liquid obtained in example 3 was better than that of examples 1 and 2.

Effect example 3 determination of polypeptide content

The polypeptide content in the products prepared in the examples 1-3 and the comparative example 1 is measured, and the test method is shown in the literature of' Wanqian, Chan, Wangcang, and the like; influence of carbon-nitrogen ratio on active ingredients and antioxidant capacity of fungus fermented ramulus mori-yellow bean [ J ] food industry science and technology, 2019, 40 (02): 93-99 ", the results are shown in Table 3.

TABLE 3

Effect example 4 human Patch test

The human body patch test is mainly used for detecting the irritation of the final cosmetic product or raw materials. The fermented royal jelly fractions obtained in examples 1 to 3 were subjected to a closed patch test for human body according to the "hygiene standards for cosmetics" (2015) to evaluate the skin irritation.

1. Test subjects:

according to the requirements of 'cosmetic contact dermatitis diagnostic standard and treatment principle', the selected test subject cannot participate in the test, and the test subject and the person with high physique sensitivity who have scars, nevus flammeus and other influences on the result judgment at the part to be tested of the skin cannot participate in the test. In the experiment, 30 suitable volunteers are selected, and the age range is 18-60 years.

2. Experimental methods

0.02mL of each of the products prepared in examples 1-3 above was dropped onto a filter paper sheet, which was then placed in a plaque tester. The test period lasted 24 h. In order to ensure the accuracy, credibility and scientificity of test results, the volunteers cannot remove the spot tester or make the tested part contact water according to the requirements during the test. The plaque tester was removed after 24h, left to stand for 30min, and the skin reaction was observed after 24h, after waiting for the disappearance of the indentation. The grading standards of the adverse skin reactions in the body patch test are shown in Table 4.

TABLE 4 grading Standard of adverse skin reactions

3. Test results

See table 5 for results, from which it can be seen; the results of the sensitization test of the fermentation products of the royal jelly separation liquid obtained in examples 1 to 3 are all negative reactions, which indicates that the fermentation products of the royal jelly separation liquid obtained in examples 1 to 3 are safe and do not bring adverse reactions to human bodies.

TABLE 5

Effect example 5

Taking logarithmic phase HaCat cells (human immortalized epidermal cells) at 2X 10⁶Inoculating the cell suspension on a 6-hole cell culture plate at the density of one/mL, adding 1mL of cell suspension into each hole, culturing for 24h in an incubator, and carefully sucking the supernatant;

adding 1mL of DMEM solution into the blank group and the model group respectively; the test solution diluted 1000 times by 1mL of the fermentation product of the royal jelly separation solution prepared in example 3 is added into the test solution;

after adding, continuing culturing for 6h, and irradiating the model group and the example group by ultraviolet light (UVA) with the illumination power of 9J/cm²The irradiation time was 1.5h, and the blank control group was not irradiated with light. Incubation was continued for 24h after illumination and each set of samples was repeated 3 replicates and each replicate was tested 3 times. Treating cells, collecting cell lysate, specifically breaking cells, centrifuging at 10000r/min and 4 ℃ for 10min, and taking supernatant to obtain cell lysis supernatant. Taking 20 mu L of cell lysate, and detecting the total protein content in the sample by using a BCA kit;

testing the release levels of KLK1 inflammatory factor and IL-1 inflammatory factor in the blank control group, model group and example group;

the test method comprises the following steps: measurement of KLK1 inflammatory factor after experimental operation according to the ELISA kit specification, each OD value was measured at 450nm, and based on the OD values, the release level of KLK1 inflammatory factor was calculated, the BCA content correction was required for the release amount of KLK1 inflammatory factor, and the relative expression amount of KLK1 inflammatory factor was calculated by the following formula, and the results are shown in Table 6 and FIG. 3; IL-1 inflammatory factor was measured using ELISA kit in the same manner as KLK1 inflammatory factor, and the results are shown in Table 6 and FIG. 4;

relative expression level of inflammatory factor is expressed by the amount of inflammatory factor expressed in experimental group/the amount of inflammatory factor expressed in blank control group.

TABLE 6

The results show that the royal jelly isolate fermentation product prepared in example 3 can effectively inhibit the expression level of KLK7 inflammatory factors and IL-1 inflammatory factors, and has ideal antibacterial performance.

Effect example 6

The experiment adopts human skin fibroblasts from a Chinese scientific cell bank to verify the cytotoxicity of the royal jelly isolate fermentation product prepared in the example 3.

1. The experimental steps are as follows:

the fermented product of the royal jelly isolate prepared in example 3 was prepared into test solutions of 40%, 20%, 10%, 5%, 2.5%, 1.25%, 0.625% and 0.3125% by volume using serum-free DMEM medium.

Human skin fibroblasts were cultured in a medium containing 10% fetal bovine serum and 1% double antibody (1X 10)⁵U/L penicillin, 100mg/L streptomycin). Cells were grown at 37 ℃ with 5% CO₂In the incubator saturated with humidity, when the cell fusion reached 85% or more, the cells in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing DMEM. Counting with cell counting plate, adjusting cell suspension concentration to 7 × 10⁴one/mL, the cell suspension was inoculated into a 96-well plate at a rate of 100. mu.L/well, at 37 ℃ with 5% CO₂Incubate for 12h under conditions.

The old medium was removed and the cells were washed twice with phosphate buffered saline. Adding 100 mu L of the prepared experimental group to-be-tested liquid with different concentrations and subjected to filtration sterilization into each hole of the experimental group, and making 6 multiple holes for each to-be-tested liquid; the control group contains cells, and serum-free DMEM medium is added; the blank control group was cell-free and 100. mu.L of PBS was added. Then at 37 ℃ and 5% CO₂Incubate for 24h under conditions. Then 10. mu.L of CCK-8 solution was added to each well, and incubated for 3 hours, and the absorbance value was measured at a wavelength of 450 nm.

The cell viability was calculated as follows:

cell survival rate (%) ═ a_{Experimental group}-A_{Blank control group})/(A_{Control group}-A_{Blank control group})×100％；

The experimental results are shown in table 7 and fig. 5, and the results show that the royal jelly isolate fermentation product prepared in example 3 has no obvious toxicity to human fibroblasts, and has a promoting effect on the growth of human skin fibroblasts.

TABLE 7

Finally, it should be further noted that, in the present invention, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

While the disclosure has been disclosed above by the description of specific embodiments thereof, it should be understood that various modifications, improvements or equivalents of the disclosure may be devised by those skilled in the art within the spirit and scope of the appended claims. Such modifications, improvements and equivalents are intended to be included within the scope of the present disclosure as claimed.

Claims

1. A preparation method of a royal jelly separation liquid leavening is characterized by comprising the following steps: inoculating lactobacillus into the fermentation substrate, fermenting, culturing, and sterilizing; the fermentation substrate comprises a royal jelly separating liquid, and the royal jelly separating liquid is a material prepared by removing protein from fresh royal jelly.

2. The method for preparing a fermented product of royal jelly isolate according to claim 1, wherein the lactic acid bacteria comprise streptococcus thermophilus and/or lactobacillus;

and/or the lactic acid bacteria are added in the form of lactic acid bacteria liquid, and the concentration of the lactic acid bacteria in the lactic acid bacteria liquid is 10⁶～10⁸CFU/mL；

And/or the royal jelly separation liquid is royal jelly micromolecule water liquid of Apis cerana health science and technology company;

and/or, the preparation method of the royal jelly separation liquid comprises the following steps: extracting fresh Lac Regis Apis with water, and removing proteins by membrane separation;

and/or the royal jelly separation liquid accounts for 1 to 3 percent, such as 1.50 percent, of the mass of the fermentation substrate;

and/or, the fermentation substrate further comprises water;

and/or the fermentation culture time is 10-15 h;

and/or the temperature of the fermentation culture is 37-45 ℃;

and/or the sterilization method is high-pressure steam sterilization.

3. The method for preparing a fermented product of a royal jelly isolate according to claim 2, wherein the Lactobacillus comprises any one or more of Lactobacillus rhamnosus (Lactobacillus rhamnosus), Lactobacillus bulgaricus (Lactobacillus bulgaricus) and Lactobacillus johnsonii (Lactobacillus johnsonii);

and/or the lactic acid bacteria are added in the form of lactic acid bacteria liquid, and the concentration of the lactic acid bacteria in the lactic acid bacteria liquid is 10⁶～10⁷CFU/mL；

And/or the mass ratio of the lactobacillus liquid to the fermentation substrate is (1-5): 100, respectively;

and/or the fermentation culture time is 13-15 h;

and/or the temperature of the fermentation culture is 37-43 ℃;

and/or when the high-pressure steam sterilization method is adopted for sterilization, the sterilization temperature is 90-100 ℃;

and/or when the high-pressure steam sterilization method is adopted for sterilization, the sterilization time is 20-30 min;

and/or when the high-pressure steam sterilization method is adopted for sterilization, the sterilization pressure is 0.09-0.15 MPa.

4. The method for preparing a fermented product of royal jelly isolate according to claim 3, wherein the Lactobacillus rhamnosus includes Lactobacillus rhamnosus with strain number PB-LR76, manufactured by Zhengzhou and Synbiotic engineering technologies, Inc.;

and/or, the Lactobacillus johnsonii comprises Lactobacillus johnsonii with strain number LBJ 456 produced by Zhengzhou and Synbiotic engineering technology, Inc.;

and/or the lactobacillus bulgaricus comprises lactobacillus bulgaricus with product number of BMZ124644, produced by Ningbom Biotechnology Limited;

and/or the streptococcus thermophilus comprises streptococcus thermophilus produced by Ningbo boat Biotechnology Co., Ltd, with product number BMZ 132094;

and/or when the high-pressure steam sterilization method is adopted for sterilization, the sterilization temperature is 95-100 ℃.

5. The method for preparing a fermented product of a royal jelly-isolated liquid according to claim 3 or 4, wherein said lactic acid bacteria are a mixed strain of said Streptococcus thermophilus and said Lactobacillus bulgaricus, said Lactobacillus rhamnosus, or said Lactobacillus johnsonii;

preferably, when the lactic acid bacteria are mixed strains of the streptococcus thermophilus and the lactobacillus bulgaricus, the number ratio of the streptococcus thermophilus to the lactobacillus bulgaricus is (1-3): 1.

6. the method for preparing a fermented product of a royal jelly-isolated liquid according to any one of claims 1 to 5, wherein the fermentation substrate further comprises a sterilization operation before use;

and/or after the sterilization operation, any one or more of filtration, secondary sterilization and mixing with a preservative are further included.

7. The method for preparing a fermented product of a royal jelly isolate according to claim 6, wherein the method of sterilizing the fermentation substrate before use is autoclaving; the sterilization temperature is preferably 90-100 ℃, more preferably 95-100 ℃; the sterilization time is preferably 20-30 min; the sterilization pressure is preferably 0.09-0.15 MPa;

and/or the filtration is carried out in a filter, the pore size of the filter is 0.4-0.6 μm, preferably 0.45 μm;

and/or the secondary sterilization method is a high-pressure steam sterilization method; the temperature of the secondary sterilization is preferably 90 to 100 ℃, more preferably 95 to 100 ℃; the secondary sterilization time is preferably 20-30 min; the pressure of the secondary sterilization is preferably 0.09-0.15 MPa;

and/or, in the process of mixing with the preservative, the mixing temperature is 65-80 ℃, preferably 75-80 ℃;

and/or, the preservative comprises 1, 2-hexanediol and/or p-hydroxyacetophenone; when the preservative comprises p-hydroxyacetophenone and 1, 2-hexanediol, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.3% -0.5%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 0.5% -1%; preferably, the mass percent of the p-hydroxyacetophenone in the sterilized material is 0.5%, and the mass percent of the 1, 2-hexanediol in the sterilized material is 0.5%.

8. A fermented product of a royal jelly-separated liquid, which is produced by the method for producing a fermented product of a royal jelly-separated liquid according to any one of claims 1 to 7.

9. Use of the fermented product of royal jelly-isolated liquid according to claim 8 as a product, a product additive or a product base for the preparation of an external preparation for skin;

preferably, the fermentation product of the royal jelly isolate is used as an antioxidant active ingredient and/or an anti-inflammatory active ingredient in the skin external preparation;

preferably, the antioxidant active ingredient is an antioxidant active ingredient with DPPH free radical scavenging capacity;

preferably, the anti-inflammatory active ingredient is an anti-inflammatory active ingredient having an inhibitory effect on the expression of KLK7 inflammatory factor and/or IL-1 inflammatory factor.

10. An external preparation for skin, comprising the fermented product of the royal jelly-isolated liquid according to claim 8;

preferably, the skin external agent comprises a mask, essence or toner;

preferably, the mass percentage of the fermented product of the royal jelly separation liquid in the skin external preparation is 5-100%, and more preferably 50-100%.