CN115154393A

CN115154393A - Aloe fermentation liquor and preparation method and application thereof

Info

Publication number: CN115154393A
Application number: CN202210780117.0A
Authority: CN
Inventors: 李晓敏; 苏敦; 任红红; 陈文杰; 闫永涛; 许佳璇; 梁志强
Original assignee: Perfect Guangdong Commodity Co Ltd
Current assignee: Perfect Guangdong Commodity Co Ltd
Priority date: 2022-07-04
Filing date: 2022-07-04
Publication date: 2022-10-11
Anticipated expiration: 2042-07-04
Also published as: CN115154393B

Abstract

The invention relates to an aloe fermentation broth and a preparation method and application thereof, belonging to the technical field of biology. The preparation method of the aloe fermentation liquor comprises the following steps: (1) Inoculating the zymophyte liquid into a mixture of the aloe liquid and a culture medium to obtain a fermentation stock solution; (2) Fermenting the fermentation stock solution to obtain a fermentation product, namely the aloe fermentation solution; the zymophyte liquid consists of lactobacillus liquid, bead bacteria liquid and lactococcus liquid. According to the aloe fermentation liquid, the type and the content of the fermentation bacteria are optimized, and the synergistic interaction effect among the composite fermentation bacteria is realized, so that the fermentation efficiency and the whitening and antioxidant effects of the fermentation liquid are improved.

Description

Aloe fermentation liquor and preparation method and application thereof

Technical Field

The invention relates to an aloe fermentation broth and a preparation method and application thereof, belonging to the technical field of biology.

Background

Aloe is perennial evergreen herb of Liliales, liliaceae, also known as Aloe barbadensis Miller, aloe china, allium fistulosum, aloe vera, aloe barbadensis Miller, and Aloe americana. Aloe is native to arid regions of the tropical regions of Africa and is distributed almost worldwide. Wild aloe vera is distributed in both the Indian and Malaysia zones, the African continent and in tropical regions. The wild aloe can be cultivated in Fujian, guangdong, guangxi, sichuan and Yunnan provinces in China. Aloe (Aloe barbadensis Miller) is one of the few edible species in Aloe, and its products are widely used in food, skin care, health care, medicine, etc.

Aloe is a pure natural green plant which integrates medicine, beauty treatment, health care, food and appreciation, and various medicinal values and treatment effects of aloe cause wide attention in the medical field. Since the 80's of the 20 th century, aloe has been increasingly sought after by consumers and rapidly raised the "aloe" hot tide worldwide. Aloe contains abundant amino acids, vitamins, polysaccharides, anthraquinone compounds, various enzymes, minerals, etc., especially contains strontium and germanium, and these nutrients can supplement human body. Aloe has well known name, "plant doctor" for its antibacterial, anti-inflammatory, immune function enhancing, wound healing, heat clearing, bowel relaxing, colitis preventing, blood lipid, blood glucose and blood pressure lowering, circulatory system and sleep quality improving, digestive system diseases preventing and treating, and appetite stimulating effects. Aloe and yellow juice of Aloe of gastroenteropathy have effects of diminishing inflammation, sterilizing, invigorating stomach, relaxing bowels, etc., and have remarkable therapeutic effect on acute gastritis. In addition, because the rich mucus of the aloe can be adhered to the damaged ulcer surface, not only can the regeneration of living cell tissues be realized, but also new tissues can grow out of the ulcer part and the surrounding tissues, so the aloe also has great help for treating gastric ulcer caused by gastric acid. Aloe and constipation research has proved that anthraquinone compound derivatives contained in Aloe release aloe-emodin in intestinal canal, and can effectively stimulate large intestine peristalsis, thereby effectively improving gastrointestinal function, constipation, etc., removing toxic substance, clearing pathogenic fire, tonifying deficiency, and caring skin.

Aloe active ingredients have long been prepared in the form of aloe vera gel. Firstly, cutting aloe leaves, collecting leaf juice, placing the leaf juice in a pot, decocting the leaf juice into thick paste, soaking the thick paste in a container, cooling and solidifying the thick paste to obtain the aloe leaf tea. However, the aloe gel prepared by this method is coarse, and most of the natural active ingredients are decomposed by heating in a pot. Based on the traditional preparation process, cobble firstly proposes a method for preparing stable and transparent aloe gel, namely, antioxidant and preservative are added in the preparation process, the extraction quality of the aloe gel is improved, but the aloe gel extracted by the extraction method can cause skin allergy, and the processes of heating, oxidation and the like can accelerate the degradation of effective components, and the oxidation can cause the oxidation of the effective components. In addition, the extraction of the effective components of aloe can be carried out by acid extraction, alkali extraction and enzyme extraction. With the development of science and technology, other new extraction methods are derived.

Fermentation is one of the traditional processing methods of Chinese medicinal materials. As early as thousands of years ago, china already uses fermentation methods to prepare medicines, such as fermented soybean, agilawood koji, chinese gall, red yeast rice and the like, which are all medicines formed by fermentation. These are achieved by using microorganisms having a strong bioconversion ability. Modern fermentation process research shows that the microorganism has multiple capacities of oxidation, esterification, methylation, hydroxylation, reduction and the like, the effective components of the Chinese herbal medicine can be fully separated and extracted after the Chinese herbal medicine is fermented, so that the Chinese herbal medicine has more biological activity, contains a large amount of active enzyme, can be quickly absorbed by organism tissue cells after being taken, achieves the functions of disease prevention, body building and immunity enhancement, and better plays the efficacy of the Chinese herbal medicine, namely a natural medicine. Meanwhile, some components can be decomposed and converted into new components, and the new components become the material basis for the appearance of new pharmacological effects or enhancement of the pharmacological effects. Therefore, the compound can be used for fermentation pharmacy, thereby reducing the medication cost and improving the efficacy.

In the reports related to the extraction of the aloe effective components by fermentation technology, the effect of the effective components is poor, and the effective components of aloe are difficult to be fully exerted.

Disclosure of Invention

The invention aims to provide aloe fermentation liquor and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that: a preparation method of aloe fermentation liquor comprises the following steps: (1) Inoculating the zymophyte liquid into a mixture of the aloe liquid and a culture medium to obtain a fermentation stock solution; (2) Fermenting the fermentation stock solution to obtain a fermentation product, namely the aloe fermentation solution; the zymocyte liquid consists of lactobacillus liquid, bead bacteria liquid and lactosphere bacteria liquid.

The fresh juice of Aloe contains aloe-emodin glycoside, p-coumaric acid, glucose, etc., and Aloe has effects of moisturizing and moistening. However, it is wrong to apply fresh aloe juice directly to the face in order to achieve a skin-beautifying effect because the aloe juice is directly applied to the skin and causes adverse reactions such as irritation, skin allergy and the like, and the aloe juice is easily oxidized into black when encountering air. Through a large number of experiments, the inventor of the application finds that the aloe fermented liquid obtained by fermenting the freshly squeezed aloe liquid by using the zymophyte liquid has stronger whitening and antioxidant effects than the unfermented aloe liquid, and does not generate irritation to the skin.

As a preferred embodiment of the method for preparing the aloe fermentation broth, the lactobacillus solution is obtained by activating lactobacillus in a liquid culture medium, and the concentration of the lactobacillus in the lactobacillus solution is 10 ⁸ ～10 ⁹ CFU/mL; the moniliforme liquid is obtained by activating moniliforme in a liquid culture medium, and the concentration of the moniliforme in the moniliforme liquid is 10 ⁸ ～10 ⁹ CFU/mL; the lactococcus liquid is obtained by activating lactococcus in a liquid culture medium, and the concentration of the lactococcus in the lactococcus liquid is 10 ⁸ ～10 ⁹ CFU/mL。

In a preferred embodiment of the method for preparing the aloe vera broth according to the present invention, the fermented broth is composed of a paracasei lactobacillus broth, a leuconostoc mesenteroides broth, and a lactococcus lactis broth.

Research shows that the lactobacillus paracasei belongs to lactobacillus casei flora in lactobacillus, is widely present in fermented foods such as cheese, pickled vegetables and the like, has probiotic functions of lactobacillus casei in regulating intestinal tracts, enhancing immunity and the like, and bacteriocin generated by metabolism can inhibit harmful bacteria in the intestinal tracts and plays an important role in inhibiting pathogenic bacteria and regulating intestinal tract balance; leuconostoc mesenteroides is an important strain of Leuconostoc, generally exists on the body surface of plants, and can ferment citric acid to generate characteristic flavor substances, and is commonly used in fermented dairy products, silage, pickles and fruit wine; lactococcus lactis is one of the commonly used leavening agents in the fermentation industry, and particularly in fermented dairy products, lactococcus lactis grows rapidly and is separated from anabolism; the genome is small but contains enough biological information, and the intracellular and extracellular proteins are easy to separate and purify, so that the advantages make the lactococcus lactis have great research value in the aspect of metabolic regulation. The nature of the fermentation bacteria is various, the fermentation mechanisms of different fermentation bacteria are different, and the properties and the efficacy of fermentation products generated by fermenting different fermentation substrates are also different. Through a large amount of experimental screening and verification, the inventor discovers that the aloe liquid is fermented by adopting composite zymophyte consisting of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, and the three strains are mutually promoted through a synergistic interaction effect, so that the finally prepared aloe fermentation liquid has strong whitening and antioxidant effects.

In a preferred embodiment of the method for preparing an aloe fermentation broth according to the present invention, the volume percentage of the lactobacillus paracasei liquid is 1 to 30%, the volume percentage of the leuconostoc mesenteroides liquid is 1 to 30%, and the volume percentage of the lactococcus lactis liquid is 1 to 30%, based on the fermentation stock solution. The inventor of the present application has found through a great deal of experiments that the interaction between lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis is stronger in the volume percentage range.

In a preferred embodiment of the method for preparing the aloe fermentation broth, the volume percentage of the lactobacillus paracasei liquid is 2-10%, the volume percentage of the leuconostoc mesenteroides liquid is 2-10%, and the volume percentage of the lactococcus lactis liquid is 2-10%, based on the fermentation stock solution. Through a large number of experiments, the inventor of the application finds that the growth and fermentation effects of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis are better in the preferred volume percentage range.

In a preferred embodiment of the method for preparing an aloe vera broth according to the present invention, the volume percentage of the lactobacillus paracasei liquid is 5%, the volume percentage of the leuconostoc mesenteroides liquid is 5%, and the volume percentage of the lactic acid lactococcus liquid is 5%, based on the fermentation stock solution. When the volume percentage of the paracasei lactobacillus liquid, the leuconostoc mesenteroides liquid and the lactic acid lactococcus liquid is 5 percent, the growth and fermentation effects of the three bacteria are the best.

As a preferred embodiment of the method for preparing the aloe fermentation broth, the culture medium is MRS medium. Through a large number of experiments, the inventor of the application discovers that the MRS culture medium is adopted as the culture medium for fermenting the aloe, so that the environment for the survival of the composite fermentation strain is more favorably created, the content of the effective substances is further improved, and the whitening and antioxidant effects of the aloe fermentation liquor are better.

In a preferred embodiment of the method for preparing the aloe fermentation liquid, the aloe liquid is 1-40% by volume of the fermentation stock solution.

In a preferred embodiment of the method for preparing the aloe fermentation liquid, the aloe liquid is 2-30% by volume of the fermentation stock solution.

In a preferred embodiment of the method for preparing the aloe fermentation broth, the aloe broth is 5% by volume based on the fermentation stock solution. Through a large number of experiments, the inventor of the application finds that the addition amount of the aloe liquid has great influence on the fermentation of the composite fermentation strain, and when the volume percentage of the aloe liquid in the fermentation stock solution is 5%, the fermentation of the composite fermentation strain is the best.

As a preferred embodiment of the method for preparing the aloe fermentation broth, in the step (2), the fermentation temperature is 25 to 37 ℃, the fermentation time is 12 to 48 hours, and the pH of the fermentation stock solution is 4.1 to 4.7.

As a preferred embodiment of the preparation method of the aloe fermentation liquor, the method further comprises the steps of sequentially sterilizing, centrifuging and collecting supernatant of the fermentation product.

As a preferred embodiment of the method for preparing the aloe fermentation broth, the sterilization conditions are as follows: the sterilization temperature is 120-125 ℃, and the sterilization time is 10-20 min; the centrifugation conditions were: the centrifugal temperature is 15-25 ℃, the centrifugal speed is 5500-6500 r/min, and the centrifugal time is 25-35 min.

Through a large amount of experimental researches, the inventor optimizes the fermentation conditions for fermenting the aloe liquid by using zymophyte, and finally finds that when the fermentation temperature, the fermentation time and the specific preparation steps are adopted for fermentation, the macro-molecular effective ingredients of the aloe liquid can be converted into micro-molecules, so that the content of effective substances in the fermentation product is higher, and the whitening and antioxidant effects of the finally prepared aloe fermentation liquid are better.

The invention also provides the aloe fermentation liquor obtained by the preparation method of the aloe fermentation liquor.

The invention also provides a skin care product which comprises the aloe fermentation liquor.

The invention also provides application of the aloe fermentation liquid in preparing skin care products with whitening and anti-aging effects.

As a preferred embodiment of the application of the invention, the skin care product comprises face cream, eye cream, toner or essence.

Compared with the prior art, the invention has the beneficial effects that: the invention provides an aloe fermentation broth, which improves the fermentation efficiency and the whitening and antioxidant effects of the fermentation broth by optimizing the type and content of zymophyte and by the synergistic interaction between the zymophyte; by fermenting the aloe juice with the composite zymophyte, the active ingredients of macromolecules can be changed into micromolecules, and the effect of whitening and antioxidant active substances is improved; by optimizing the type of the fermentation medium and the content of the aloe juice, a better growth environment is created for the zymophyte, the fermentation efficiency is improved, and the whitening and antioxidation effects of the aloe are improved.

Drawings

FIG. 1 is a graph showing the growth of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis in different fermentation media.

FIG. 2 is a graph showing the growth of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis at different fermentation temperatures.

FIG. 3 is the growth curve of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis in MRS medium, respectively.

FIG. 4 is a graph showing the growth of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis in 5% aloe juice, respectively.

FIG. 5 is a graph showing the growth of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis in MRS medium +5% aloe vera juice, respectively.

FIG. 6 is a graph of the growth of complex species in MRS medium +5% aloe vera juice.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to specific examples.

The aloe juice used in the following examples and comparative examples was prepared by juicing and filtering aloe leaves; the aloe leaves are commercially available.

The preparation method of the zymophyte liquid used in the following examples and comparative examples is as follows: respectively picking bacterial colonies of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, looping the bacterial colonies into a liquid culture medium, activating the bacterial colonies in a shaking table at the temperature of 30 ℃ to respectively obtain activated lactobacillus paracasei bacterial liquid, leuconostoc mesenteroides bacterial liquid and lactococcus lactis bacterial liquid; the concentrations of the lactobacillus paracasei liquid, the leuconostoc mesenteroides liquid and the lactococcus lactis liquid are all 10 ⁸ ～10 ⁹ CFU/mL。

Example 1

The preparation method of the aloe fermentation liquor provided by the embodiment of the invention comprises the following steps of:

(1) Inoculating 5% of lactobacillus paracasei, 5% of leuconostoc mesenteroides and 5% of lactococcus lactis zymocyte liquid into 5% of aloe juice and a culture medium, and uniformly stirring and mixing to obtain a fermentation stock solution;

(2) Placing the fermentation stock solution in an oven at 30 ℃ for fermentation for 28h to obtain a fermentation product;

(3) Sterilizing the fermentation product at 121 deg.C for 15min, centrifuging the sterilized fermentation liquid at 6000r/min and 20 deg.C for 30min, and collecting supernatant to obtain aloe fermentation liquid.

Comparative example 1

The preparation method of the aloe fermentation liquor comprises the following steps:

(1) Inoculating 5% of paracasei lactobacillus liquid into 5% of aloe juice and MRS culture medium, and stirring and mixing uniformly to obtain fermentation stock solution;

Comparative example 2

The invention relates to aloe fermentation liquor, and a preparation method of the aloe fermentation liquor comprises the following steps:

(1) Inoculating 5% Leuconostoc mesenteroides liquid into 5% aloe juice and MRS culture medium, stirring and mixing well to obtain fermentation stock solution;

Comparative example 3

(1) Inoculating 5% lactococcus lactis liquid into 5% aloe juice and MRS culture medium, and stirring and mixing uniformly to obtain fermentation stock solution;

Comparative example 4

(1) Inoculating 5% of lactobacillus paracasei and 5% of leuconostoc mesenteroides zymocyte liquid into 5% of aloe juice and MRS culture medium, and uniformly stirring and mixing to obtain fermentation stock solution;

Comparative example 5

(1) Inoculating 5% of lactobacillus paracasei and 5% of lactobacillus lactis zymocyte liquid into 5% of aloe juice and a culture medium, and uniformly stirring and mixing to obtain a fermentation stock solution;

Comparative example 6

The preparation method of the aloe fermentation liquor provided by the embodiment of the invention comprises the following steps:

(1) Inoculating 5% leuconostoc mesenteroides and 5% lactococcus lactis zymocyte liquid into 5% aloe juice and a culture medium, and uniformly stirring and mixing to obtain a fermentation stock solution;

(2) Fermenting the fermentation stock solution in an oven at 30 ℃ for 28h to obtain a fermentation product;

Test examples

1. The test example researches the influence of different types and concentrations of culture media on the growth and fermentation of zymophyte, and comprises the following specific steps:

(1) And (3) strain separation and purification: respectively picking bacterial colonies of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, looping the bacterial colonies into a liquid culture medium, activating the bacterial colonies in a shaking table at the temperature of 30 ℃, and respectively obtaining activated lactobacillus paracasei bacterial liquid, leuconostoc mesenteroides bacterial liquid and lactococcus lactis bacterial liquid, wherein the concentrations of the lactobacillus paracasei bacterial liquid, the leuconostoc mesenteroides bacterial liquid and the lactococcus lactis bacterial liquid are all 10 ⁸ ～10 ⁹ CFU/mL；

(2) Preparation of fermentation stock solution: preparing 100ml of MRS, MRS +2% aloe juice, MRS +5% aloe juice, 2% glucose +2% aloe juice, 2% glucose +5% aloe juice, 4% glucose +2% aloe juice, 4% glucose +5% aloe juice, and sterilizing at 121 deg.C for 15min;

(3) Fermentation culture: respectively taking 5ml of 7 culture mediums, putting into a 5ml test tube, respectively taking 250ul of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, respectively inoculating into the culture mediums, and placing at 30 ℃ for culturing for 12h;

(4) And (3) detection: 200uL of each cultured fermentation liquid is put into a 96-well plate, and the OD value is detected by a microplate reader under the wavelength of 600 nm.

As shown in Table 1 and FIG. 1, it is understood from Table 1 and FIG. 1 that the growth of Lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis is more preferable when MRS medium +5% aloe vera juice is used as the fermentation medium.

TABLE 1

2. The test example researches the influence of different fermentation temperatures on the growth and fermentation of zymophyte, and comprises the following specific steps:

(1) And (3) strain separation and purification: respectively picking bacterial colonies of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, looping the bacterial colonies into a liquid culture medium, activating the bacterial colonies in a shaking table at the temperature of 30 ℃ to respectively obtain activated lactobacillus paracasei bacterial liquid, leuconostoc mesenteroides bacterial liquid and lactococcus lactis bacterial liquid;

(2) Preparation of fermentation stock solution: preparing 100ml MRS culture medium and 5% Aloe juice fermentation stock solution, respectively, placing in autoclave, and sterilizing at 121 deg.C for 15min;

(3) Fermentation culture: taking 5ml of the culture medium in the step (2), putting the 5ml of the culture medium into a 5ml test tube, respectively inoculating 250ul of each of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis into the culture medium, and respectively culturing at the temperature of 25 ℃, 30 ℃ and 37 ℃ for 12 hours;

As shown in Table 2 and FIG. 2, it is understood from Table 2 and FIG. 2 that the growth of Lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis is more favorable when the fermentation temperature is 30 ℃.

TABLE 2

3. The experimental example researches the influence of different fermentation time on the growth and fermentation of zymophyte and the mutual promotion effect among lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, and the specific experimental steps are as follows:

(1) And (3) strain separation and purification: respectively picking bacterial colonies of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, looping the bacterial colonies into a liquid culture medium, activating the strains in a shaking table at the temperature of 30 ℃, respectively taking 250ul of each of the three activated bacteria to activate in 5ml of the liquid culture medium, and respectively obtaining activated lactobacillus paracasei liquid, leuconostoc mesenteroides liquid and lactococcus lactis liquid;

(2) Preparation of fermentation stock solution: preparing 100ml MRS culture medium, aloe juice, MRS culture medium +5% Aloe juice, and sterilizing at 121 deg.C for 15min;

(3) Fermentation culture: taking 5ml of the culture medium obtained in the step (2), putting the 5ml of the culture medium into a 5ml test tube, respectively taking 250ul of each of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis, respectively inoculating any two strains of 5% of lactobacillus paracasei, 5% of leuconostoc mesenteroides and 5% of lactococcus lactis into an MRS culture medium and 5% of aloe juice culture medium, inoculating the 5% of lactobacillus paracasei, 5% of leuconostoc mesenteroides and 5% of lactococcus lactis into the MRS culture medium and 5% of aloe juice culture medium, and respectively placing the culture stock solution at the temperature of 30 ℃ for culturing for 48 hours; wherein L represents lactobacillus paracasei; c represents leuconostoc mesenteroides; r represents lactococcus lactis.

(4) And (3) detection: and (4) taking 200uL of fermentation liquor obtained after fermentation of each group of culture stock solution in the step (3) every other hour into a 96-well plate, detecting by using an enzyme-labeling instrument at the wavelength of 600nm, and detecting the OD value.

As shown in tables 3 to 4 and fig. 3 to 6, it is understood from tables 3 to 4 and fig. 3 to 6 that lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis hardly grow in a simple aloe juice fermentation raw liquid, and thus the aloe juice alone cannot be used as the fermentation raw liquid; the optimal culture time of lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis in a simple MRS culture medium fermentation stock solution is 24 hours, and the growth OD value of the lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis in the MRS culture medium +5% aloe juice culture stock solution is not increased any more after the three bacteria are independently cultured or are cultured in a combined way for 28 hours, and for the production cost, the production cost is increased by continuously increasing the culture time. Therefore, the optimal culture time of the lactobacillus paracasei, the leuconostoc mesenteroides and the lactococcus lactis cultured in the MRS culture medium and the 5 percent aloe juice culture stock solution independently or in a combined way is 28 hours.

TABLE 3

TABLE 4

Examples of effects

1. Safety evaluation of Aloe fermentation broth

The aloe fermentation liquids prepared in example 1 and comparative examples 1 to 3 were evaluated for irritation of cosmetics.

(1) The tested population is female, 40 people, and the age is between 16 and 65 years. The skin of the subject is healthy, has no allergic history of the skin disease, and meets the voluntary entry standard of the subject. Randomly grouped into 4 groups of 10 people each.

The test method is a human body patch test. Selecting a qualified spot tester, placing 0.020-0.025g of a tested object in the spot tester, sticking the spot tester with the tested object on the back or the bent side of the forearm of the tested object by using a non-irritating adhesive tape, and lightly pressing with a palm to uniformly stick the spot tester on the skin for 24 hours. And (5) removing the tested spot tester for 30min, and observing skin reaction after the indentation disappears. If the result is negative, the test is observed once more at 24h and 48h after the patch test. The results were recorded according to the reference standards of cosmetic hygiene Specifications (2007) and cosmetic safety Specifications (2015).

Evaluation criteria: level 0: negative reaction; level 1: suspicious reactions, only faint erythema; and 2, stage: weak positive reaction, erythema, infiltration, edema, and possibly pimple; and 3, level: strong positive reaction, erythema, infiltration, edema, pimple and reaction beyond the tested area; 4, level: strong positive reaction, obvious erythema, serious infiltration, edema and fusion herpes, and reaction beyond the tested area.

(2) The results of the patch test on the skin of a human body show that: skin reactions of all the test subjects were observed 24h and 48h after the test by the patch test of the aloe fermentation broth prepared in example 1 and comparative examples 1 to 3, and all the test subjects showed negative skin reactions, which indicates that the anti-wrinkle and anti-firmness compositions of the aloe fermentation broth prepared in example 1 and comparative examples 1 to 3 are safe to use.

2. DPPH radical scavenging test for Aloe fermentation broth

DPPH free radical is a very stable free radical of oxygen center, is one of important indexes of sample oxidation resistance, and is widely applied to oxidation-resistant food. In the research of health products and medicines. DPPH free radical has single electron, its alcoholic solution is purple, and has strong absorption at 515 nm. When antioxidant exists, DPPH free radicals are eliminated, the color of the solution is lightened, the absorbance at 515nm is reduced, and the change of the absorbance is in direct proportion to the degree of the elimination of the free radicals in a certain range. The ability of the sample to scavenge DPPH radicals is reflected by the degree of absorbance decrease.

The specific experimental steps of the DPPH free radical scavenging experiment are as follows:

(1) Taking equal volume (generally 3 mL) of solution to be detected and 2x10 ⁴ mixing the solution of DPPH in mol/L (tube A1);

(2) Taking equal volume of anhydrous ethanol (solvent of substance to be detected) and 2x10 ⁴ mixing mo1/L DPPH solution (A2 tube);

(3) Taking absolute ethyl alcohol with the same volume and uniformly mixing the absolute ethyl alcohol with a solution to be detected (A3 tube);

(4) After 30min of reaction, the absorbance values of tubes A1, A2 and A3 were measured at 517 nm.

The clearance calculation formula is: clearance (%) = [ (A2 + A3) -A1]/A2.

And (3) dissolving the aloe fermentation liquor prepared in the example 1 and the comparative examples 1 to 6 in deionized water to prepare the aloe fermentation liquor to-be-detected liquid with the volume percentage of 5%.

And respectively measuring the DPPH free radical clearance rate of the aloe fermentation broth to-be-measured liquid with the volume percentage of 5 percent according to a DPPH free radical clearance rate test method. The test results are shown in table 5.

TABLE 5

As can be seen from Table 5, the aloe vera broth fermented by the complex fermentation broth consisting of Lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis can significantly improve the DPPH radical clearance rate.

4. Tyrosinase activity inhibition assay for aloe fermentation broth

The synthesis of melanin is affected by tyrosinase, which catalyzes the hydroxylation of L-tyrosine to dopa and oxidizes dopa to dopaquinone, which forms the final reaction product melanin. The test object with tyrosinase activity inhibition effect can slow down the catalytic conversion of L-tyrosine into dopaquinone by tyrosinase, and the inhibition effect of the test object on the tyrosinase activity is evaluated according to the change of absorbance by measuring the absorbance of the dopaquinone at 475 nm.

The specific experimental steps of the tyrosinase activity inhibition experiment are as follows:

(1) Sequentially adding a tyrosine solution, a sample solution and a PBS solution into a 96-well plate according to the addition of the reagents in the table 6, and fully and uniformly mixing;

(2) Incubating for 10min at the constant temperature of 37 ℃, and sequentially adding 20uL of tyrosinase solution;

(3) Mixing uniformly at 37 ℃ for reaction for 5min, placing the mixture into an enzyme-labeling instrument, and measuring at the wavelength of 475 nm.

Tyrosinase activity inhibition was calculated as: inhibition ratio (%) = [1- (Td-Tc)/(Tb-Ta) ]. Times.100%

TABLE 6

And respectively measuring the tyrosinase activity inhibition rate of the aloe fermentation liquid to be measured with the volume percentage of 5 percent according to a tyrosinase activity inhibition rate test method. The test results are shown in Table 7.

TABLE 7

As can be seen from Table 7, the aloe fermentation broth fermented by the composite fermentation broth consisting of Lactobacillus paracasei, leuconostoc mesenteroides and lactococcus lactis can significantly improve the tyrosinase activity inhibition rate.

5. Whitening and anti-aging efficacy test of aloe fermentation liquid

Skin aging is caused by damage of external environment such as ultraviolet ray and light pollution, and not only various oxidation free radicals are formed to destroy collagen synthesis, but also DNA damage of skin cells is caused, so that skin aging is caused, wrinkles appear, skin elasticity is reduced, and pigmentation forms color spots. This test measures the level of wrinkles and freckles on the facial skin by a VISA-CR facial image analyzer, scores by the instrument's cloud database and its algorithm, lower scores indicating more wrinkles and freckles.

(1) The test method comprises the following steps: 80 women of 30-65 years old are selected as trial objects and divided into 4 groups of 20 persons, and the aloe fermentation liquid of the example 1 and the comparative examples 1-3 are respectively tried in each group. Before test, after cleaning face, the tester is placed in constant temperature and humidity environment (temperature 21-23 deg.C, humidity 55-65%) for 30min, and then VISA-CR is used to take facial photograph as primary data, and the tested person coats the aloe fermentation liquid of each test group on face, uses 1 time per day, continuously tests for 60 days, and tests for 0 dayVISA-CR was used to test facial wrinkles and spots, each marked as D, after washing the face on days 30 and 60 ₀ 、D ₃₀ And D ₆₀ . It is noted that each group D ₀ 、D ₃₀ And D ₆₀ Values are the average of the set of values, and the improvement of facial wrinkles and spots before and after use is calculated by the instrument software, with lower scores indicating more wrinkles and spots.

(2) The test results are shown in Table 8.

TABLE 8

As can be seen from table 8, the subjects who tried the aloe fermentation broth of example 1 had the best improvement of facial wrinkles and color spots, which indicates that the aloe fermentation broth of example 1 had the best whitening anti-aging effect, and experimental data indicate that lactobacillus paracasei, leuconostoc mesenteroides, and lactococcus lactis have a synergistic effect, and the aloe fermentation broth obtained by fermenting the composite fermentation broth of the lactobacillus paracasei, the leuconostoc mesenteroides, and the lactococcus lactis can improve the whitening anti-aging effect of the aloe fermentation broth.

6. Melanin inhibition efficacy test of Aloe fermentation broth

The early zebrafish embryos are transparent throughout the body, and melanin is deposited on the body surface when the zebrafish develops to 72hpf (hours post-fertilization), and can be clearly observed under a microscope. If the test substance is administered before 72hpf, it can be seen whether the melanin formation is reduced. The generation and inhibition of melanin in the body surface of the zebra fish have higher similarity with human beings, the main action path of the product with the whitening effect is to reduce the generation of the melanin by inhibiting the activity of tyrosinase, and the condition of the melanin of a tested object can be quantified by an image processing technology, so that the evaluation of the whitening effect is realized. The specific experimental method is as follows: a24-hour (24 hpf) embryo from the fertilization of an AB-line wild-type zebrafish was selected. Zebrafish were divided into normal control and experimental groups. The normal control group was not treated, and the experimental group used the diluted aloe fermentation broth (diluted to a concentration of 0.01 g/ml) prepared in example 1, and after incubation for a period of time, photographed the head of zebrafish, and analyzed the melanin signal intensity of the zebrafish head using advanced image processing software. The results are shown in Table 9.

TABLE 9

Group of	Melanin signal intensity
		Normal control group	46799.25
Experimental group	28464**

In contrast to the blank group, "+" indicates P < 0.05, "+" indicates P < 0.01, and "+" indicates P < 0.0001.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. The preparation method of the aloe fermentation liquor is characterized by comprising the following steps:

(1) Inoculating the zymophyte liquid into a mixture of the aloe liquid and a culture medium to obtain a fermentation stock solution;

(2) Fermenting the fermentation stock solution to obtain a fermentation product, namely the aloe fermentation solution;

the zymophyte liquid consists of lactobacillus liquid, bead bacteria liquid and lactococcus liquid.

2. The method of claim 1, wherein the lactobacillus solution is obtained by activating lactobacillus in a liquid medium, wherein the concentration of lactobacillus in the lactobacillus solution is 10% ⁸ ～10 ⁹ CFU/mL; the moniliforme liquid is obtained by activating moniliforme in a liquid culture medium, and the concentration of the moniliforme in the moniliforme liquid is 10 ⁸ ～10 ⁹ CFU/mL; the lactococcus liquid is obtained by activating lactococcus in a liquid culture medium, and the concentration of the lactococcus in the lactococcus liquid is 10 ⁸ ～10 ⁹ CFU/mL。

3. The method of claim 1, wherein the fermented liquid is selected from the group consisting of paracasei lactobacillus liquid, leuconostoc mesenteroides liquid, and lactococcus lactis liquid.

4. The method of claim 3, wherein the volume percentage of the lactobacillus paracasei liquid is 1-30%, the volume percentage of the leuconostoc mesenteroides liquid is 1-30%, and the volume percentage of the lactococcus lactis liquid is 1-30%, based on the fermentation stock solution.

5. The method of claim 4, wherein the volume percentage of the lactobacillus paracasei liquid is 2-10%, the volume percentage of the leuconostoc mesenteroides liquid is 2-10%, and the volume percentage of the lactococcus lactis liquid is 2-10%, based on the fermentation stock solution.

6. The method of claim 5, wherein the volume percentage of the lactobacillus paracasei solution is 5%, the volume percentage of the leuconostoc mesenteroides solution is 5%, and the volume percentage of the lactococcus lactis solution is 5%, based on the fermentation stock solution.

7. The method of claim 1, wherein the culture medium is MRS medium.

8. The method of claim 1, wherein the volume percentage of the aloe vera broth based on the fermentation broth is 1-40%.

9. The method of preparing an aloe vera broth according to claim 8, wherein the aloe vera broth is present in an amount of 2-30% by volume of the broth.

10. The method of preparing an aloe vera broth according to claim 9 wherein the aloe vera broth comprises 5% by volume of the broth.

11. The method for preparing aloe fermentation broth according to claim 1, wherein in step (2), the fermentation temperature is 25-37 ℃, the fermentation time is 12-48 h, and the pH value of the fermentation stock solution is 4.1-4.7.

12. The method of preparing an aloe fermentation broth according to claim 1, further comprising the steps of sequentially sterilizing, centrifuging, and collecting the supernatant of the fermentation product.

13. The method of preparing an aloe vera broth as claimed in claim 12 wherein the sterilization conditions are: the sterilization temperature is 120-125 ℃, and the sterilization time is 10-20 min; the centrifugation conditions were: the centrifugal temperature is 15-25 ℃, the centrifugal speed is 5500-6500 r/min, and the centrifugal time is 25-35 min.

14. An aloe vera broth obtained by the method of making an aloe vera broth according to any one of claims 1-13.

15. A skin care product comprising the aloe vera broth of claim 14.

16. The use of the aloe vera broth according to claim 15 in the preparation of a skin care product having whitening anti-aging effects.

17. Use according to claim 16, wherein the skin-care product comprises a cream, an eye cream, a toner or a serum.