CN116999374A

CN116999374A - Bifidobacterium adolescentis milk ferment, product containing same and preparation and application thereof

Info

Publication number: CN116999374A
Application number: CN202310086320.2A
Authority: CN
Inventors: 张炽坚; 何廷刚; 唐明慧; 张文云; 王昌涛; 张智婷; 艾勇; 罗剑芳; 林广欣; 耿林; 蔡少纯; 李佩晶; 荣宝珊
Original assignee: Hua An Tang Biotech Group Co ltd
Current assignee: Hua An Tang Biotech Group Co ltd
Priority date: 2023-01-19
Filing date: 2023-01-19
Publication date: 2023-11-07

Abstract

The application provides a bifidobacterium adolescentis milk ferment, a product containing the bifidobacterium adolescentis milk ferment, and preparation and application thereof. The preparation method of the bifidobacterium adolescentis milk ferment comprises the following steps: inoculating bifidobacterium adolescentis into a fermentation substrate, fermenting and culturing for 20-36 h, sterilizing, centrifuging and collecting supernatant; wherein the fermentation substrate comprises milk, and no carbohydrate carbon source is added. The application prepares the milk through fermenting the milk by the specific microorganism, has the antioxidation effect, can improve the content of cell elastin, improve the activity of cell lysosomes and promote the migration of cells; the bifidobacterium adolescentis milk ferment is high in use safety, has no stimulation to skin, can improve the skin barrier effect, is good in stability, easy to store, simple in preparation process, energy-saving and environment-friendly, and has a good application prospect in the field of cosmetics.

Description

Bifidobacterium adolescentis milk ferment, product containing same and preparation and application thereof

Technical Field

The application belongs to the field of cosmetics, and particularly relates to a bifidobacterium adolescentis milk ferment, a product containing the bifidobacterium adolescentis milk ferment, and preparation and application of the bifidobacterium adolescentis milk ferment.

Background

Milk is the oldest natural beverage, is rich in nutrient substances such as calcium, vitamin D, protein and the like, has the effects of promoting the growth and development of human bodies, promoting the development of brains, promoting digestion, protecting intestines and stomach, preventing constipation, improving immunity, beautifying and the like, and is widely applied to the field of foods. It is also reported in the cosmetic field that milk is rich in macromolecular proteins, which is liable to cause allergy, especially sensitive skin, in long-term use, for example, in the face washing, face pack and bathing processes. The milk is rich in grease, and the grease in the milk is dispersed in the emulsion in an emulsified state in the form of granular fat balls, so that pores can be blocked by using the grease in the milk for a long time, and folliculitis is easy to induce. Milk has poor stability, and after one week of storage at room temperature, obvious delamination and floccules occur, and unpleasant odor and the like are generated, and the milk cannot be used as cosmetics which can be stored for a long time.

With the improvement of living standard and the development of science and technology, cosmetics become necessary products in the life of people, and raw materials of cosmetics are divided into two major categories of artificial synthesis and natural extraction. Although the synthetic raw materials have remarkable effects on certain effects, the synthetic raw materials have obvious side effects, can irritate skin, damage skin barrier, cause the skin to become sensitive muscle and the like, and have relatively high preparation cost. Compared with the artificial raw materials, the natural extract has the advantages of improved safety and greatly reduced preparation cost, so that the raw materials of cosmetics containing natural active ingredients derived from plants become research hotspots in the field, but due to the complex components in the natural extract, when the extraction process is selected improperly, the efficacy of the active ingredients can be affected, and skin allergy can be caused.

Therefore, there is a need in the art to develop a method for efficiently extracting active ingredients from milk, and promote the prepared active ingredients to have ideal cosmetic effects, high use safety, no irritation to skin, and broad application range of milk.

Disclosure of Invention

The application aims to solve the technical problem of overcoming the defects in the prior art and providing a bifidobacterium adolescentis milk ferment, a product containing the bifidobacterium adolescentis milk ferment and preparation and application thereof. The fermentation treatment of the milk by the specific microorganism not only widens the application field of the milk, but also can prepare the bifidobacterium adolescentis milk ferment which has the oxidation resistance effect, can improve the content of cell elastin, improve the activity of cell lysosomes, promote cell migration, has high use safety, has no stimulation to skin, can improve the skin barrier effect, has good stability, is easy to store, has simple preparation process, saves energy and protects environment, and has good application prospect in the cosmetic field.

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

the application provides a preparation method of a bifidobacterium adolescentis milk ferment, which comprises the following steps: inoculating bifidobacterium adolescentis into a fermentation substrate, fermenting and culturing for 20-36 h, sterilizing, centrifuging and collecting supernatant; wherein the fermentation substrate comprises milk, and no carbohydrate carbon source is added.

The non-added carbohydrate carbon source means that there is no need to add any additional carbohydrate carbon source conventionally used in the art, such as glucose, sucrose or starch, in addition to the milk. During the research and development process, it is found that when the carbohydrate carbon source is additionally added, although sufficient nutrition is provided for the strain, the performance of the final product is seriously affected, for example, the oxidation resistance is reduced, the skin irritation is improved, and the use safety is poor.

In some embodiments, a nitrogen source may also be included in the fermentation substrate. The nitrogen source used in the present application is a nitrogen source conventionally used in the art in addition to the milk.

Wherein the nitrogen source may comprise L-cysteine hydrochloride.

Wherein the nitrogen source accounts for 0.01 to 0.05 percent of the mass of the milk, and preferably 0.03 to 0.05 percent.

In some embodiments, the fermentation substrate may also be subjected to procedures conventional in the art including sterilization prior to use.

Wherein the conditions and methods of sterilization may be those conventional in the art for such procedures and may generally be high temperature sterilization.

When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization temperature may be a temperature conventional in the art for such an operation, and preferably is 95 to 100 ℃.

When the sterilization of the fermentation substrate is performed by the high temperature sterilization method, the sterilization time may be a time conventional in this type of operation in the art, preferably 15 to 35 minutes, more preferably 30 minutes.

Wherein the sterilization operation may further comprise a cooling operation, typically to room temperature, as is conventional in the art.

In some embodiments, the bifidobacterium adolescentis may comprise bifidobacterium adolescentis model BBF-06, manufactured by the bioengineering company of jaundice, shandong and/or bifidobacterium adolescentis model SF-B40, manufactured by the bioengineering company of shandong sunflower.

In some embodiments, the bifidobacterium adolescentis can be added in the form of a bifidobacterium adolescentis bacterial liquid according to the routine in the art, wherein the number of viable bacteria in the bifidobacterium adolescentis bacterial liquid is 10 ⁶ ～10 ¹⁰ CFU/mL, preferably 10 ⁷ ～10 ⁹ CFU/mL。

In some embodiments, the amount of bifidobacterium adolescentis inoculated per unit volume of the fermentation substrate may be conventional in the art, preferably 10 ⁵ ～10 ⁹ CFU/mL, more preferably 10 ⁶ ～10 ⁸ CFU/mL。

In some embodiments, the conditions and methods of fermentation culture may be conventional in the art, and typically may be stationary cultured in an incubator.

In some embodiments, the fermentation time is preferably 24 to 30 hours.

In some embodiments, the temperature of the fermentation culture may be 37-43 ℃.

In some embodiments, the conditions and methods of sterilization may be conventional in the art, and may generally be high temperature sterilization.

When the sterilization is performed using the high temperature sterilization method, the sterilization temperature may be a temperature conventional in such an operation in the art, and preferably is 95 to 100 ℃.

When the sterilization is performed by the high temperature sterilization method, the sterilization time may be a time conventional in the art for such an operation, preferably 20 to 40 minutes, more preferably 30 minutes.

In some embodiments, the rotational speed of the centrifuge may be conventional in the art, preferably 4000 to 8000rpm, more preferably 4000 to 6000rpm, for example 4800rpm.

In some embodiments, the radius of the centrifugation may be a radius conventional in the art for such operations, preferably 8-15 cm.

In some embodiments, the centrifugation time may be conventional in the art, preferably 10 to 40 minutes, more preferably 20 to 40 minutes, for example 30 minutes.

In some embodiments, the centrifugation may further comprise filtering and collecting the filtrate.

Wherein the filter membrane used for the filtration may have a pore size of 0.22 to 0.8. Mu.m, preferably 0.22 to 0.45. Mu.m.

In a preferred embodiment, the filtering operation may further include a secondary sterilization and/or mixing with a preservative.

Wherein, the secondary sterilization method can be a high temperature sterilization method conventionally used in the art.

When the secondary sterilization is performed using the high temperature sterilization method, the temperature of the secondary sterilization may be a temperature conventional in the art for such an operation, and preferably is 95 to 100 ℃.

When the secondary sterilization is performed by the high temperature sterilization method, the time for the secondary sterilization may be a time conventional in the art for such an operation, preferably 20 to 40 minutes, more preferably 30 to 40 minutes.

Wherein the temperature of the mixing during the mixing with the preservative may be a temperature conventional in the art for such operations, preferably 50-80 ℃, more preferably 70-80 ℃.

Wherein the preservative may comprise p-hydroxyacetophenone and/or 1, 2-hexanediol as conventional in the art.

When the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the mass percentage of the p-hydroxyacetophenone to the filtrate prepared by filtering can be 0.2-0.6%, and the mass percentage of the 1, 2-hexanediol to the filtrate prepared by filtering can be 0.5-2%; preferably, the p-hydroxyacetophenone accounts for 0.2 to 0.5 percent of the mass of the filtrate obtained by filtering, and the 1, 2-hexanediol accounts for 0.5 to 1 percent of the mass of the filtrate obtained by filtering.

The application also provides the bifidobacterium adolescentis milk ferment which is prepared by the preparation method of the bifidobacterium adolescentis milk ferment.

The application also provides an application of the bifidobacterium adolescentis milk ferment in preparing external skin preparations directly as a product, as an additive or as a substrate.

In some embodiments, the bifidobacterium adolescentis milk fermentate may be used as at least one of an antioxidant active ingredient, a skin barrier improving active ingredient, an anti-aging active ingredient, and a soothing active ingredient in the skin external preparation.

Wherein the antioxidant active ingredient may be an antioxidant active ingredient having DPPH radical scavenging ability and/or hydroxy radical scavenging ability.

Wherein the skin barrier improving active ingredient may be a skin barrier improving active ingredient having moisturizing and moisturizing effects.

Wherein the anti-aging active ingredient may be an anti-aging active ingredient having an increased skin elastin content.

Wherein the soothing active ingredient may be a soothing active ingredient having a function of inhibiting hemolysis of erythrocytes caused by SDS.

The application also provides a skin external preparation which comprises the bifidobacterium adolescentis milk ferment.

In some embodiments, the skin external preparation may include, but is not limited to, a cream, a mask, an essence, or a toner, as is conventional in the art.

In some embodiments, the external preparation for skin may further include at least one of active ingredients, preservatives, thickeners, oils, emulsifiers, and solvents conventionally used in the art.

Wherein the active ingredient may include at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient, and an anti-oxidation active ingredient.

Wherein the preservative may comprise a preservative conventionally used in the art, preferably comprising butylene glycol and/or phenoxyethanol.

The thickener may include, among others, thickeners conventionally used in the art, preferably including carbomers.

The grease may include grease conventionally used in the art, and preferably includes at least one of jojoba seed oil, polydimethylsiloxane, squalane, and cetyl alcohol.

The emulsifying agent may include, among others, emulsifying agents conventionally used in the art, preferably polysorbate-20 and/or sorbitan isostearate.

The solvent may include a solvent conventionally used in the art, preferably deionized water.

Wherein the preservative accounts for 12-15% of the weight of the skin external agent, and is preferably 13.03%.

Wherein the thickener accounts for 0.5-1% of the skin external agent by mass, and preferably 0.6%.

Wherein the grease accounts for 4-10% of the mass of the skin external agent, and is preferably 5-6%.

Wherein the emulsifier accounts for 1 to 3 percent of the mass of the skin external agent, and is preferably 1.5 to 1.6 percent.

Wherein the solvent may be used in an amount conventional in the art, based on 100% of the total amount of the external preparation for skin, in balance with the solvent.

In some embodiments, the bifidobacterium adolescentis milk ferment may be 5% -99%, preferably 60% -99% of the mass of the external skin preparation.

In some embodiments, the room temperature generally refers to 15-40 ℃.

On the basis of conforming to the common knowledge in the field, the above preferred conditions can be arbitrarily combined to obtain the preferred examples of the application.

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

The application has the positive progress effects that: the bifidobacterium adolescentis milk ferment prepared by the method has ideal oxidation resistance, can improve the content of cell elastin, improve the activity of cell lysosomes and promote cell migration; the use safety is high, the skin is not stimulated, and the skin barrier effect can be improved; the preparation method has the advantages of good stability, easy storage, simple preparation process, mild fermentation conditions, low energy consumption, cost saving, zero burden on skin, higher safety, and suitability for the requirements of modern skin external preparations on functionality and safety, and can be widely applied to the field of skin external preparations.

Drawings

The application may be better understood by reference to the following description taken in conjunction with the accompanying drawings. The accompanying drawings, which are included to provide a further illustration of the preferred embodiments of the application and together with a further understanding of the principles and advantages of the application, are incorporated in and constitute a part of this specification.

Wherein:

FIG. 1 is a graph showing the comparison of DPPH radical scavenging rates of the products obtained in examples 1 to 3 and comparative examples 1 to 3;

FIG. 2 is a graph showing comparison of the hydroxyl radical scavenging rates of the products prepared in examples 1 to 3 and comparative examples 1 to 3;

FIG. 3 is a graph showing the comparison of erythrocyte hemolysis rates of the products obtained in examples 1 to 3 and comparative examples 1 to 3;

FIG. 4 is a graph showing comparison of erythrocyte hemolysis inhibition ratios of the products prepared in examples 1 to 3 and comparative examples 1 to 3;

FIG. 5 is a graph showing the comparison of the content of elastin in fibroblasts after skin treatment by the products prepared in examples 1 to 3 and comparative examples 1 to 3;

FIG. 6 is a graph showing the comparison of protein content in the blank, the product obtained in example 1 and the products obtained in comparative examples 4 to 5;

FIG. 7 is a photograph showing the properties of the products of example 1, comparative example 4 and comparative example 5 at day 1 after the preparation;

FIG. 8 is a photograph showing the properties of the products prepared in example 1, comparative example 4 and comparative example 5 after they are stored at-20℃for 7 days under the conditions of illumination and-60 ℃;

FIG. 9 is a chart showing skin feel report comparisons of the products prepared in example 1, comparative example 4 and comparative example 5;

FIG. 10 is a graph showing the difference between the moisture content of the skin at 30min and 1h and the moisture content of the skin at 0min after the skin was treated with the cream prepared in effect example 10;

FIG. 11 is a graph showing the difference between the epidermal water loss rate at 30min and 1h and the epidermal water loss rate at 0min after the skin was treated with the cream prepared in effect example 10;

FIG. 12 is a graph showing comparison of the cell lysosomal activity after skin fibroblasts were treated with the products prepared in examples 1 to 3 and comparative examples 3 to 4;

FIG. 13 is a graph showing comparison of cell migration ability after skin fibroblasts were treated with the products prepared in examples 1 to 3 and comparative example 4.

Detailed Description

The application is further illustrated by means of the following examples, which are not intended to limit the scope of the application. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.

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

Bifidobacterium adolescentis in example 1 below was purchased from the bioengineering company, jatropha, shandong, model BBF-06;

Bifidobacterium adolescentis in example 3 below was purchased from shandong sunflower bioengineering limited under the model SF-B40;

bifidobacterium lactis in comparative example 3 below was purchased from zheng and joint biotechnology limited, product number HH-BA68.

Example 1

(1) Sterilizing 300g of milk in a high-temperature sterilizing pot at 95 ℃ for 30min, and cooling to room temperature after sterilization to obtain a fermentation substrate;

(2) Preparing Bifidobacterium adolescentis product number BBF-06 produced by Jiayi bioengineering Co., ltd. In Shandong, into viable count of 10 ¹⁰ CFU/mL of Bifidobacterium adolescentis liquid;

inoculating 3mL of bifidobacterium adolescentis bacterial liquid into the fermentation substrate prepared in the step (1), and standing and fermenting in a constant temperature incubator at 37 ℃ for 24 hours; after fermentation, transferring the materials into a high-pressure steam sterilizing pot, and sterilizing for 30min at 95 ℃; cooling to room temperature after sterilization, centrifuging for 30min at a rotation speed of 4800rpm and a centrifugation radius of 15cm, collecting supernatant, filtering the supernatant by a 0.22 mu m filter membrane, removing bifidobacterium adolescentis and impurities, performing secondary sterilization on the filtered filtrate, sterilizing for 30min at 95 ℃, adding p-hydroxyacetophenone and 1, 2-hexanediol serving as preservatives into the system when the temperature of the system is reduced to 75 ℃ after the secondary sterilization is finished, wherein the p-hydroxyacetophenone accounts for 0.5% of the mass of the filtrate, and the 1, 2-hexanediol accounts for 0.5% of the mass of the filtrate, so as to obtain the bifidobacterium adolescentis milk ferment.

Example 2

Compared with example 1, the fermentation substrate is different, in the step (1), 300g of milk and 0.15g of L-cysteine hydrochloride are mixed, sterilized in a high-temperature sterilizing pot with the temperature of 95 ℃ for 30min, and cooled to room temperature after sterilization, so as to obtain the fermentation substrate, and other condition parameters are the same as those of example 1, so as to obtain the bifidobacterium adolescentis milk fermentation product.

Example 3

Compared with example 1, the fermentation strain is purchased from Shandong sunflower bioengineering Co., ltd, and the bifidobacterium adolescentis product No. SF-B40 is obtained by preparing a milk ferment of bifidobacterium adolescentis from the same conditions and parameters as in example 1.

Comparative example 1

The difference from example 1 is that the fermentation substrate is the same amount of soybean extract, and other conditions are the same as those in example 1, to obtain soybean fermented product;

the preparation method of the soybean extract comprises the following steps: weighing soybeans, soaking for 8 hours, and then boiling the soybeans at the temperature of 100 ℃, wherein the mass ratio of the soybeans to water is 1:10, pulping, adding monosaccharide (glucose) with mass ratio of 0.5%, and sterilizing at 100deg.C for 30min to obtain fermentation substrate.

Comparative example 2

Compared with example 1, the fermentation substrate is different, in the step (1), 300g of milk and 1.5g of glucose are mixed, sterilized in a high-temperature sterilizing pot with the temperature of 100 ℃ for 30min, and cooled to room temperature after sterilization, so as to obtain the fermentation substrate, and other condition parameters are the same as those of example 1, so as to obtain the bifidobacterium adolescentis/milk glucose fermentation product.

Comparative example 3

The difference from example 1 was that the fermentation strain was bifidobacterium lactis obtained from Zheng and He bioengineering technologies Co., ltd, and had the product number HH-BA68, and the other conditions and parameters were the same as in example 1, to obtain a bifidobacterium lactis/milk fermented product.

Comparative example 4

The fermentation substrate obtained in the step (1) of example 1 was used as a fermentation substrate, which was different from example 1 only in that the fermentation treatment was not performed.

Comparative example 5

Compared with example 1, the method only differs from the method in the step (2) in that the fermentation time is 7h, and other condition parameters are the same as those of example 1, so as to prepare the bifidobacterium adolescentis milk ferment.

Effect example 1: DPPH free radical scavenging experiments

DPPH is an early synthetic organic radical, commonly used to evaluate the hydrogen donating ability of antioxidants, which is very stable in organic solvents, purple in color, and has a characteristic absorption peak at 517nm, when a radical scavenger is encountered, the lone pair of electrons of DPPH are paired to fade it, i.e., the absorbance at the maximum absorption wavelength becomes small. Therefore, the effect of the sample on DPPH radical scavenging can be evaluated by measuring the change in absorbance.

The DPPH free radical scavenging experiment comprises the following specific experimental steps:

(1) Taking an equal volume (1 mL) of the liquid to be measured and 2X 10 ^-4 mixing the DPPH solution of mol/L (A) ₁ A tube);

(2) Taking equal volume (1 mL) of absolute ethanol (solvent of the object to be detected) and 2X 10 ^-4 mixing the DPPH solution of mol/L (A) ₂ A tube);

(3) Mixing the same volume (1 mL) of absolute ethanol with the liquid to be measured (A) ₃ A tube);

(4) After reaction in the dark for 30min, A was measured at 517nm ₁ Tube A ₂ Tube A ₃ Tube absorbance values; the clearance rate calculation formula is: clearance = [ (A) ₂ +A ₃ )-A ₁ ]/A ₂ ×100％。

The products prepared in examples 1 to 3 and comparative examples 1 to 3 were subjected to the test of DPPH radical scavenging test, and the test liquids were the products prepared in the above examples and comparative examples, and the results are shown in Table 1 and FIG. 1.

TABLE 1

Numbering device	DPPH radical scavenging rate
		Example 1	88.92％
Example 2	94.33％
		Example 3	92.55％
Comparative example 1	47.52％
		Comparative example 2	79.05％
Comparative example 3	61.74％

As can be seen from the results, the DPPH radical scavenging rates of the products prepared in example 1, example 2 and example 3 were higher than those of the products prepared in comparative examples 1 to 3.

Effect example 2: hydroxyl radical scavenging rate

Hydroxyl radical is the most active radical in chemical nature in active oxygen, can react with almost any biological macromolecules in living cells, has extremely high reaction speed, and is the radical with the greatest harm to organisms. Salicylic acid is added into the reaction system, so that hydroxyl free radicals can be effectively captured, and a colored product 2, 3-dihydroxybenzoic acid is generated, wherein the colored product has a strong absorption peak at 510 nm.

If a sample with the function of removing the hydroxyl radical is added into the system, and the effect of the sample for capturing the hydroxyl radical is larger than that of salicylic acid, the hydroxyl radical can be timely removed, so that the generation amount of colored products is reduced, and the absorbance is reduced. Therefore, the absorbance of the reaction liquid containing the to-be-detected object is measured at the position of 510nm by adopting a fixed reaction time method, and compared with a blank liquid, the scavenging effect of the sample on the hydroxyl radical can be measured.

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

a1: 2mL of 6mmol/L FeSO were added sequentially to the tube ₄ ，2mL 6mmol/L H ₂ O ₂ (wherein H ₂ O ₂ The final addition, namely starting the whole reaction), 2mL of the sample to be detected, shaking uniformly and standing for 15min at room temperature. 2mL of 6mmol/L salicylic acid is added, the mixture is shaken uniformly, and the mixture is taken out after being heated in a water bath at 37 ℃ for 30min, and the absorbance A1 is measured;

a2: 2mL of 6mmol/L FeSO were added sequentially to the tube ₄ ，2mL 6mmol/L H ₂ O ₂ 2mL of a sample to be detected is shaken uniformly, kept stand at room temperature for 15min, then added with 2mL of deionized water, shaken uniformly, heated in a water bath at 37 ℃ for 30min, taken out and measured for absorbance A2;

a3: 2mL of 6mmol/L FeSO were added sequentially to the tube ₄ 2mL of deionized water, 2mL of 6mmol/L H ₂ O ₂ Shaking, standing at room temperature for 15min, adding 2mL of 6mmol/L salicylic acid, shaking, heating in water bath at 37deg.C for 30min, taking out, and measuring absorbance A3.

Hydroxyl radical scavenging = [ (a3+a2) -a1]/a3×100%.

The products prepared in the above examples and comparative examples were tested for hydroxyl radical scavenging and the results are shown in Table 2 and FIG. 2.

TABLE 2

Numbering device	Hydroxyl radical scavenging rate
		Example 1	80.21％
Example 2	55.61％
		Example 3	98.63％
Comparative example 1	17.02％
		Comparative example 2	53.98％
Comparative example 3	59.54％

As can be seen from the results, the hydroxyl radical scavengers of the products prepared in examples 1 to 3 were all much higher than those of the products prepared in comparative examples 1 and 2.

Effect example 3: erythrocyte hemolysis stimulation experiment

1. Preparation of Red cell suspensions (RBC)

1.1. Cleaning

Fresh sheep blood was diluted with PBS at a dilution ratio of 2:5 in a 50mL centrifuge tube. The centrifuge tube was gently turned upside down to allow adequate mixing of the blood and PBS. Subsequently, centrifugation at 1000 Xg for 10min at room temperature, after centrifugation, the supernatant (supernatant in the form of a pale yellow blood leukocyte layer) was discarded, and then PBS buffer was added, and repeated 1-2 times, which process removed a large amount of leukocytes, plasma and yellow debris.

1.2. Preparation of erythrocyte suspensions (RBC)

The blood cell pellet in the centrifuge tube was transferred to a new EP tube with a disposable pipette, 1mL of RBC pellet was taken in 50mL of EP tube, diluted with 49mL of PBS buffer, and mixed with gentle shaking.

250. Mu.L of RBC was placed in a 1.5mL EP tube, supplemented with 750. Mu.L of distilled water to 1mL, centrifuged at 10000 Xg for 1min to stop incubation, and the supernatant was removed after centrifugation and OD was measured at 540 nm. The OD value measured at 540nm is between 0.5 and 0.8.

2. Erythrocyte hemolysis stimulation experiment

TABLE 3 Table 3

Grouping	PBS	Water and its preparation method	Sample of	RBC
					(1) Blank group	750	---	---	250
(2) Positive group	---	750	---	250
					(3) Sample group	600	---	150	250

Preparing according to the above table 3, placing in a shaking table after sample addition, culturing at 37 deg.C for 60min at 180rpm, and taking out; after centrifugation at 10000g for 1min, the reaction was stopped. The photographs were recorded and 200. Mu.L of supernatant was aspirated into 96-well plates and absorbance was measured at 540 nm.

Erythrocyte hemolysis = (OD sample group-OD blank)/(OD positive group-OD blank) ×100%; the erythrocyte hemolysis rate of the products prepared in the above examples and comparative examples was measured, and the results are shown in Table 4 and FIG. 3.

TABLE 4 Table 4

The erythrocyte hemolysis rate can be used for detecting the irritation of the product to a certain extent, and experimental results show that the erythrocyte hemolysis rate of the products prepared in the examples 1-3 is lower than that of the products prepared in the comparative examples 1-3, so that the products prepared in the examples 1-3 are milder and safer.

Effect example 4: erythrocyte hemolysis inhibition rate

The products prepared in the above examples and comparative examples were tested for their erythrocyte hemolysis inhibition rate as follows:

1. Experimental materials and reagents

1. PBS buffer solution with pH value of 7.4 is prepared, purified water is prepared, and the mixture is stored at 4 ℃.

2.1% SDS solution configuration

SDS powder (0.5 g) was weighed and dissolved in 50mL of PBS buffer. In use, 1% SDS solution was diluted to 0.1% SDS with PBS buffer for use.

2. Experimental protocol

1. Preparation of Red cell suspensions (RBC)

1.1. Cleaning

1.2. Preparation of erythrocyte suspensions (RBC)

The experiments were performed according to table 5, the specific procedure being as follows:

TABLE 5

Grouping	PBS	Water and its preparation method	Sample of	RBC	SDS
						(1) Blank group	750	---	---	250	---
(2) Model comparison group	---	750	---	250	---
						(3) Model group	730	---	---	250	20
(4) Sample group	580	---	150	250	20
						(5) Sample control group	600	---	150	250	---

The 4 groups of samples (Nos. 2 to 5) were placed in an incubator, incubated at 180rpm and 37℃for 60 minutes, and then taken out, centrifuged at 10000 Xg for 1 minute, and the reaction was terminated. The sample hemolysis inhibition was calculated by photographing, recording and sucking 200. Mu.L of the supernatant in a 96-well plate, measuring absorbance at 540nm, and the results are shown in Table 6 and FIG. 4.

1) Model group hemolysis = (OD model-OD blank)/(OD model contrast-OD blank) ×100%;

2) Sample group hemolysis = (OD sample group-OD sample control)/(OD model contrast-OD blank) x100%;

3) Sample hemolysis inhibition = (model group hemolysis rate-sample treatment group hemolysis rate)/model group hemolysis rate × 100%.

TABLE 6

Numbering device	Erythrocyte hemolysis inhibition rate
		Example 1	45.26％
Example 2	35.17％
		Example 3	31.63％
Comparative example 1	6.49％
		Comparative example 2	40.75％
Comparative example 3	36.44％

Effect example 5

Will be 1X 10 ⁶ The fibroblasts/wells were inoculated into 6-well plates for 24 hours, the original medium was aspirated and treated with serum-free DMEM for 24 hours, respectively. After sample treatment, the original medium was aspirated, 1mL of PBS was added and the cells were exposed to UVA 7J/cm ² The blank group was not treated, DMEM (125 μl of sample to be measured; 875 μl of DMEM) containing examples 1 to 3 and comparative examples 1 to 3 was added respectively after 40min of treatment, the supernatant and cells were collected respectively after further culturing overnight, the cells were washed with PBS, 200 μl of lysate (containing PMSF at a final concentration of 1 mM) was added, and the mixture was blown uniformly with a pipette, and the cell supernatant was collected by centrifugation at 10000rpm for 5min at 4 ℃, followed by detection of the elastin content in the fibroblasts.

The content of elastin in the fibroblasts after the treatment of the products obtained in the above examples and comparative examples was tested and the results are shown in Table 7 and FIG. 5.

TABLE 7

Numbering device	Elastin content detection (ng/mg)
		Blank control group	6.6
Model group	3.48
		Example 1	6.88
Example 2	7.1
		Example 3	6.61
Comparative example 1	5.12
		Comparative example 2	4.98
Comparative example 3	2.89

The results show that the products prepared in examples 1-3 are more beneficial to promoting the secretion of elastin in fibroblasts and have more ideal repairing effect on damaged fibroblasts than comparative examples.

Effect example 6

The protein content of the products prepared in comparative example 4, comparative example 5 and example 1 was determined by a color characterization method. The deeper the purple color, the higher the protein content and the test results are shown in FIG. 6. The biological BCA kit was used for the test.

The specific test process is as follows:

(1) According to reagent A: preparing BCA working solution by the reagent B according to the ratio of 50:1;

(2) Standard substances (0, 0.025, 0.05, 0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL) with different concentrations and 20 mu L of a sample to be tested are respectively added into a 96-well plate; the blank group sample to be tested is PBS buffer solution, and the experimental group sample to be tested is the product prepared in example 1, comparative example 4 or comparative example 5;

(3) 200 mu L of prepared BCA working solution is added to each well, and after incubation for 30min at 37 ℃, OD562nm value is measured;

(4) Calculating the protein concentration in the sample according to the standard curve formula (y=0.0008x+0.016, r ² ＝0.996)。

In fig. 6, a blank control, a comparative example 4, a comparative example 5, and an example 1 are shown in this order from left to right. The deeper the purple color, the higher the protein content in the product. As can be seen from fig. 6, the products prepared in example 1 were significantly lighter in color after fermentation than those prepared in comparative examples 4 and 5, demonstrating a significant reduction in protein content.

Effect example 7 stability

The experimental method comprises the following steps: the products prepared in example 1, comparative example 4 and comparative example 5 were packaged in 15mL transparent glass bottles (the state of the products is shown in fig. 7), each product was placed at normal temperature, 60 ℃ and-20 ℃ for one week under light, and the tolerance of the product to high temperature, low temperature and light was observed, i.e. the stable state of the product was examined, and the state of the product is shown in fig. 8.

Analysis of results:

the products prepared in comparative example 4 and comparative example 5 are uniform milky white suspensions, and are all milk smell; the product obtained in example 1 was a pale yellow transparent liquid, a pale fermented taste, and no smell of raw milk.

After the products are placed for one week, the product forms are observed, and the products prepared in the comparative examples 4 and 5 are in an unstable state, and layering phenomena occur at normal temperature, illumination and 60 ℃, and white slag is attached to the bottle wall, especially in a high-temperature state; whereas the product of example 1 is more stable.

After the product is placed for one week, the product morphology is observed, and a large amount of white flocculent precipitates are found in the products prepared in the comparative examples 4 and 5 at the low temperature of-20 ℃ and are insoluble at room temperature, and only a small amount of fine precipitates are separated out in the product prepared in the example 1 and are dissolved again by shaking the fine precipitates at room temperature.

The results show that the milk is an unstable system containing suspension, layering, protein macromolecule coagulation and precipitation and the like are easy to occur in the storage process, the product prepared by fermenting the milk by adopting the specific fermentation method obviously overcomes the defects, and the product is more stable along with the extension of the fermentation time. The product of example 1 is less susceptible to light and temperature and is a more stable product system.

Effect example 8

The pH values of the products prepared in example 1, comparative example 4 and comparative example 5 were tested after preparation and after one week of storage, and the results are shown in table 8.

TABLE 8

The weak acidity is the key of skin properties, and healthy skin has a pH of about 4.2-6.5 and an average pH of about 5.5, and is in a weak acidic state. In this state, the skin can keep natural water-oil balance, has strong resistance, and is not easy to grow bacteria. The pH of the skin surface can affect the lipid content and hydration of the stratum corneum, and generally needs to be properly adjusted to maintain the pH of the skin within a reasonable range. The results show that the pH of the product prepared in example 1 is significantly lower than that of the products prepared in comparative examples 4 and 5, and is more beneficial to the maintenance of the skin slightly acidic environment.

Effect example 9

Questionnaire investigation: 3 products, the number of people: 10 persons

The products obtained in example 1 and comparative examples 4 to 5 were taken as test products, and then the test products were applied to the inner side of the arm, and the preferred products were selected for the state, smell, skin feel and absorption degree, and the statistics of the number of persons were carried out, and the results are shown in Table 9 and FIG. 9.

From the figure, 100% of the people like the state of the product prepared in example 1, 80% of the people like the smell of the product prepared in example 1, and at the same time, 90% of the people consider that the product prepared in example 1 has better skin feel and absorption degree.

TABLE 9

Effect example 10 application formulation

A cream comprising the product of example 1 described above was produced and its composition is shown in table 10.

Table 10

Effect example 11 improvement of skin barrier effect

14 healthy adults were screened, and samples (cream prepared in effect example 10) were used as required to test the moisture content and the transepidermal moisture loss rate of the test area on the inner side of the forearm, and the effect of improving skin barrier was improved 30min and 1h after the use of the samples, compared with the use before the use of the samples (T0).

Intra-group control: comparing the moisture content of the test area with the variation condition of the trans-epidermal moisture loss rate at different time points before and after using the sample;

Inter-group control: comparison Using the experimental group and the blank (the only difference compared with effect example 10 is that the product prepared in example 1 was replaced with the same amount of water), the variation of the moisture content and the trans-epidermal moisture loss rate was measured at different time points.

1) Volunteers cannot apply any product on the arm two days before and on the same day, 1-3 hours before visit, the measurement part cannot be cleaned or contacted with water, and after arriving at a laboratory, the volunteers sit still in a constant temperature and humidity room for 20min, and water and beverage cannot be drunk during the test.

2) The test area is marked on the inner sides of the left arm and the right arm by using a skin marker pen for the arms,the area of the region is 3X 3cm ² The test areas are spaced apart by at least 1cm. The sample application area and the blank control area should be randomly distributed in the test area to ensure that all sample and blank area positions are statistically balanced.

3) After the volunteers rest for 20min, data acquisition of the basic value (T0) of the trans-epidermal water loss rate is carried out on each test area on the inner side of the arm, and each area is tested once; the test areas were each cleaned with a dry tissue and data were collected for the basal moisture content (T0) of the skin stratum corneum, each area was tested 3 times in parallel and averaged.

4) Smearing samples on the corresponding test areas according to the requirements, respectively collecting data of the percutaneous moisture loss rate and the skin moisture content value of each test area after 30min, and making relevant records (T30 min);

5) After 1h, the data of the percutaneous moisture loss value and the skin moisture content value of each test area are respectively acquired, and relevant records (T1 h) are made

The skin moisture content test results are shown in Table 11, and the skin moisture content change values at 30min and 1h are calculated as compared with 0min, respectively, and the calculation results are shown in Table 11 and FIG. 10.

Table 11 skin moisture content (a.u.) (n=14

After using the face cream prepared in effect example 10, the skin moisture content was significantly increased (p=0.001 < 0.05). After 30min and 1h using the cream prepared in example 10, the moisture content was increased by 62.40% and 68.81%, respectively (the calculation formula is shown below). The results show that the cream comprising the product of example 1 (effect example 10) has the desired moisturizing effect.

Compared with the blank area, the calculation formula of the water content improvement rate at different time points is as follows:

sample side change rate= (sample side Tx time test mean-sample side T0 time test mean)/sample side T0 time test mean x 100%;

Blank side change rate= (blank side Tx time test mean-blank side T0 time test mean)/blank side T0 time test mean x 100%;

rate of moisture content increase at different time points = sample side rate of change-blank side rate of change.

Where T0 is before using the product, tx is the x-th hour after using the product.

The results of the skin transdermal moisture loss rate test are shown in Table 12, and the values of the skin transdermal moisture loss rate change at 30min and 1h are calculated as compared with those at 0min, respectively, and the calculation results are shown in Table 12 and FIG. 11.

TABLE 12 variation of skin Transcutaneous Water loss Rate (TEWL) (g/m ² h)

After 30min and 1h using the cream prepared in effect example 10, TEWL was significantly reduced (P < 0.05) compared to the blank area. Compared with the blank area, after 30min and 1h of the face cream prepared in the application effect example 10, the skin percutaneous moisture loss rate is respectively reduced by 18.10 percent and 17.93 percent (a calculation formula for calculating the formula and the moisture content improvement rate); shows that the cream product containing the bifidobacterium adolescentis milk ferment prepared in example 1 has ideal water-retaining effect.

As is clear from the above experiments, the cream prepared in effect example 10 has ideal moisturizing and water-retaining effects, i.e., has good effect of improving skin barrier.

Effect example 12 lysosomal activity assay

The lysosome contains various hydrolases, can degrade macromolecular substances, and the activity and the quantity of the lysosome can indirectly reflect the metabolic updating capacity of cells, and the higher the activity of the lysosome is, the stronger the metabolic capacity of the cells is.

At physiological pH, the net charge of neutral red dye is almost zero, thus allowing it to penetrate the cell membrane into the cell by way of non-ionic passive diffusion. The proton gradient in the lysosome causes the pH in the lysosome to be lower than the cytoplasm, which can charge the neutral red and accumulate in the lysosome, thereby detecting lysosomal activity.

The testing method comprises the following steps: skin fibroblasts were plated in 96-well plates, 8000 cells per well. After the cells were attached, a blank control group and a sample group were set, a serum-free culture medium was added to the blank control group, and a serum-free culture medium containing 1.25% of the samples (the products produced in examples 1 to 3 and comparative examples 3 to 4 described above) was added to the sample group. After 24h of treatment, the cell culture broth was discarded, 200. Mu.L of fresh serum-free broth was exchanged, 20. Mu.L of neutral red was added for incubation for 2h, then the broth containing neutral red was removed, washed 2 times with PBS, then 200. Mu.L of neutral red detection lysate was added, lysed for 10min on a shaking table at room temperature, and A540 per well was determined.

Sample group lysosomal activity was calculated using a blank control as a reference. Lysosomal activity% = (a 540 sample group/a 540 blank) ×100%, the results are shown in table 13 and fig. 12.

TABLE 13

Numbering device	Sample group lysosomal Activity
		Blank control group	100％
Example 1	108.39％
		Example 2	115.30％
Example 3	114.63％
		Comparative example3	86.14％
Comparative example 4	81.73％

The results show that: compared with the blank control group, the products prepared in the examples 1-3 have obviously raised lysosome activity after the cells are treated, and the samples in the comparative examples 3 and 4 are adopted to treat the cells, so that the lysosome activity is not improved, but is reduced. From these results, it was found that the products prepared in examples 1 to 3 promote catabolism of nutrients by lysosomes and promote cell metabolism renewal.

Effect example 13 cell migration experiments

Skin fibroblasts were plated on 12-well plates and cultured overnight in an incubator. The culture medium was then removed, streaked with a sterile gun head, and the untreated state (labeled 0h in the figure) was recorded by photographing, followed by adding a serum-free culture medium containing 1.25% of the sample (the products produced in examples 1 to 3 and comparative example 4 described above), and after 24h of treatment, photographing was performed (labeled 24h in the figure), and the results are shown in FIG. 13.

As is clear from the results of FIG. 13, the products prepared in examples 1 to 3 of the present application significantly promoted migration of cells, especially the product prepared in example 2, which had the best effect of promoting migration of cells, while the product prepared in comparative example 4 showed no significant migration of cells.

Finally, it is also noted that 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 application has been disclosed by the foregoing description of specific embodiments thereof, it will be appreciated that those skilled in the art may devise various modifications, adaptations, or equivalents of the application within the spirit and scope of the appended claims. Such modifications, improvements, or equivalents are intended to be included within the scope of this application as claimed.

Claims

1. A preparation method of a bifidobacterium adolescentis milk ferment is characterized by comprising the following steps: inoculating bifidobacterium adolescentis into a fermentation substrate, fermenting and culturing for 20-36 h, sterilizing, centrifuging and collecting supernatant; wherein the fermentation substrate comprises milk, and no carbohydrate carbon source is added.

2. The method for preparing a milk ferment of bifidobacterium adolescentis according to claim 1, wherein the fermentation substrate further comprises a nitrogen source;

preferably, the nitrogen source comprises L-cysteine hydrochloride;

preferably, the nitrogen source accounts for 0.01 to 0.05 percent of the mass of the milk, and more preferably 0.03 to 0.05 percent of the mass of the milk.

3. A method of preparing a bifidobacterium adolescentis milk ferment as claimed in claim 1 wherein the ferment substrate comprises a sterilising operation prior to use;

preferably, the sterilization method is a high-temperature sterilization method;

when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization temperature is 95-100 ℃;

when the high-temperature sterilization method is adopted to sterilize the fermentation substrate, the sterilization time is 15-35 min, preferably 30min;

preferably, the sterilization operation is further followed by cooling to room temperature.

4. The method for preparing a bifidobacterium adolescentis milk ferment according to claim 1, wherein the method for preparing a bifidobacterium adolescentis milk ferment satisfies at least one of the following conditions:

The bifidobacterium adolescentis comprises bifidobacterium adolescentis model BBF-06 manufactured by Shandong Zhongke Jiayi bioengineering Co., ltd and/or bifidobacterium adolescentis model SF-B40 manufactured by Shandong Helianthus annuus bioengineering Co., ltd;

the Bifidobacterium adolescentis is added in the form of Bifidobacterium adolescentis bacterial liquid, and the number of viable bacteria in the Bifidobacterium adolescentis bacterial liquid is 10 ⁶ ～10 ¹⁰ CFU/mL, preferably 10 ⁷ ～10 ⁹ CFU/mL；

The number of the bifidobacterium adolescentis inoculated into the fermentation substrate per unit volume is 10 ⁵ ～10 ⁹ CFU/mL, preferably 10 ⁶ ～10 ⁸ CFU/mL。

5. The method for preparing a milk ferment of bifidobacterium adolescentis according to any one of claims 1-4, characterized in that the method for preparing a milk ferment of bifidobacterium adolescentis satisfies at least one of the following conditions:

the fermentation culture is carried out in a constant temperature incubator;

the fermentation culture time is 24-30 hours;

the temperature of the fermentation culture is 37-43 ℃;

the sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is adopted for the sterilization, the sterilization temperature is 95-100 ℃; when the high-temperature sterilization method is adopted for the sterilization, the sterilization time is 20-40 min, preferably 30min;

The rotational speed of the centrifugation is 4000-8000 rpm, preferably 4000-6000 rpm;

the radius of the centrifugation is 8-15 cm;

the centrifugation time is 10 to 40 minutes, preferably 20 to 40 minutes.

6. The method for preparing a milk ferment of bifidobacterium adolescentis according to claim 1, characterized in that the centrifugation operation is followed by a filtration operation, in which the filtrate is collected;

preferably, the pore size of the filter membrane used for the filtration is 0.22-0.8 μm, preferably 0.22-0.45 μm;

preferably, the filtering operation is further followed by a secondary sterilization and/or mixing with a preservative.

7. The method for preparing a bifidobacterium adolescentis milk ferment according to claim 6, wherein the method for preparing a bifidobacterium adolescentis milk ferment satisfies at least one of the following conditions:

the secondary sterilization method is a high-temperature sterilization method; when the high-temperature sterilization method is adopted for the secondary sterilization, the temperature of the secondary sterilization is 95-100 ℃; when the high-temperature sterilization method is adopted for the secondary sterilization, the time of the secondary sterilization is 20-40 min, preferably 30-40 min;

in the mixing process with the preservative, the mixing temperature is 50-80 ℃, preferably 70-80 ℃;

The preservative comprises p-hydroxyacetophenone and/or 1, 2-hexanediol; when the preservative comprises the p-hydroxyacetophenone and the 1, 2-hexanediol, the p-hydroxyacetophenone accounts for 0.2-0.6% of the mass of the filtrate obtained by filtering, and the 1, 2-hexanediol accounts for 0.5-2% of the mass of the filtrate obtained by filtering; preferably, the p-hydroxyacetophenone accounts for 0.2 to 0.5 percent of the mass of the filtrate obtained by filtering, and the 1, 2-hexanediol accounts for 0.5 to 1 percent of the mass of the filtrate obtained by filtering.

8. A bifidobacterium adolescentis milk ferment characterized by being prepared by the method of preparing a bifidobacterium adolescentis milk ferment as claimed in any one of claims 1 to 7.

9. Use of a bifidobacterium adolescentis milk ferment as claimed in claim 8 directly as a product, as an additive or as a substrate in the preparation of a skin external preparation;

preferably, the bifidobacterium adolescentis milk ferment is used as at least one of an antioxidant active ingredient, a skin barrier improving active ingredient, an anti-aging active ingredient and a soothing active ingredient in the skin external agent;

more preferably, the antioxidant active ingredient is an antioxidant active ingredient having DPPH radical scavenging ability and/or hydroxy radical scavenging ability;

More preferably, the skin barrier improving active ingredient is a skin barrier improving active ingredient having moisturizing and moisturizing effects;

more preferably, the anti-aging active ingredient is an anti-aging active ingredient having an increased skin elastin content;

more preferably, the soothing active ingredient is a soothing active ingredient having a function of inhibiting hemolysis of erythrocytes caused by SDS.

10. A skin external preparation comprising the bifidobacterium adolescentis milk ferment according to claim 8;

preferably, the skin external agent further comprises at least one of active ingredient, preservative, thickener, grease, emulsifier and solvent; more preferably, the active ingredient comprises at least one of a moisturizing active ingredient, a whitening active ingredient, an anti-inflammatory active ingredient, an anti-allergic active ingredient, and an anti-oxidative active ingredient; more preferably, the preservative comprises butylene glycol and/or phenoxyethanol; more preferably, the thickener comprises carbomers; more preferably, the grease comprises at least one of jojoba seed oil, polydimethylsiloxane, squalane, and cetyl alcohol; more preferably, the emulsifier comprises polysorbate-20 and/or sorbitan isostearate; more preferably, the solvent comprises deionized water; more preferably, the preservative accounts for 12-15% of the weight of the skin external agent, and even more preferably 13.03%; more preferably, the thickener accounts for 0.5-1% of the weight of the skin external agent, and even more preferably 0.6%; more preferably, the grease accounts for 4-10% of the mass of the skin external agent, and even more preferably 5-6%; more preferably, the emulsifier accounts for 1 to 3 percent of the mass of the skin external agent, and even more preferably, 1.5 to 1.6 percent;

Preferably, the skin external agent comprises a face cream, a face pack, essence or toner;

preferably, the bifidobacterium adolescentis milk ferment accounts for 5% -99% of the mass of the skin external agent, and more preferably 60% -99%.