Freeze-dried powder of probiotic fermented complex with protection against ultraviolet radiation damage, sunscreen cream and method for its preparation

Abstract

The application discloses a freeze-dried powder of probiotic fermentation compound with anti-ultraviolet radiation damage, a sunscreen cream and a preparation method thereof. The method is to ferment probiotics in blueberry whey protein fermentation medium, and obtain a complex concentrate of protein and polyphenol wrapped with probiotics through membrane separation. The complex concentrate of the application can still maintain a certain survival rate of bacterial bodies, polyphenols and flavonoid components under ultraviolet irradiation. After being applied to the skin of mice induced by UVB and being treated by gavage, the complex concentrate shows good protection ability against ultraviolet damage, relieves the degradation of skin type III collagen caused by ultraviolet irradiation, reduces the overexpression of pro-inflammatory cytokines, and regulates the immune system of the body and improves the health of the skin by adjusting the intestinal flora structure of mice and up-regulating the abundance of dominant flora producing short-chain fatty acids. The freeze-dried powder prepared from the complex concentrate can be used to prepare a live bacteria type sunscreen cream, and has high ultraviolet reflection ability and reduced potential skin irritation risk.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of microbial fermentation, and relates to a probiotic fermentation compound freeze-dried powder with the function of preventing ultraviolet radiation damage, a sunscreen cream and a preparation method thereof. BACKGROUND

[0002] It is well known that skin damage caused by excessive exposure to ultraviolet radiation, ranging from sunburn, redness and sunburn to the development of skin photoaging, and ultimately may cause skin cancer. The damage of ultraviolet rays in sunlight to human skin has a cumulative effect, which is related to the intensity, frequency and duration of exposure. According to the length of the ultraviolet wavelength, it is generally divided into long-wave ultraviolet (UVA: 320-400 nm), medium-wave ultraviolet (UVB: 290-320 nm) and short-wave ultraviolet (UVC: 200-290 nm). The shorter the wavelength of ultraviolet rays, the greater the radiation energy, and the greater the damage. Under a certain radiation dose, the sunburn caused by medium-wave ultraviolet is 1000 times that of long-wave ultraviolet.

[0003] The damage of ultraviolet rays to human body is mainly divided into direct damage, indirect damage and circulatory effect. Direct damage is the damage of DNA induced by ultraviolet radiation, and the accumulation of damaged DNA will activate p53 protein, and then induce the apoptosis of keratinocytes. Indirect damage is mainly the oxidative stress caused by the accumulation of excessive reactive oxygen species (ROS). A large number of ROS will cause oxidative damage to elastin and collagen, protein conformation change and unfolding, and the change of protein structure will lead to loss of tensile strength, wrinkle formation, dryness and increase of skin fragility. At the same time, ROS will also damage the integrity of lipids and nucleic acids, and induce various human diseases such as cataract and respiratory diseases. And the circulatory effect is mainly caused by the interaction between mitochondrial DNA and ROS. Mitochondrial DNA is prone to mutation under the oxidative stress induced by ROS, which will affect the cell metabolism such as ATP synthesis process, and finally cause the ROS to surge. High levels of ROS will cause damage to cell components such as proteins, nucleic acids and lipids, and tissues and organs.

[0004] According to the sunscreen mechanism, sunscreen agents are mainly divided into chemical sunscreen agents and physical sunscreen agents. Chemical sunscreen agents can absorb ultraviolet rays and convert them into other forms of energy and release, thereby reducing the ultraviolet energy irradiated to the skin. Chemical sunscreen agents have good sunscreen effect, but are easy to cause skin irritation, produce side effects such as allergy and inflammation. Physical sunscreen agents protect the skin by reflecting or scattering ultraviolet rays, and are safe and stable, but are easy to block pores and affect sweat gland secretion. Natural sunscreen agents generally refer to plant, animal and microbial extracts or derivatives with the function of preventing ultraviolet radiation damage. The after-sun repair function of natural sunscreen agents, that is, reducing ROS, anti-inflammatory, improving human skin barrier function and the like, is not possessed by chemical sunscreen agents and physical sunscreen agents, so natural sunscreen agents can complement and perfect the functions of traditional sunscreen agents.

[0005] Although extensive research has been carried out on the use of probiotics to defend against skin ultraviolet damage, they show good application prospect in protecting skin photodamage, however, the bioavailability is difficult to maintain in the production, processing, storage and intake stages, especially in the harsh gastrointestinal environment after oral administration, which makes it difficult to guarantee the anti-photoaging effect. Therefore, it has great application value to actively seek a material with stability and anti-photoaging activity to prevent ultraviolet damage.

[0006] Blueberries have high nutritional value and are rich in flavonoids and some polyphenolic compounds, which have excellent antioxidant activity and antioxidant activity derived in vitro ultraviolet protection ability. However, the biological activity of these active ingredients is limited by the harsh digestive environment, especially the intestinal digestive tract. High-activity natural products are easily affected by processing environment, light and heat, and even lose the original biological function after being digested by gastrointestinal fluid. Probiotic fermentation and microcapsule technology can greatly improve the activity of natural products entering the human body. How to make the fermentation products even probiotics efficiently play their biological functions after reaching the intestinal tract is the focus of attention at present. SUMMARY

[0007] The purpose of the present application is to provide a kind of probiotic fermentation complex freeze-dried powder with the function of preventing ultraviolet radiation damage, sunscreen cream and its preparation method. The method is to ferment probiotics with blueberry concentrate and whey protein mixture as culture medium, and then separate by membrane separation technology to obtain probiotic fermented blueberry whey protein concentrate component. The concentrate component is a complex concentrate of protein and polyphenol wrapped with probiotics. The complex concentrate not only has high stability and anti-photoaging activity, but also can regulate the immune system and improve skin health by adjusting the intestinal flora structure of mice and up-regulating the abundance of short-chain fatty acid-producing dominant flora.

[0008] The technical solution to achieve the purpose of the present application is as follows:

[0009] The preparation method of the freeze-dried powder of the probiotic fermentation compound with the function of preventing ultraviolet radiation damage comprises the following steps:

[0010] (1) Preparation of blueberry concentrate: wash the blueberries and squeeze the juice, first filter through 200 mesh gauze, collect the filtrate, then filter again through 150 mesh gauze, collect the filtrate, dilute the collected filtrate with water, and then concentrate by membrane concentration method, the concentration time is 40-60 min, the molecular weight cut-off of the membrane is 1 kDa, the transmembrane pressure is 1.0-1.5 Mpa, and the volume ratio of blueberries before and after concentration is 2-3:1, to obtain the blueberry concentrate, wherein the mass-volume ratio of blueberries to blueberry concentrate is 1g:2-3mL;

[0011] (2) Preparation of blueberry whey protein fermentation medium: add blueberry concentrate, sucrose and whey protein to water, stir until mixed evenly, adjust to the appropriate pH for the strain, and pasteurize to obtain the blueberry whey protein fermentation medium, wherein the addition amount of blueberry concentrate is 3-15 wt.%, the addition amount of whey protein is 3-9 wt.%, and the addition amount of sucrose is 3-5 wt.%;

[0012] (3) Fermentation: add the activated probiotic strain to the blueberry whey protein fermentation medium for fermentation, wherein the probiotic strain is a mixture of L. plantarum 67 and L. paracasei grx 701 with the preservation number of CGMCC No. 27785;

[0013] (4) Ultrafiltration concentration: separate and concentrate the fermentation mixture obtained in step (3) by membrane separation method, the molecular weight cut-off of the ultrafiltration membrane is 100 kDa, and the probiotic fermentation blueberry whey protein concentrate component with a molecular weight of >100 kDa is obtained;

[0014] (5) Freeze-drying: pre-freeze the probiotic fermentation blueberry whey protein concentrate component and then vacuum freeze-dry to obtain the probiotic fermentation compound freeze-dried powder.

[0015] Preferably, in step (2), the addition amount of blueberry concentrate in the blueberry whey protein fermentation medium is 11 wt.%, the addition amount of whey protein is 6 wt.%, and the addition amount of sucrose is 3 wt.%.

[0016] The L. paracasei grx 701 described in the present application was preserved in the China General Microbiological Culture Collection Center on July 3, 2023, with the preservation number of CGMCC No. 27785, and the preservation address is No. 3, Beichen West Road, Haidian District, Beijing, China.

[0017] Preferably, in step (2), the total inoculation amount of the probiotic bacterial strains is 2-5%, more preferably 3%, and the inoculation ratio of Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 is equal.

[0018] Preferably, in step (3), the fermentation time is 10-18 h, and the fermentation temperature is 37-42℃.

[0019] Preferably, in step (4), the membrane separation conditions are room temperature, a transmembrane pressure of 0.25 MPa, and a concentration time of 20-30 min.

[0020] Preferably, in step (5), the pre-freezing temperature is -20 to -80℃, preferably -80℃, and the pre-freezing time is 24-36 h; the vacuum freeze-drying temperature is -50 to -80℃, and the vacuum freeze-drying time is 24-36 h.

[0021] The present application also provides a probiotic bacterial fermentation complex freeze-dried powder prepared by the above preparation method.

[0022] Further, the present application provides the use of the above probiotic bacterial fermentation complex freeze-dried powder in the preparation of an anti-UV radiation damage product.

[0023] Preferably, the anti-UV radiation damage product is a food or a skin care product.

[0024] The present application provides an anti-UV radiation damage cream, which is composed of the following ingredients in parts by weight: 1 part of squalane, 3 parts of white oil, 3 parts of vaseline, 3 parts of lanolin, 0.5 parts of glycerol monostearate, 1 part of sorbitan monostearate, 3 parts of stearic acid, 2 parts of cetyl alcohol, 2 parts of Tween-80, 4 parts of glycerol, 2 parts of propylene glycol, 1 part of betaine, 74 parts of water, an appropriate amount of preservative, 0.5 parts of hyaluronic acid stock solution, and 5 parts of probiotic bacterial fermentation complex freeze-dried powder.

[0025] The preparation method of the above anti-UV radiation damage cream is as follows:

[0026] (1) According to the formula, mix squalane, white oil, vaseline, lanolin, glycerol monostearate, sorbitan monostearate, stearic acid, and cetyl alcohol as the oil phase, mix Tween-80, glycerol, propylene glycol, betaine, and water as the water phase, use the preservative as the C phase, and mix the hyaluronic acid stock solution and the probiotic bacterial fermentation complex freeze-dried powder as the D phase.

[0027] (2) In a sterile environment, heat the A phase to 75-85℃ until completely dissolved, heat the B phase to 85-90℃, add the A phase to the B phase while stirring the B phase, and high-speed shear and stir to fully mix the two, then cool to 40±5℃, add the C phase and the D phase, homogenously stir, and finally cool to room temperature and seal and package.

[0028] Compared with the prior art, the present application has the following advantages:

[0029] (1) The present application uses blueberry concentrated solution and whey protein mixed solution as fermentation culture solution, obtains a blueberry and whey protein mixed solution fermented by probiotics, and then separates a concentrated component with a molecular weight > 100 kDa by membrane separation technology. Compared with other components, the blueberry whey protein concentrated component fermented by Lactobacillus plantarum 67 and Paracaseicola grx 701 shows better ability to prevent skin from ultraviolet damage during gastrointestinal digestion, which is manifested as significantly reducing the redness, desquamation, scabbing and wrinkle of the back of the mouse skin caused by ultraviolet irradiation, and significantly reversing the skin dehydration, epidermal thickening, MDA level increase, ROS surge and excessive expression of pro-inflammatory cytokines IL-1beta and TNF-alpha of the back skin, and can also regulate the immune system and improve skin health by adjusting the intestinal flora structure of the mouse and up-regulating the abundance of the dominant flora producing short-chain fatty acids.

[0030] (2) The cream added with the probiotic fermented compound freeze-dried powder has high ultraviolet reflection ability, and as a kind of live bacteria type sunscreen cream, can reduce the potential skin irritation risk compared with chemical sunscreen cream. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 Effect of ultraviolet UVB irradiation for 0-30 min on the survival rate of lactic acid bacteria.

[0032] Figure 2 Effect of probiotic fermentation of different substrates on the content of polyphenols under ultraviolet irradiation for 0-30 min.

[0033] Figure 3 Effect of probiotic fermentation of different substrates on the content of total flavonoids under ultraviolet irradiation for 0-30 min.

[0034] Figure 4 Macrograph of the back of the mouse.

[0035] Figure 5 Score chart of the back of the mouse.

[0036] Figure 6 Chart of the determination results of the water content of the skin of the mouse.

[0037] Figure 7 H&E staining chart of the skin of the back of the mouse.

[0038] Figure 8 Chart of the determination results of type I collagen in the skin of the mouse.

[0039] Figure 9 Chart of the determination results of type III collagen in the skin of the mouse.

[0040] Figure 10 Figure for the change of ROS in the skin tissue cells of mice.

[0041] Figure 11 Figure for the determination results of GSH content in the skin tissue of mice.

[0042] Figure 12 Figure for the determination results of MDA content in the skin tissue of mice.

[0043] Figure 13 Figure for the determination results of the level of pro-inflammatory cytokine IL-1β in the blood of mice.

[0044] Figure 14 Figure for the determination results of the level of pro-inflammatory cytokine TNF-α in the blood of mice.

[0045] Figure 15 Figure for the species abundance of the phylum level of the intestinal flora of mice.

[0046] Figure 16 Figure for the species abundance of the class level of the intestinal flora of mice.

[0047] Figure 17 Figure for the species abundance of the family level of the intestinal flora of mice.

[0048] Figure 18 Figure for the species abundance of the genus level of the intestinal flora of mice.

[0049] Figure 19 Figure for the species abundance of the species level of the intestinal flora of mice.

[0050] Figure 20 Figure for the microscope images of the cream containing probiotic fermented blueberry whey protein freeze-dried powder dyed with water-soluble methylene blue and oil-soluble dye Sudan red, magnification 4x, 10x, 40x, 100x.

[0051] Figure 21 Figure for the transmittance of the sunscreen cream determined.

[0052] Figure 22 Figure for the reflectance of the sunscreen cream determined.

[0053] Figure 23 Figure for the tyrosinase inhibition rate of probiotic fermentation of different substrates with different contents.

[0054] Figure 24 Figure for the surface moisture of pig skin after applying the cream with 10% different products and ultraviolet irradiation.

[0055] Figure 25 Figure for the surface oil of pig skin after applying the cream with 10% different products and ultraviolet irradiation.

[0056] Figure 26The surface skin color of the pig skin after applying the cream with 10% different products under UV irradiation.

[0057] Figure 27 The surface pigment of the pig skin after applying the cream with 10% different products under UV irradiation.

[0058] Figure 28 A physical map of the application of sunscreen on the surface of the skin, where a represents the base cream; b represents the cream prepared from the freeze-dried powder of the probiotic fermented whey protein; c represents the cream prepared from the freeze-dried powder of the probiotic fermented blueberry whey protein; d represents the cream prepared from the freeze-dried powder of the probiotic fermented blueberry. DETAILED DESCRIPTION

[0059] The relevant culture medium, reagent formula and test method in the following examples are as follows.

[0060] The Lactobacillus plantarum 67 of the application is disclosed in the published paper [Chen D, Guo C, Ren C, et al. Effects of vacuum freeze-drying protectants on the adhesion ability of Lactobacillus plantarum 67 [J]. Food and Fermentation Industries, 2023, 49(9): 16-26.] in the laboratory.

[0061] The Lactobacillus paracasei grx 701 of the application was preserved in the China General Microbiological Culture Collection Center on July 3, 2023, with the preservation number CGMCC No. 27785 and the preservation address being No. 3, Beichen West Road, Chaoyang District, Beijing, China.

[0062] The applicant Yangzhou University declares to guarantee to issue Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 to the public within 20 years from the application date.

[0063] 1. Experimental raw materials: whey protein (protein content: 52%), blueberry raw materials (produced in China Daxing'anling), Nestle commercial product (Commerial product, CP): Nestle Life Garden 90 billion skin probiotic capsules.

[0064] 2. MRS culture medium: glucose 20.0 g, peptone 10.0 g, anhydrous sodium acetate 5.0 g, ammonium citrate 2.0 g, potassium phosphate dibasic 2.0 g, magnesium sulfate heptahydrate 0.2 g, manganese sulfate tetrahydrate 0.05 g, Tween-80 1.0 mL, beef extract 10.0 g, yeast extract 5.0 g, deionized water 1.0 L, sterilized at 121℃ for 15 min, and cooled to room temperature for use. The MRS solid culture medium is prepared by adding 15.0 g of agar.

[0065] 3. Preparation of fermented samples:

[0066] (1) Blueberry concentrate:

[0067] (1.1) Juice, filtration: fresh and plump Daxing'anling wild blueberries were washed with clean water and juiced, filtered with 200 mesh gauze, the filtrate was collected, and then filtered with 150 mesh gauze, the filtrate was collected.

[0068] (1.2) Concentration: the filtrate was concentrated by membrane concentration method, using a membrane with a molecular weight cut-off of 1 kDa, a transmembrane pressure of 1.0 Mpa, a concentration time of 40 min, and a volume ratio of blueberry before and after concentration of 2-3:1, to obtain blueberry concentrate, which was pasteurized at 95°C for 5 min, wherein the mass volume ratio of blueberry to blueberry concentrate was 1g:2-3mL.

[0069] (2) Blueberry whey protein fermentation medium:

[0070] Blueberry concentrate, sucrose and whey protein were added to water, stirred until mixed evenly, adjusted to the appropriate pH for the strain, 95°C for 5 min, pasteurized to obtain a blueberry whey protein fermentation medium, wherein the addition amount of blueberry concentrate was 11wt.%, the addition amount of whey protein was 6wt.%, and the addition amount of sucrose was 3wt.%.

[0071] (3) Whey protein fermentation medium: sucrose and whey protein were added to water, stirred until mixed evenly, adjusted to the appropriate pH for the strain, 95°C for 5 min, pasteurized to obtain a whey protein fermentation medium, wherein the addition amount of whey protein was 6wt.% and the addition amount of sucrose was 3wt.%.

[0072] (4) Blueberry fermentation medium: blueberry concentrate and sucrose were added to water, stirred until mixed evenly, adjusted to the appropriate pH for the strain, 95°C for 5 min, pasteurized to obtain a blueberry fermentation medium, wherein the addition amount of blueberry concentrate was 11wt.% and the addition amount of sucrose was 3wt.%.

[0073] The optimum pH of Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 was 6.5.

[0074] 4. Macroscopic observation and scoring of mouse back skin:

[0075] Before the mice were sacrificed, the irradiated area of the mouse back was photographed, and the degree of wrinkle formation on the mouse back skin was evaluated using a scoring table.

[0076] Table 1 Skin wrinkle scoring criteria

[0077]

[0078] 5. Determination of water content in the skin of the mouse back:

[0079] Shearing a piece of skin tissue, after stripping the connective tissue and adipose tissue, accurately weighing the wet weight and quickly loading it into a sterile 1.5 mL centrifuge tube. Open the centrifuge tube and place it in the oven, dry at 105℃ for 4h, and calculate the water content of the skin according to formula (2):

[0080] The percentage of skin water content (%) = (wet weight - dry weight) / wet weight x 100% (2).

[0081] 6. Mouse skin epidermis thickness: H&E staining was used.

[0082] 7. Determination of type I and type III collagen in mouse skin: determined by ELISA kit respectively.

[0083] 8. Changes of ROS in mouse skin tissue cells: frozen section observation of ROS changes.

[0084] 9. Determination of antioxidant capacity of mouse skin: according to the instructions of MDA and GSH kit.

[0085] 10. Determination of pro-inflammatory cytokines TNF-α and IL-1β levels in mouse blood: according to the instructions of ELISA.

[0086] The present application will be further described in detail below in conjunction with specific examples and drawings.

[0087] Example 1

[0088] Preparation of probiotic fermented freeze-dried powder

[0089] 1. Activation of strains: Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 were inoculated into MRS medium respectively and cultured at 37℃ for 24h.

[0090] 2. Preparation of different fermentation media: blueberry fermentation medium, whey protein fermentation medium and blueberry whey protein fermentation medium were prepared respectively.

[0091] 3. Inoculation and fermentation: Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 bacterial liquid obtained in step (1) were added to different fermentation media respectively, and the total bacterial liquid inoculation amount was 3%, among which the inoculation amount of Lactobacillus plantarum 67 bacterial liquid was 1.5mL / 100mL, and the inoculation amount of Lactobacillus paracasei grx 701 bacterial liquid was 1.5mL / 100mL, and mixed and fermented for 12h.

[0092] 4. Preparation of the probiotic fermented concentrate component samples: The probiotic fermented mixture and the blueberry whey protein fermented medium were separated and concentrated by membrane separation technology, respectively. The membrane separation conditions were as follows: room temperature, transmembrane pressure of 0.25 MPa, concentration time of 25 min, and ultrafiltration membrane molecular weight cut-off of 100 kDa. The probiotic fermented blueberry concentrate component, the probiotic fermented whey protein concentrate component, the probiotic fermented blueberry whey protein concentrate component, and the blueberry whey protein unfermented concentrate component with a molecular weight of >100 kDa were obtained, respectively.

[0093] 5. Freeze-drying: The probiotic fermented blueberry concentrate component, the probiotic fermented whey protein concentrate component, and the probiotic fermented blueberry whey protein concentrate component were pre-frozen at -80°C for 24 h, and then were placed in a vacuum freeze-drying machine for freeze-drying for 48 h at a freeze-drying temperature of -80°C. The probiotic fermented blueberry freeze-dried powder, the probiotic fermented whey protein freeze-dried powder, and the probiotic fermented blueberry whey protein freeze-dried powder were obtained, respectively.

[0094] Example 2

[0095] The viable cell count, the polyphenol content, and the flavonoid content of the probiotic fermented blueberry concentrate component, the probiotic fermented whey protein concentrate component, the probiotic fermented blueberry whey protein concentrate component, and the blueberry whey protein unfermented concentrate component obtained in Example 1 under ultraviolet irradiation for different times were determined.

[0096] Figure 1 The viable cell count of the different probiotic fermented concentrate components under ultraviolet UVB irradiation at an intensity of 0.2 mw / cm 2 for 0 to 30 min indicated the viable cell count of the probiotic fermented concentrate components under ultraviolet UVB irradiation at an intensity of 0.2 mw / cm 2Radiation intensity under the cell viability, wherein L. plantarum, L. paracasei (grx 701) is a concentrated component sample obtained by taking MRS medium as the fermentation substrate; Whey + blueberry, L. plantarum, L. paracasei (grx 701) is a probiotic fermented blueberry whey protein concentrate component sample; Whey, L. plantarum, L. paracasei (grx 701) is a probiotic fermented whey protein concentrate component sample; blueberry, L. plantarum, L. paracasei (grx 701) probiotic fermented blueberry concentrate component sample; the results are expressed as mean ± standard error of mean, n = 3. It can be seen from the figure that before ultraviolet irradiation, the viable cell content of the probiotic fermented blueberry whey protein concentrate component sample is the highest, which shows that the blueberry whey protein fermentation medium as the fermentation substrate has a positive promoting and protective effect on the growth and reproduction of probiotics. With the increase of irradiation time, the survival rate of different probiotic fermented concentrate component samples is decreasing, because under the action of ultraviolet, the DNA and cell membrane structure of the bacteria may be damaged, resulting in the death of the bacteria. The viable cell count of the probiotic fermented blueberry whey protein concentrate component sample is the highest, followed by the probiotic fermented whey protein concentrate component sample, the concentrated component sample obtained by taking MRS medium as the fermentation substrate, and the probiotic fermented blueberry concentrate component sample. This further confirms that the blueberry whey protein fermentation medium has a good protective effect on probiotics. Whey protein also has a certain ability to protect probiotics from ultraviolet damage.

[0097] Figure 2The polyphenol content of the concentrated components fermented by different probiotics under UV irradiation for 0-30 min was determined. As shown in the figure, the polyphenol content of the concentrated component sample obtained by using MRS medium as the fermentation substrate was the lowest, and its polyphenol content showed a decreasing trend with the increase of irradiation time. This may be because without the presence of fermentation substrate, polyphenols cannot form complexes or are not protected in any way, and are more susceptible to damage by UV light. The polyphenol content of the concentrated component sample fermented by probiotics from blueberry was relatively high, which was because blueberry itself was rich in polyphenol substances. The decrease in the first 10 min of UV irradiation was due to the destructive effect of UV on the active substances in blueberry. The increase at 15 min was probably because the irradiation of UV promoted the release or transformation of polyphenols in blueberry. The decrease in the next 15-30 min was due to further damage to polyphenols caused by long-term UV irradiation. The polyphenol content of the concentrated component sample fermented by probiotics from blueberry and whey protein was stable, indicating that the components in whey protein may form complexes with polyphenols, thereby enhancing the stability of polyphenols. In addition, whey protein may also help to resist the damage of external factors such as UV light to polyphenols through its own properties. Both may have a synergistic effect. The polyphenols in blueberry interact with whey protein to form more stable complexes. This complex not only improves the stability of polyphenols, but also may enhance their biological activity. Therefore, under the irradiation of UV light, the polyphenol content of the concentrated component sample fermented by probiotics from blueberry and whey protein can remain stable. Therefore, the stable polyphenol content in the concentrated component sample fermented by probiotics from blueberry and whey protein indicates that the combination of blueberry and whey protein has a good protective effect on polyphenols. In addition, the fermentation process itself may have a positive impact on the stability of polyphenols. Fermentation can change the chemical structure of the material, making polyphenols more easily combined with whey protein, or producing new polyphenol substances through the action of microorganisms.

[0098] Figure 3 The total flavonoid content of the concentrated components fermented by different probiotics under UV irradiation for 0-30 min was determined. As shown in the figure, the total flavonoid content of the concentrated component sample fermented by probiotics from blueberry and whey protein was relatively high, indicating that the combination of blueberry and whey protein had a significant protective and promoting effect on total flavonoids. Blueberry itself is rich in flavonoids, and whey protein may form complexes with flavonoids or provide protection, allowing the total flavonoid content to remain at a high level. In the early stage of UV irradiation, the total flavonoid content increased, which may be because the irradiation of UV promoted the release of total flavonoid substances. The total flavonoid content of the concentrated component sample fermented by probiotics from blueberry was also relatively high, but showed a decreasing trend, indicating that under long-term UV irradiation, its structure may be damaged, leading to a decrease in content. The total flavonoid content of the concentrated component obtained by using MRS medium as the fermentation substrate was low and stable. This indicates that without fermentation substrate, the content of flavonoids is already low and is not easily affected by external factors.

[0099] Therefore, the blueberry whey protein fermentation medium as a substrate has a protective and promoting effect on the total flavonoid content.

[0100] Example 3

[0101] Protective effect of probiotic fermented blueberry whey protein concentrate component on ultraviolet-damaged mouse skin

[0102] 1. Grouping and administration of experimental animals

[0103] (1) Experimental animals: 64 female ICR mice (SPF level, 6-8 weeks old) were provided by the Comparative Medicine Center of Yangzhou University.

[0104] (2) Experimental grouping: randomly divided into 8 groups, including normal group (NC), model group (MC), positive control group (PC), blueberry group (BC), whey protein group (WC), blueberry whey protein unfermented group (BW), blueberry whey protein fermented group (BWF), and commercial product group (CP), and the range of hair removal of mice in each group was 4x3cm 2 .

[0105] Normal group: no ultraviolet irradiation, 800mg / kg·bw of 0.9% physiological saline was administered by gavage before each irradiation, and 2mg / cm 2 of basic cream was applied externally;

[0106] Model group: ultraviolet irradiation was performed, 800mg / kg·bw of 0.9% physiological saline was administered by gavage before each irradiation, and 2mg / cm 2 of basic cream was applied externally;

[0107] Positive control group: ultraviolet irradiation was performed, 800mg / kg·bw of vitamin C (VC) was administered by gavage before each irradiation, and 2mg / cm 2 of the corresponding cream was applied externally;

[0108] Blueberry group: ultraviolet irradiation was performed, 800mg / kg·bw of probiotic fermented blueberry concentrate component was administered by gavage before each irradiation, and 2mg / cm 2 of the corresponding cream was applied externally;

[0109] Whey protein group: ultraviolet irradiation was performed, 800mg / kg·bw of probiotic fermented whey protein concentrate component was administered by gavage before each irradiation, and 2mg / cm 2 of the corresponding cream was applied externally;

[0110] Blueberry whey protein unfermented group: ultraviolet irradiation was performed, 800mg / kg·bw of blueberry whey protein unfermented concentrate component was administered by gavage before each irradiation, and 2mg / cm 2 of the corresponding cream was applied externally;

[0111] Blueberry whey protein fermentation group: UV irradiation was performed, and 800 mg / kg·bw of probiotic fermented blueberry whey protein concentrate components was orally administered before each irradiation, and 2 mg / cm 2 The corresponding cream was applied;

[0112] Commercial product group (Commerial product, CP): UV irradiation was performed, and 800 mg / kg·bw of Nestle Life Garden 90 billion skin probiotic capsules was orally administered before each irradiation.

[0113] 2. Preparation of a UV-damaged mouse model

[0114] A Philips UVB lamp with a power of 20 W was installed in parallel at the top of a light-proof UV lamp box, and the straight-line distance from the bottom where the mice were fixed was 20 cm. Before UV irradiation, the UV lamp was preheated for 10 min, and the irradiation intensity was measured using a UV radiation instrument during irradiation. According to the results of the pre-experiment and the corresponding reference literature, the minimum erythema dose (MED) was determined, the irradiation intensity was 1 MED in the first week, and 1 MED was added every week starting from the second week, until it increased to 4 MED in the fourth week. Irradiation was performed 3 times a week for a total of 4 weeks, and 1 h before each irradiation, the mice were orally administered and the cream was applied. At the end of the experiment, the eyeball was bled, and the skin at the modeling site on the back of the mice was taken. The administration and irradiation of the mice in each group are shown in Table 2.

[0115] Table 2 Administration and irradiation of mice in each group

[0116]

[0117] 3. Preventive and therapeutic effects of probiotic fermented blueberry whey protein concentrate components on UVB-induced skin damage in mice

[0118] (1) Macroscopic effects of probiotic fermented blueberry whey protein concentrate components on UV-damaged skin in mice

[0119] Before the mice were sacrificed, the irradiated area on the back of the mice was photographed, and the skin on the back of the mice before sacrifice was evaluated using a scoring table to determine the degree of wrinkle formation.

[0120] (2) Effects of probiotic fermented blueberry whey protein concentrate components on the water content of mouse skin

[0121] After the cut skin tissue was stripped of connective tissue and adipose tissue, its wet weight was accurately measured and quickly placed in a sterile 1.5 mL centrifuge tube. The centrifuge tube was uncapped and placed in an oven at 105℃ for 4 h, and the water content of the skin was calculated according to the formula.

[0122] (3) Effects of probiotic fermented blueberry whey protein concentrate components on the thickness of mouse skin

[0123] Mouse skin epidermis thickness: H&E staining was used.

[0124] (4) Effect of probiotic fermented blueberry whey protein concentrate components on the collagen content of mouse skin

[0125] Determination of collagen type I and collagen type III in mouse skin: ELISA kits were used respectively.

[0126] (5) Effect of probiotic fermented blueberry whey protein concentrate components on ROS in mouse skin

[0127] Changes in ROS in mouse skin tissue cells: frozen sections were used to observe changes in ROS.

[0128] (6) Effect of probiotic fermented blueberry whey protein concentrate components on the antioxidant capacity of mouse skin

[0129] According to the MDA and GSH kit instructions.

[0130] (7) Effect of probiotic fermented blueberry whey protein concentrate components on the levels of pro-inflammatory cytokines in mouse serum

[0131] Determination of pro-inflammatory cytokines TNF-α and IL-1β levels in mouse serum: according to the ELISA instructions.

[0132] (8) Determination of mouse intestinal flora species abundance

[0133] On the day before the mice were sacrificed, fresh feces from each replicate of mice in each group were collected in sterile cryogenic tubes and quickly placed in liquid nitrogen. After 3 minutes of rapid freezing, the cryogenic tubes were quickly placed in -80°C for later use. Dry ice was transported to Beijing Nuowu Ziyuan Biological Information Technology Co., Ltd. on the same day, and samples were selected for 16S rRNA gene V3-V4 region high-throughput sequencing to analyze changes in mouse intestinal flora. Genomic DNA of the samples was extracted using CTAB or SDS method, and V34 region primer sequences 341F: CCTAYGGGRBGCASCAG and 806R: GGACTACNNGGGTATCTAAT were used for PCR amplification. The qualified PCR amplification products were mixed and purified, and finally the gel recovery kit provided by qiagen company was used to recover the products. Then the recovered products were used for library construction, and after the library was detected, NovaSeq6000 was used for sequencing.

[0134] Figure 4For the macrograph of the back of the mice, it can be seen that after the experiment, the skin of the back of the mice in the NC group was smooth and ruddy, showing normal skin texture; after ultraviolet irradiation, the back of the mice in the MC group appeared red, swelling, blisters and scabbing, etc.; after the intervention of the samples in each group, the skin of the back of the mice irradiated by ultraviolet was obviously improved, especially for PC and BWF, a small amount of scabbing was visible on the back of the mice, and the mice in the BWF group began to grow fluff and quickly covered the back the next day after being smeared; while a large number of sun scars still existed in the WC and CP groups. Figure 5 For the score graph of the back of the mice, it can be seen that compared with the mice in the NC group, the wrinkle score of the back of the mice in the MC group was significantly reduced (p<0.05) after ultraviolet irradiation, and the skin of the back of the mice in the MC group was rough and deep wrinkles appeared. Figure 4 The intervention of each group alleviated the wrinkle proliferation induced by ultraviolet irradiation, especially for PC and BWF, although the mice in the BWF group still appeared slight redness, the skin was relatively smooth, and the wrinkle score was significantly higher than that in the NC group (p<0.05), which was close to the effect of the positive control. The above results show that BWF has a good effect on preventing skin ultraviolet damage.

[0135] Figure 6 For the determination result graph of the water content of the skin of the mice, it can be seen that compared with the mice in the NC group, the water content of the skin of the mice in the MC group was significantly reduced (p<0.001) after ultraviolet irradiation, indicating that ultraviolet irradiation caused dryness and water loss of the skin. After the intervention of PC, BC, BWF and CP, the water loss of the skin of the mice was improved, which had a significant difference (p<0.01) compared with the mice in the MC group, especially for BC, BWF and CP, which were close to the NC group.

[0136] Figure 7 For the H&E staining graph of the back skin of the mice, it can be seen from the graph that the epidermal layer of the mice in the NC group was thin and connected closely with the dermal layer, at the same time, the cells in the epidermal layer were arranged in order, and the cells changed from vertical to flat and ordered arrangement from the basal layer to the transparent layer; the epidermis of the mice in the MC group was obviously thickened, and the thickness was uneven, and the keratinization was serious, the epidermis was accompanied by exfoliation, a few cells in the basal layer were vacuolated, and inflammatory cells (lymphocytes, mononuclear cells) infiltration, etc., indicating that the mouse skin damage model induced by ultraviolet radiation was successfully established. Compared with the MC group, the thickness of the epidermal layer and the dermal layer of the mice in each group was reduced after the intervention, but the thickness was uneven, among which the mice in the PC and BWF groups had better effect, the structure of each layer of the epidermis was clear, the epidermis and the dermal layer were connected closely, the collagen fibers arranged relatively in order, and the fracture accumulation phenomenon was not found, indicating that PC and BWF had the effect of reversing the ultraviolet-induced thickening of the epidermal layer of the mice.

[0137] Figure 8For the results of the determination of type I collagen in the skin of mice, it can be seen from the figure that the content of type I collagen in the skin of mice in the MC group was significantly lower than that in the NC group (p<0.001), indicating that ultraviolet irradiation can degrade type I collagen in the skin tissue of mice. Compared with the MC group, the content of type I collagen in the skin of mice in the PC group was significantly increased (p<0.01), and the same was true for the BC and WC groups (both p<0.01), indicating that they can prevent the degradation of collagen caused by ultraviolet irradiation, and the effect of the PC group is closest to that of the NC group. Similarly, it can be seen from the figure that the content of type III collagen in the skin of mice in the MC group was significantly lower than that in the NC group (p<0.001), indicating that ultraviolet irradiation can degrade type III collagen in the skin tissue of mice. The content of type III collagen in the skin of mice in the PC, BC, WC and BWF groups was higher than that in the MC group, and there was a significant difference (p<0.01), and the content of type III collagen in the skin of mice in the BW and CP groups was also higher than that in the MC group (p<0.05), indicating that they can all alleviate the degradation of collagen caused by ultraviolet irradiation, and at the same time, the intervention effect of the PC, BC, WC and BWF groups is better than that of the BW and CP groups. Figure 9

[0138] Figure 10 For the changes of ROS in the tissue cells of mice. Excessive ROS can cause the degradation of collagen and elastin, reduce their synthesis, and cause wrinkles in the skin. The commonly used method for detecting ROS is to use ROS fluorescent probe-dihydroethidium, which can freely penetrate the cell membrane of living cells, enter the cells, be oxidized to ethidium bromide by the superoxide anion produced in the cells, and then interact with DNA. Red fluorescence can be observed by fluorescence microscope and rhodamine filter, so that the distribution of ROS can be observed. From the figure, it can be seen that compared with the NC group, after ultraviolet irradiation, the ROS in the skin cells of the MC group increased significantly, mainly distributed in the epidermis, followed by the dermis; the ROS density in the intervention groups was significantly reduced, especially in the BC and BWF groups, indicating that they can capture ROS directly or remove ROS indirectly. Figure 10

[0139] Figure 11 and Figure 12 For the results of the determination of antioxidant capacity in the skin tissue of mice. From the figure, it can be seen that compared with the NC group, the antioxidant capacity in the skin tissue of mice in the MC group was significantly lower than that in the NC group (p<0.001), indicating that ultraviolet irradiation can reduce the antioxidant capacity in the skin tissue of mice. Compared with the MC group, the antioxidant capacity in the skin tissue of mice in the PC, BC, WC and BWF groups was significantly increased (p<0.01), and the same was true for the BW and CP groups (both p<0.05), indicating that they can all alleviate the degradation of collagen caused by ultraviolet irradiation, and at the same time, the intervention effect of the PC, BC, WC and BWF groups is better than that of the BW and CP groups. Figure 12 ​​The results of determination of GSH content in skin tissue of mice showed that, compared with the NC group, ultraviolet irradiation significantly reduced the GSH level in skin tissue (p<0.001), from 62.16±9.54 μmol / g protein to 22.80±5.39 μmol / g protein; PC and CP intervention restored the GSH level, which had a statistical difference compared with the MC group (p<0.001), and was close to the GSH level of the NC group; and the intervention effect of BW and BWF was only inferior to that of the PC group (60.95±12.48 μmol / g protein). From the results of determination of MDA content in skin tissue of mice, Figure 12 The results of determination of MDA content in skin tissue of mice showed that, compared with the NC group, the MDA level of the MC group was significantly increased (p<0.01), from 11.04±7.68 nmol / mg protein to 24.16±7.03 nmol / mg protein, and the MDA level after the intervention of PC, WC, BW and CP almost returned to the normal level (p<0.01). Compared with the MC group, the MDA level of the BC group was also reduced, but it did not reach a statistical significance, which may be caused by a small number of experimental samples or experimental errors. The BWF group significantly reduced the increase of the MDA level caused by ultraviolet irradiation (p<0.001), and was even lower than the MDA level of the PC group, indicating that the anti-lipid oxidation effect of BWF was superior to that of the positive control.

[0140] Figure 13 and 14 The results of determination of pro-inflammatory cytokines IL-1β and TNF-α are shown in the following figures. From the results of determination of IL-1β level in blood of mice, Figure 13 The results of determination of IL-1β level in blood of mice showed that, when ultraviolet irradiation, the keratinocytes in the MC group released the cytokine IL-1β, which had a significant difference compared with the NC group (p<0.01); after the intervention of the other groups except the CP group, the expression level of IL-1β was significantly reduced (p<0.01), and was close to the level of the NC group. From the results of determination of TNF-α level in blood of mice, Figure 14 The results of determination of TNF-α level in blood of mice showed that, compared with the NC group, the expression level of TNF-α in the MC group was significantly higher than that in the NC group (p<0.01); the PC, WC, BWF and CP groups significantly reduced the increase of the expression level of TNF-α induced by ultraviolet irradiation (p<0.01), especially the BWF, indicating that the BWF was more effective in reducing the overexpression of pro-inflammatory cytokines such as IL-1β and TNF-α.

[0141] At each classification level (Phylum, Class, Family, Genus, Species), the species annotation and abundance information of each sample were first analyzed, and then the top 10 phyla and classes with the highest abundance and the top 35 families, genera and species with the highest abundance were selected. Using the abundance information in each sample, a species relative abundance column accumulation chart was generated to visually view the species with high relative abundance and their proportion at different classification levels in each sample.

[0142] As shown in Figure 15 Firmicutes (Firmicutes), Bacteroidota (Bacteroidetes), Campylobacterota (Campylobacter), Verrucomicrobiota (Verrucomicrobia) and Proteobacteria (Proteobacteria) are dominant in all groups at the phylum level, and the sum of their relative abundance is more than 90%. Compared with the NC group, after ultraviolet irradiation, the relative abundance of Bacteroidota and Campylobacterota in the MC group increased (0.593±0.164 to 0.773±0.057, 0.010±0.006 to 0.039±0.054, respectively), while the relative abundance of Firmicutes and Verrucomicrobiota decreased (0.311±0.219 to 0.154±0.051, 0.034±0.059 to 0.018±0.031, respectively), indicating that ultraviolet radiation can cause intestinal flora disorder in mice. Each group intervention can improve the intestinal flora disorder induced by ultraviolet radiation, among which BW and BWF have better and more significant effects. For example, BWF intervention significantly reduces the relative abundance of Bacteroidota and Campylobacterota (0.771±0.120, 0.0180±0.0163, respectively), and significantly increases the relative abundance of Verrucomicrobiota (relative abundance 0.858±0.101), even higher than that in the NC group.

[0143] As shown in Figure 16As shown, at the class level, Bacteroidia (Bacteroidia class), Bacilli (Bacilli class), Campylobacteria (Campylobacteria class), Verrucomicrobiae (Verrucomicrobiae class) and Clostridia (Clostridia class) are dominant, and the sum of their relative abundance is more than 92%. Compared with the NC group, after ultraviolet irradiation, the relative abundance of Bacteroidia, Campylobacteria and Clostridia in the MC group increased (0.593±0.164 to 0.773±0.057, 0.010±0.006 to 0.039±0.054, and 0.047±0.009 to 0.088±0.043, respectively), while the relative abundance of Bacilli and Verrucomicrobiae decreased (0.263±0.222 to 0.064±0.059, and 0.0349±0.059 to 0.018±0.031, respectively). Each group intervention can improve the intestinal flora disorder induced by ultraviolet radiation, among which BWF intervention significantly reduces the relative abundance of Bacteroidia and Campylobacteria (0.0771±0.012 and 0.0179±0.0163, respectively), and significantly increases the relative abundance of Verrucomicrobiae (relative abundance of 0.858±0.101), which is even higher than that of the NC group.

[0144] As shown in FIG. 6B, at the genus level, the relative abundance of Bacteroides, Clostridium, Lachnospira, Helicobacter, Prevotella, Akkermansia, and Erysipelatoclostridium in the MC group increased after ultraviolet irradiation, while the relative abundance of Bifidobacterium, Roseburia, and Tannerella decreased, indicating that ultraviolet radiation will cause intestinal flora disorder in mice. BW and BWF can improve the abnormal changes of the above flora, among which BWF has the best effect. Figure 17

[0145] As shown in FIG. 6B, at the genus level, the relative abundance of Bacteroides, Clostridium, Lachnospira, Helicobacter, Prevotella, Akkermansia, and Erysipelatoclostridium in the MC group increased after ultraviolet irradiation, while the relative abundance of Bifidobacterium, Roseburia, and Tannerella decreased, indicating that ultraviolet radiation will cause intestinal flora disorder in mice. BW and BWF can improve the abnormal changes of the above flora, among which BWF has the best effect. Figure 18 ​As shown, at the genus level, the Helicobacter, Lachnospiraceae_NK4A136_group, Odoribacter, Candidatus_Stoquefichus, Intestinimonas, Lachnoclostridium, Oscillibacter, Rikenella and Cetobacterium were more abundant in the MC group than in the NC group after UV irradiation, while Parabacteroides, Parasutterella, [Clostridium]_innocuum_group, Cetobacterium, Akkermansia and Paeniclostridium were less abundant in the MC group than in the NC group, indicating that UV irradiation destroyed the intestinal flora of the mice and caused the flora to be disordered. The intervention of each group improved the species at the genus level, especially the BWF group. Compared with the MC group, the BWF intervention improved the increase in the above flora abundance and was close to the NC group, while the abundance of Ligilactobacillu, Alloprevotella, Candidatus_Saccharimonas, Rikenellaceae_RC9_gut_group, Akkermansia, [Eubacterium]_xylanophium_group, Prevotellaceae_UCG_001 and Bacteroides increased significantly, among which Prevotellaceae_UCG_001, Alloprevotella, Rikenellaceae_RC9_gut_group and Akkermansia were the main intestinal flora contributors involved in inflammation regulation and short-chain fatty acid production, indicating that BWF had a regulatory effect on the UV-induced imbalance of the flora.

[0146] As Figure 19As shown, at a certain level, the relative abundance of Bacteroides_acidifaciens, Akkermansia_muciniphila, Parabacteroides_goldsteinii, Burkholderiales_bacterium_YL45 and Bacteroides_caecimuris in the MC group of mice decreased significantly after ultraviolet irradiation, and each group could improve the abnormal decrease of the above flora to varying degrees. Among them, PC, BW and BWF can significantly increase the relative abundance of Akkermansia_muciniphila (0.018 ± 0.031 up-regulated to 0.0208 ± 0.011, 0.0296 ± 0.046 and 0.0858 ± 0.101, respectively); BC can significantly increase the relative abundance of Parabacteroides_goldsteinii (0.0253 ± 0.0187 up-regulated to 0.0833 ± 0.0839). Related studies have shown that Akkermansia_muciniphila is a very important intestinal microorganism, which has multiple effects such as regulating intestinal flora diversity, improving intestinal mucosal barrier, regulating body metabolism and anti-inflammatory; Parabacteroides_goldsteinii can be involved in the treatment of obesity, and has the effects of enhancing intestinal integrity and anti-inflammatory.

[0147] Example 4

[0148] 1. Preparation of sunscreen cream base: oil phase: squalane 1 g, white oil 3 g, vaseline 3 g, lanolin 3 g, glyceryl monostearate 0.5 g, sorbitan monostearate 1 g, stearic acid 3 g, cetyl alcohol 2 g; water phase: Tween-80 2 g, glycerol 4 g, propylene glycol 2 g, betaine 1 g, water 74 g; C phase: preservative: composite polyol 0.8 g; D phase: hyaluronic acid stock solution 0.5 g, probiotic freeze-dried powder 5 g. Heat the oil phase to 75-85°C in a sterile environment, and completely dissolve. Heat the water phase to 85-90°C. While stirring the water phase, add the oil phase to the water phase, high-speed shear and stir to fully mix for 30 min at 80°C. Cool to 40°C and homogenize until C and D phases are added. Finally, cool to room temperature to prepare the cream. The probiotic freeze-dried powder is probiotic fermented blueberry whey protein freeze-dried powder, probiotic fermented blueberry freeze-dried powder, and probiotic fermented whey protein freeze-dried powder, respectively, and different cream formulations are prepared by adding different ingredients. By changing the amount of probiotic freeze-dried powder added to the cream, creams with different concentrations of probiotic freeze-dried powder are obtained.

[0149] 2. Weigh 5g of face cream containing probiotic-fermented blueberry whey protein freeze-dried powder, add 100mL of distilled water, heat to a gentle boil, stir well and let cool naturally, centrifuge, and measure the pH to be less than 7, which meets the requirement of pH less than 8.3 for topical emulsions.

[0150] 3. Staining method: Add water-soluble methylene blue dye to the face cream emulsion containing probiotic-fermented blueberry whey protein freeze-dried powder. Observe under a microscope. If the external phase appears blue, it is an oil-in-water (O / W) emulsion. Add oil-soluble Sudan Red. If the internal phase appears red under a microscope, it is an oil-in-water (O / W) emulsion. Otherwise, it is a water-in-oil emulsion.

[0151] Figure 20 This is a microscopic image of the face cream stained with water-soluble methylene blue and oil-soluble Sudan Red. As shown in the image, this product is an oil-in-water emulsion, with a refreshing feel and no greasy residue. It is suitable for various skin types, including dry, oily, sensitive, and combination skin. Its gentle nature makes it suitable for all skin types and less likely to cause allergic reactions; it has excellent moisturizing effects, increases skin's water retention, and promotes healthier skin.

[0152] 4. Using a Cary 610 / 390 micro-infrared spectrometer, the infrared spectra were measured with wavenumbers ranging from 500 to 4000 / cm². -1 The study investigated the UV transmittance and reflectance of face creams containing different concentrations of probiotic-fermented blueberry whey protein freeze-dried powder, probiotic-fermented blueberry freeze-dried powder, and probiotic-fermented whey protein freeze-dried powder, and compared them with ZnO2, the most common sunscreen additive on the market.

[0153] Depend on Figure 21 It was found that a face cream with 10% probiotic-fermented blueberry freeze-dried powder had lower transmittance (T%) in both the UV and blue light regions, lower than a sunscreen face cream with 2% ZnO2. A face cream with 15% probiotic-fermented whey protein freeze-dried powder had higher UV transmittance, with the latter being slightly higher than the former. However, the transmittance in the blue light region was not significantly different from the latter.

[0154] Depend on Figure 22It can be seen that the reflectivity (R%) of the sunscreen cream added with the probiotic fermented blueberry freeze-dried powder, the probiotic fermented blueberry whey protein freeze-dried powder is higher than that of the sunscreen cream added with ZnO2 in the ultraviolet region. At the same time, in the blue light region, when the addition amount of the probiotic fermented blueberry whey protein freeze-dried powder, the probiotic fermented blueberry freeze-dried powder and the probiotic fermented whey protein freeze-dried powder is 5%, the sunscreen cream has the same reflection ability as the sunscreen cream added with 2% ZnO2. When the addition amount of the probiotic fermented blueberry whey protein freeze-dried powder is 5%, the sunscreen cream has a higher ultraviolet reflection ability, which is higher than that of the sunscreen cream added with the probiotic fermented blueberry freeze-dried powder with an addition amount of 5%. The above results show that the probiotic fermented blueberry whey protein freeze-dried powder has a strong ultraviolet protection ability, and also has a blue light protection ability.

[0155] Figure 23The addition amount of probiotic fermented blueberry whey protein freeze-dried powder, probiotic fermented blueberry freeze-dried powder, probiotic fermented whey protein freeze-dried powder for the tyrosinase inhibition rate of blueberry whey protein freeze-dried powder is 5%, 10%, 15%, L represents the face cream prepared by probiotic fermented blueberry freeze-dried powder, R represents the face cream prepared by probiotic fermented whey protein freeze-dried powder, and LR represents the face cream prepared by probiotic fermented blueberry whey protein freeze-dried powder. The method for determining the tyrosinase inhibition rate is described in the reference

Wang Xumei, Shen Xuemei, Wu Wenqin, Zhang Na, Wang Rongqing. Comparison of 19 kinds of Chinese herbal medicine whitening and antioxidant activity. Journal of Anhui University, 2017, 41(1), 86-88.

[0156] Figures 24-27A skin analyzer was used to test the surface moisture, oil content, skin tone, and pigmentation of pigskin after applying face creams containing 10% of different probiotic fermented freeze-dried powders and then exposing them to ultraviolet light. Here, L represents face creams prepared with probiotic fermented blueberry freeze-dried powder, R represents face creams prepared with probiotic fermented whey protein freeze-dried powder, and LR represents face creams prepared with probiotic fermented blueberry whey protein freeze-dried powder. The changes in moisture, oil content, pigmentation, and skin tone of pigskin after 0–3.5 hours relative to 0 hours were observed when different additives (10% concentration) were applied and exposed to ultraviolet light.

[0157] Depend on Figure 24 It can be seen that, with the increase of time, the face cream containing probiotic-fermented blueberry whey protein freeze-dried powder showed a higher and relatively stable moisture score compared to the face cream without any cream applied, indicating better performance. Conversely, the face creams containing both probiotic-fermented blueberry freeze-dried powder and probiotic-fermented whey protein freeze-dried powder showed less change in moisture value compared to the face cream without any cream applied, and even showed a decreasing trend. This suggests that face creams containing fermented products have a good moisturizing effect on the skin, with the probiotic-fermented blueberry whey protein freeze-dried powder showing the best effect.

[0158] Depend on Figure 25 It was observed that, over time, the oil content of face creams containing different probiotic fermented freeze-dried powders was significantly lower than that of areas without face cream, indicating that all face creams have oil-controlling effects. Certain components in the probiotic fermented freeze-dried powders may help regulate sebum secretion, keeping the skin refreshed. Furthermore, the probiotic fermented freeze-dried powders may help maintain the balance of the skin's microecology, further enhancing the oil-controlling effect and keeping the skin stable.

[0159] Depend on Figure 26 It is evident that over time, skin tone was affected to some extent in both areas where face creams containing different probiotic fermented freeze-dried powders were applied and areas without face cream. Ultraviolet (UV) radiation is one of the main external factors causing skin tone changes; it can penetrate the skin's surface, damaging skin cells and leading to darkening and age spots. However, compared to areas without face cream, skin treated with face creams containing probiotic fermented freeze-dried powder showed better skin tone retention. In particular, face creams containing probiotic fermented blueberry whey protein freeze-dried powder and probiotic fermented whey protein freeze-dried powder demonstrated especially outstanding performance in maintaining skin tone. This may be because specific components in these fermented products effectively resist UV damage to the skin, thus helping to maintain a healthy complexion.

[0160] Depend on Figure 27It can be seen that the pigment value decreases with the increase of time, indicating that ultraviolet light can cause melanocytes in the skin to be active, leading to pigment deposition, thereby increasing the pigment value of the skin. The pigment value of the skin without applying cream decreases more, while the pigment value of the skin applying the cream containing the probiotic fermented blueberry whey protein freeze-dried powder decreases less. This shows that the cream containing the probiotic fermented blueberry whey protein freeze-dried powder may contain antioxidant ingredients and other active substances beneficial to skin health, which plays a positive role in reducing the damage of ultraviolet light to the skin.

[0161] Figure 28 A physical diagram of the application of sunscreen on the skin surface. L represents the cream prepared from the probiotic fermented blueberry freeze-dried powder, R represents the cream prepared from the probiotic fermented whey protein freeze-dried powder, and LR represents the cream prepared from the probiotic fermented blueberry whey protein freeze-dried powder. It can be seen from the figure that different creams have good skin application.

Claims

1. A method for the preparation of a freeze-dried powder of a probiotic fermented complex with protection against UV radiation damage, characterized in that, The method comprises the following steps: (1) Preparation of blueberry concentrate: wash blueberries and squeeze juice, first filter through 200 mesh gauze, collect the filtrate, then filter again through 150 mesh gauze, collect the filtrate, dilute the collected filtrate with water, and concentrate by membrane concentration method, the concentration time is 40-60 min, the molecular weight cut-off of the membrane is 1 kDa, the transmembrane pressure is 1.0-1.5 Mpa, and the volume ratio of blueberry before and after concentration is 2-3:1, to obtain blueberry concentrate, wherein the mass-volume ratio of blueberry to blueberry concentrate is 1g:2-3 mL; (2) Preparation of blueberry whey protein fermentation medium: add blueberry concentrate, sucrose and whey protein to water, stir until mixed evenly, adjust to the appropriate pH for the strain, and pasteurize to obtain a blueberry whey protein fermentation medium, wherein the addition amount of blueberry concentrate is 3-15 wt.%, the addition amount of whey protein is 3-9 wt.%, and the addition amount of sucrose is 3-5 wt.%; (3) Fermentation: add the activated probiotic strain to the blueberry whey protein fermentation medium for fermentation, wherein the probiotic strain is a mixture of Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 with a preservation number of CGMCC No. 27785; (4) Ultrafiltration concentration: separate and concentrate the fermentation mixture obtained in step (3) by membrane separation method, the molecular weight cut-off of the ultrafiltration membrane is 100 kDa, and a blueberry whey protein concentrate component fermented by probiotics with a molecular weight of >100 kDa is obtained; (5) Freeze-drying: pre-freeze the blueberry whey protein concentrate component fermented by probiotics, and then vacuum freeze-dry to obtain a freeze-dried powder of probiotic fermentation complex.

2. The production method according to claim 1, characterized by, In step (2), the addition amount of blueberry concentrate in the blueberry whey protein fermentation medium is 11 wt.%, the addition amount of whey protein is 6 wt.%, and the addition amount of sucrose is 3 wt.%; the total inoculation amount of probiotic strains is 2-5%, and the inoculation ratio of Lactobacillus plantarum 67 and Lactobacillus paracasei grx 701 is equal.

3. The preparation method according to claim 1, characterized in that, In step (3), the total inoculation amount of probiotic strains is 3%.

4. The production method according to claim 1, characterized by, In step (3), the fermentation time is 10-18 h, and the fermentation temperature is 37-42 ℃.

5. The production method according to claim 1, characterized by, In step (4), the membrane separation conditions are room temperature, the transmembrane pressure is 0.25 MPa, and the concentration time is 20-30 min.

6. The production method according to claim 1, characterized by, In step (5), the pre-freezing temperature is -20 to -80 ℃, the pre-freezing time is 24-36 h, the vacuum freeze-drying temperature is -50 to -80 ℃, and the vacuum freeze-drying time is 24-36 h.

7. The freeze-dried powder of probiotic fermentation complex prepared by the preparation method of any one of claims 1-6.

8. The use of the freeze-dried powder of probiotic fermentation complex according to claim 7 in the preparation of skin care products for preventing ultraviolet radiation damage.

9. A sunscreen cream, characterized by By weight parts, consisting of: squalane 1 part, white oil 3 parts, vaseline 3 parts, lanolin 3 parts, glycerol monostearate 0.5 parts, sorbitan monostearate 1 part, stearic acid 3 parts, cetyl alcohol 2 parts, Tween-80 2 parts, glycerol 4 parts, propylene glycol 2 parts, betaine 1 part, water 74 parts, preservative amount, hyaluronic acid stock solution 0.5 parts, the probiotic bacteria fermentation complex freeze-dried powder of claim 7 5 parts.

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