Application of roxburgh rose normal juice or probiotic fermentation product thereof in preparation of functional food or medicine for resisting light aging or regulating intestinal flora disorder

Abstract

The invention discloses application of roxburgh rose normal juice or probiotic fermentation products thereof in preparation of functional food or medicine for resisting light aging or regulating intestinal flora disorder, stable delivery of roxburgh rose active components in a dairy product multi-phase matrix is optimized, a delicious, accessible and standardized daily intervention carrier is constructed, and the roxburgh rose normal juice or the probiotic fermentation products thereof are applied to preparation of functional food or medicine for resisting light aging or regulating intestinal flora disorder. UVB / UVA / induced skin photoaging is intervened in multiple ways, collagen degradation is slowed down, pigment and inflammation phenotypes are improved, and the defects that existing prevention and treatment means are mainly used for external sunscreen and cosmeceuticals, and compliance and visible light protection are insufficient are overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the application of a roxburgh rose original juice or its probiotic fermentation product in the preparation of a functional food or medicine for resisting photoaging or regulating intestinal flora disorders, and belongs to the field of microbial fermentation. BACKGROUND

[0002] Skin photoaging can lead to wrinkles, decreased skin elasticity, pigment disorders, and damaged skin barrier, and significantly increases the risk of photocarcinogenesis and skin cancer, affecting appearance and skin health, and causing long-term medical and social psychological burden. Skin photoaging is mainly caused by long-term sunlight exposure, involving the synergistic effects of UVA, UVB, visible light, and infrared light, leading to collagen degradation and abnormal matrix remodeling through mechanisms such as reactive oxygen species generation, DNA photodamage, MMP upregulation, and TGF-β pathway inhibition. Existing methods for improving skin photoaging include topical retinoids, antioxidants, and fruit acids, which can promote collagen neogenesis, inhibit MMPs, and improve pigmentation and texture, but are limited in terms of compliance and irritation. Means such as chemical peeling, laser / intense pulsed light, and photodynamic therapy can remodel the dermis, improve wrinkles and pigmentation, but are associated with downtime, recurrence, and an increased risk of post-inflammatory hyperpigmentation in dark skin. Emerging stem cell exosomes and combination therapies show repair potential, but long-term safety, cost, and standardization are still lacking. Oral non-drug interventions are important in the management of photoaging, and can reduce ROS and MMP activity through antioxidant and anti-inflammatory pathways, support collagen homeostasis, and make up for the lack of compliance and resistance to topical and procedural treatments, serving as a long-term, accessible, and systemic adjunct strategy to improve prevention and repair benefits.

[0003] Functional dairy products, as carriers of active ingredients, offer advantages such as mild taste, suitability for daily intake, good matrix stability, and improved bioavailability. Their protein-lipid multiphase system protects antioxidant molecules from oxidation and photodegradation. The fermentation process also generates small peptides and organic acids, synergistically improving intestinal absorption and tolerance. Furthermore, they facilitate standardized production and quality control, making them suitable for developing daily anti-photoaging foods. Rosa roxburghii Tratt, a fruit of the Rosaceae family, is rich in vitamin C, flavonoids, and polyphenols, possessing antioxidant, anti-inflammatory, and immunomodulatory functions. It can be developed and applied as a functional dairy product and fermentation matrix. The main mechanisms of Rosa roxburghii's antioxidant activity include its high vitamin C, flavonoid, and polyphenol content scavenging ROS, chelating metal ions, and inhibiting lipid peroxidation and protein oxidation; and upregulating antioxidant enzyme systems (such as SOD, CAT, and GSH-Px) to synergistically alleviate the release of inflammatory mediators. This provides a physicochemical and biological basis for developing Rosa roxburghii into functional milk and fermented milk. However, despite the biological basis and food application potential of prickly pear in terms of antioxidation, anti-inflammation, and matrix homeostasis, existing research has mostly focused on overall antioxidant or metabolic health, and there are no direct reports or mechanistic explanations of using prickly pear to combat skin photoaging. Utilizing the antioxidant activity of prickly pear in synergy with milk / fermented milk carriers to develop functional foods for combating skin photoaging holds promise for inhibiting ROS and MMPs, maintaining collagen homeostasis, forming an accessible dietary protection pathway, and enhancing the skin barrier and resistance to photodamage. Summary of the Invention

[0004] Objectives of this invention: The first objective of this invention is to provide the application of prickly pear juice or its probiotic fermentation product in the preparation of functional foods or pharmaceuticals that combat photoaging or regulate intestinal flora imbalance. The second objective of this invention is to provide a prickly pear flavored milk with anti-photoaging properties.

[0005] Technical solution: The application of the prickly pear juice or its probiotic fermentation product described in this invention in the preparation of functional foods or medicines that resist photoaging or regulate intestinal flora imbalance.

[0006] Furthermore, the probiotic strain is *Lactobacillus fermentum* grx938.

[0007] Furthermore, the amount of prickly pear juice added to the flavored milk is 5-15%.

[0008] Furthermore, the functional food or medicine is used to improve skin oxidative stress, inflammation, and water retention.

[0009] Furthermore, the functional food is fermented milk.

[0010] Furthermore, the gut microbiota that the functional food or drug is used to regulate includes... Limosilactobacillus , Corynebacterium, norank_f_ Paracaedibacteraceae , Mammaliicoccus, Alistipesand Prevotellaceae_UCG-001.

[0011] A photoaging-resistant roxburgh rose flavor milk, the preparation steps of which comprise: high-temperature sterilization of whole milk, mixing with sterilized roxburgh rose stock after cooling, and mixing.

[0012] Further, the flavor milk further comprises the addition of probiotic fermentation after the addition of sterilized roxburgh rose stock.

[0013] Further, the probiotic strain is fermented Lactobacillus mucus grx938.

[0014] Further, the inoculation amount of fermented Lactobacillus mucus grx938 is 1-5%.

[0015] Further, the fermentation condition is 37-42°C for 16-24 h.

[0016] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application realizes stable delivery of roxburgh rose active components in a multi-phase matrix of dairy products by optimization, constructs a delicious, accessible and standardized daily intervention carrier, intervenes in UVB / UVA / induced skin photoaging in multiple ways, slows down collagen degradation, improves pigment and inflammation phenotype, and makes up for the defects of existing prevention and treatment means, mainly external sunscreens and cosmeceuticals, poor compliance and insufficient visible light protection. And it can also be used to improve intestinal flora disorder. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The effect of the addition amount of roxburgh rose stock on the antioxidant capacity of roxburgh rose milk.

[0018] Figure 2 The effect of the addition amount of roxburgh rose stock on the stability of roxburgh rose milk.

[0019] Figure 3 The macroscopic score of skin condition.

[0020] Figure 4 The appearance of the irradiated area of the mouse skin.

[0021] Figure 5 The apoptosis of skin cells.

[0022] Figure 6 The improvement of the oxidation index ROS.

[0023] Figure 7 The improvement of the oxidation index MDA.

[0024] Figure 8 The improvement of the oxidation index SOD.

[0025] Figure 9 The improvement of the skin inflammation factor IL-1β.

[0026] Figure 10 Improvement of skin inflammation factor IL-6.

[0027] Figure 11 Improvement of skin inflammation factor TNFα.

[0028] Figure 12 Improvement of skin water retention factor HYAL1.

[0029] Figure 13 Improvement of skin water retention factor HA.

[0030] Figure 14 Improvement of intestinal flora. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are further described below in combination with the drawings.

[0032] Example 1

[0033] 1. Preparation of rosehip flavor milk Full-fat milk was sterilized at 95℃ for 5 min, cooled to 37℃, and 0, 5, 10, 15, 20, 25, and 30% of sterilized rosehip juice was added respectively and mixed uniformly, and placed in a refrigerator at 4℃ for standby.

[0034] 2. Preparation of rosehip flavor fermented milk The fermentation bacteria used in the present application are fermented Lactobacillus mucus grx938 (patent application CN202410777612.5, antioxidant functional fermented Lactobacillus mucus and its application in rosehip flavor fermented milk).

[0035] Full-fat milk was sterilized at 95℃ for 5 min, cooled to 37℃, and 0, 5, 10, 15, 20, 25, and 30% of sterilized rosehip juice was added respectively and mixed uniformly, and 1×10 9 cfu / mL of fermented Lactobacillus mucus grx938 was inoculated at an inoculation amount of 3%, and fermented at 37℃ for 24 h. After fermentation, it was placed at 4℃ for post-ripening for 24 h. After post-ripening, it was placed in a refrigerator at 4℃ for standby.

[0036] 3. Detection method The prepared fermented milk samples were determined for vitamin C (Shanghai Yuanye Biological Technology Co., Ltd., R22193-50T) and total antioxidant capacity (Nanjing Jiancheng Biological Engineering Institute, A015-2-1) using a kit.

[0037] 4. Result analysis The antioxidant capacity results are as follows Figure 1As shown, when no rose apple original juice is added, the vitamin C content in the fermented L. gasseri grx938 fermented milk is below the detection limit, and the total antioxidant capacity is only 1.337 mM Trolox. With the increase of the amount of rose apple original juice added, the vitamin C content and total antioxidant capacity in the rose apple fermented milk are both increased. When the amount of rose apple original juice added is 10%, the vitamin C content in the rose apple fermented milk is 357.5 mg / 100 mL, and the total antioxidant capacity is 119.36 mM Trolox. When the amount of rose apple original juice added is increased to 15%, the vitamin C content in the rose apple fermented milk is 473.3 mg / 100 mL, and the total antioxidant capacity is 127.87 mM Trolox.

[0038] The effect of the amount of rose apple original juice added on the centrifugal precipitation rate of milk protein is shown in Table 3. Figure 2 As shown, when the amount of rose apple original juice added is greater than 15%, the centrifugal precipitation rate of milk is >40%, so the amount of rose apple original juice added should be between 5% and 15%.

[0039] Example 2

[0040] 1. Experimental grouping and modeling 30 SPF athymic nude mice (Nude Mouse) weighing 18-22 g were purchased from Liaoning Changsheng Biotechnology Co., Ltd. After the experimental mice were adaptively fed for one week, they were randomly divided into 5 groups, each group of 6, namely a blank group, a model group, a positive drug group, a rose apple milk group, and a rose apple fermented milk group. The specific grouping and treatment methods of the animal experiment are as follows: every other day, the mice were irradiated with ultraviolet light according to the determined dose (the blank group was not irradiated); the mice in the blank group were given physiological saline 15 mL / kg / d by gavage every day, the mice in the model group were given physiological saline 15 mL / kg / d by gavage every day, the mice in the positive drug group were given vitamin E solution by gavage every day, the mice in the rose apple milk group were given milk containing 15% rose apple original juice by gavage every day, and the mice in the fermented rose apple milk group were given fermented milk containing 15% rose apple original juice by gavage.

[0041] Table 1. Information on the grouping of the animal experiment

[0042] Modeling method: the ultraviolet radiation source was about 30 cm away from the back skin of the mice. The experiment lasted for 8 weeks, and the ultraviolet radiation was performed every other day, at 9 am every day, 1 hour before administration. The minimum erythema dose incremental method, which is currently internationally recognized and scientific, was used. The irradiation dose was 1 MED (100 mJ / cm 2 ) in the first week, and then increased by 1 MED every week until the irradiation dose stopped increasing at 4 MED in the fourth week. The irradiation dose of 4 MED was continued until the end of the experiment in the eighth week, and the total irradiation dose reached 9 J / cm 2 .

[0043] Before the end of the experiment, mice were euthanized by dislocation after blood collection, and 1 cm of skin tissue from their backs was taken. 2 The tissue was fixed in 10% paraformaldehyde for skin histopathological studies, and the remaining skin tissue was stored at -80°C for later use. Feces were collected from the colon under aseptic conditions.

[0044] 2. Macroscopic evaluation of the skin Every Wednesday, the irradiated areas on the backs of mice were photographed, and the appearance of the irradiated skin was recorded. Observations of the irradiated areas included skin roughness, presence of erythema, degree of wrinkling, changes in thickness, and retention of elasticity—all indicative of photoaging. The skin on the backs of the mice was macroscopically scored from 0 to 6 (0 representing normal skin, 6 representing severely photoaged skin). The examination was conducted for a total of 8 weeks.

[0045] Table 2 Macroscopic evaluation criteria for skin damage

[0046] Eight weeks after UVB modeling, the model group mice developed deep wrinkles and mild skin laxity, accompanied by erythema. Mice treated with prickly pear milk, fermented prickly pear milk, or vitamin E via gavage showed only a few shallow wrinkles in their skin tissue, without significant erythema. The scoring results are shown below. Figure 3 The appearance of the skin in the irradiated area of ​​the mouse is shown in the figure. Figure 4 This indicates that intervention with prickly pear milk or fermented prickly pear milk can significantly improve skin damage caused by UVB.

[0047] 3. Evaluation of skin cell apoptosis Fixed tissue samples were dehydrated for 60 min each in 75%, 85%, 95% I, 95% II, 100% I, and 100% II ethanol solutions, then cleared in xylene I and II for 45 min each, followed by permeabilization in paraffin I, paraffin II, and paraffin III for 60 min each. The samples were then embedded in paraffin and sectioned. The paraffin sections were baked, then immersed in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 3 min, anhydrous ethanol II for 3 min, 95% ethanol for 3 min, 80% ethanol for 3 min, and pure water for 2 min. The sections were then transferred to a humidified chamber, and 20 μg / ml Proteinase K working solution was added to each sample. The mixture was incubated at 37°C for 30 min. The samples were washed thoroughly with PBS three times, 5 min each time. The PBS around the tissue was blotted off with absorbent paper, and a sufficient amount of TUNEL assay solution was added to each slide. The slides were incubated at 42°C in the dark for 1 h. Wash away excess detection solution with PBS, add DAPI and incubate in the dark for 3 minutes to stain the nuclei, then rinse with PBS to remove excess DAPI. Blot the slide dry with absorbent paper, mount with mounting solution containing an anti-fluorescence quencher, and then observe and acquire images under a fluorescence microscope. (DAPI UV excitation wavelength 330-380nm, emission wavelength 420nm, emits blue light; TUNEL excitation wavelength 510-560nm, emission wavelength 590nm, emits red light) Eight weeks after UVB modeling, the number of apoptotic skin cells in the model group was significantly higher than that in other groups (p < 0.05). After intervention with prickly pear milk, fermented prickly pear milk, or vitamin E via gavage, there was no significant difference in the number of apoptotic cells in the skin tissue of mice compared to the control group. (See attached results). Figure 5 (Red represents dead cells, blue represents live cells). This indicates that prickly pear milk or fermented prickly pear milk can significantly improve skin cell apoptosis induced by UVB.

[0048] 4. Improvement in oxidation indicators To prepare a 10% skin tissue homogenate, 0.1 g of skin tissue was rapidly and accurately weighed and placed into a pre-cooled 5 mL EP tube. The tissue was minced on ice and homogenized at a ratio of tissue weight to physiological saline volume of 1:9 (m:v). The sample extract was homogenized in a high-speed low-temperature tissue homogenizer at a homogenization frequency of 70 Hz and a temperature of -10℃, with a 20-second pause every 60 s, for a total of 20 homogenization cycles. The homogenate was centrifuged at 12000 rpm, 4℃, for 10 min, and the supernatant was aliquoted for use. The contents of ROS (Beyotime Biotechnology Research Institute, A376250826), superoxide dismutase activity (Nanjing Jiancheng Bioengineering Institute, A031-3-2), and malondialdehyde (Wuhan E-EL-0060) in the skin tissue were determined according to the assay kit instructions.

[0049] The results are as follows Figure 6~8 As shown, after 8 weeks of UVB modeling, the ROS concentration in the skin tissue of mice in the model group was significantly higher than that in the control group (p < 0.05). After intervention with prickly pear milk and fermented prickly pear milk, the ROS concentration in the skin tissue of mice was significantly lower than that in the model group (p < 0.05), and there was no significant difference between the model group and the control group (p > 0.05). The SOD concentration in the skin tissue of mice in the model group was significantly lower than that in the control group (p < 0.05). After intervention with prickly pear milk and fermented prickly pear milk, the ROS concentration in the skin tissue of mice was significantly higher than that in the model group (p < 0.05). Among them, there was no significant difference between the prickly pear milk group and the control group (p > 0.05), and the SOD concentration in the fermented prickly pear milk group was significantly higher than that in the control group (p < 0.05). The MDA concentration in the skin tissue of mice in the model group was significantly higher than that in the control group (p < 0.05). After intervention with prickly pear milk and fermented prickly pear milk, the MDA concentration in the skin tissue of mice was significantly lower than that in the model group (p < 0.05). Among them, there was no significant difference between the fermented prickly pear milk group and the control group (p > 0.05). The results indicate that both prickly pear milk and fermented prickly pear milk can significantly reduce ROS and MDA concentrations and increase SOD concentrations, thereby combating UVB-induced skin oxidative stress and improving skin photoaging.

[0050] 5. Improvement of skin inflammation Take out the prepared serum sample and use an ELISA kit to measure the levels of IL-1β (Wuhan Elite Biotechnology Co., Ltd., E-MSEL-M0003), IL-6 (Wuhan Elite Biotechnology Co., Ltd., E-MSEL-M0003), and TNF-α (Wuhan Elite Biotechnology Co., Ltd., E-MSEL-M0001) in the serum sample.

[0051] The results are as follows Figure 9~11As shown, after 8 weeks of UVB modeling, the concentrations of IL-1β, IL-6, and TNFα in the skin tissue of mice in the model group were significantly higher than those in the control group (p < 0.05). After intervention with prickly pear milk and fermented prickly pear milk, the concentrations of IL-1β, IL-6, and TNFα in the skin tissue of mice were significantly lower than those in the model group. However, the concentrations of IL-1β and IL-6 in the fermented prickly pear milk group showed no significant difference compared to the control group. Both prickly pear milk and fermented prickly pear milk significantly reduced key pro-inflammatory mediators and alleviated skin inflammation induced by UVB.

[0052] 6. Improvement in skin's water retention The contents of HA (Kelu (Wuhan) Biotechnology Co., Ltd., ELK10864) and HYAL1 (Jiangsu Enzyme Immunization Industry Co., Ltd., 49326M1) in mouse skin tissue were determined using an ELISA kit.

[0053] The results are as follows Figure 12~13 As shown, after 8 weeks of UVB modeling, the concentration of HA in the skin tissue of mice in the model group was significantly lower than that in the control group, while the concentration of HYAL1 was significantly higher (p < 0.05). After intervention with prickly pear milk and fermented prickly pear milk, the concentration of HA in the skin tissue of mice was significantly higher than that in the model group, while the concentration of HYAL1 was significantly lower (p < 0.05), and there was no significant difference between the two groups and the control group. This indicates that both prickly pear milk and fermented prickly pear milk can significantly improve UVB-induced abnormal HA metabolism. By increasing HA and inhibiting HYAL1, they can restore the skin matrix and moisturizing status to physiological levels, suggesting their potential to maintain extracellular matrix homeostasis and promote barrier and elasticity recovery in anti-photoaging.

[0054] 7. Improvement in colonic flora diversity Microbial DNA was extracted from fecal samples of designated mice using the QIAamp Fast DNA Stool Mini Kit (QIAGEN) according to the manufacturer's instructions. DNA quality and concentration were assessed by 1.0% agarose gel electrophoresis and a NanoDrop® ND-2000 spectrophotometer (Thermo Scientific Inc.). The V3-V4 region of the bacterial 16S ribosomal RNA gene was amplified using an ABI GeneAmp® 9700 PCR thermal cycler (ABI) and the following primers: 338F: 5'-barcode-ACTCCTACGGGAGGCAGCA-3' and 806R: 5'-GGACTACHVGGGTWTCTAAT-3' (the barcode is an 8-base sequence unique to each sample). After separation by 2% agarose gel electrophoresis, the PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences) according to the manufacturer's instructions and quantified using a Quantus™ fluorometer (Promega). Purified amplified fragments were mixed in equimolar amounts and subjected to paired-end sequencing on an Illumina MiSeq PE300 / NovaSeq PE250 platform using standard procedures at MajorBio Biotechnology Co., Ltd. (Shanghai, China). After demultiplexing, the sequences were quality filtered using fastp (version 0.19.6) and assembled using FLASH (version 1.2.11). Subsequently, the DADA2 plugin in the Qiime2 (version 2020.2) pipeline was used to denoise high-quality sequences with recommended parameters, achieving single-nucleotide resolution based on the in-sample error spectrum. The DADA2-denoised sequences are typically referred to as amplicon sequence variants (ASVs). The taxonomic classification of ASVs was performed using the Naive Bayes consensus classification system implemented in Qiime2, combined with the SILVA 16S rRNA database (v138). Data analysis was performed online on the MajorBio cloud platform (https: / / www.majorbio.com / ).

[0055] The results are as follows Figure 14 As shown, after 8 weeks of UVB modeling, species difference analysis of the colonic microbiota in mice revealed that the colonic microbiota of the model group mice (UVB group) was significantly different. Limosilactobacillus , Corynebacterium, norank_f_ Paracaedibacteraceae and Mammaliicoccus The levels of bacteria in the group were significantly higher than those in the control group (p < 0.05), while Alistipes , Prevotellaceae_UCG-001 The levels of bacteria such as *Rhizopus* and *Rhizopus* were significantly lower than those in the control group (p < 0.05). After intervention with prickly pear milk (RM) and fermented prickly pear milk (FRM), Limosilactobacillus ,Corynebacterium, norank_f_ Paracaedibacteraceae and Mammaliicoccus The levels of bacteria in the *Isperidium* genus were significantly lower than those in the control group and the model group (p < 0.05), while... Alistipes and Prevotellaceae_UCG-001 The levels of *Ischemicum* spp. were significantly higher than those in the control group and the model group (p < 0.05), indicating that both *Prickly Pear* milk and fermented *Prickly Pear* milk can significantly improve UVB-induced gut microbiota dysbiosis and can significantly regulate... Limosilactobacillus , Corynebacterium, norank_f_ Paracaedibacteraceae , Mammaliicoccus, Alistipes and Prevotellaceae_UCG-001 At the level of bacteria genus.

Claims

1. The application of a prickly pear juice or its probiotic fermentation product in the preparation of functional foods or medicines that resist photoaging or regulate intestinal flora imbalance.

2. The application according to claim 1, characterized in that, The probiotic strain is Lactobacillus fermentum grx938.

3. The application according to claim 1, characterized in that, The amount of prickly pear juice added to the flavored milk is 5-15%.

4. The application according to claim 1, characterized in that, The functional foods or medicines mentioned above are used to improve skin oxidative stress, inflammation, and water retention.

5. The application according to claim 1, characterized in that, The functional foods or drugs used to regulate gut microbiota include Limosilactobacillus , Corynebacterium, norank_f_Paracaedibacteraceae , Mammaliicoccus, Alistipes and Prevotellaceae_UCG-001.

6. The application according to claim 1, characterized in that, The functional food is fermented milk.

7. A prickly pear flavored milk with anti-photoaging properties, characterized in that, The preparation steps include: sterilizing whole milk at high temperature, cooling it, adding sterilized prickly pear juice and mixing well.

8. The anti-photoaging prickly pear flavored milk according to claim 7, characterized in that, The flavored milk also includes fermentation with probiotics after adding sterilized prickly pear juice.

9. The anti-photoaging prickly pear flavored milk according to claim 8, characterized in that, The probiotic strain is Lactobacillus fermentum grx938.

10. The anti-photoaging prickly pear flavored milk according to claim 9, characterized in that, The inoculum size of *Lactobacillus fermentum* grx938 is 1-5%.

Images