Preparation method and application of schizochytrium sp. and dioscorea zingiberensis tuber ferment with enhanced moisturizing and anti-aging effects

Abstract

This invention relates to a method for preparing and applying a fermented product of Bifida Ferment Lysate and Dioscorea Oolong with enhanced moisturizing and anti-aging effects, belonging to the field of microbial fermentation technology. This invention discloses a *Bifidobacterium longum*, the nucleotide sequence of which is shown in SEQ ID NO: 1. This invention utilizes *Bifidobacterium longum* HJ-005 to ferment *Dioscorea opposita* and *Dioscorea opposita* to prepare a *Bifida Ferment Lysate* fermented product of *Dioscorea opposita*, simultaneously increasing the content of absorbable diosgenin, anthocyanins, arginine, and organic selenium, with better effects compared to other lactic acid bacteria. It can significantly reduce the water loss and shrinkage of zebrafish embryos, exhibiting a moisturizing effect, and can significantly promote the expression of zebrafish type I collagen and Elna genes, promoting collagen and elastin regeneration, thus possessing anti-aging effects and can be used as a new raw material for cosmetics.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation technology, and in particular to a method for preparing and applying a fermented product of Bifida Ferment Lysate and Dioscorea tuber that enhances moisturizing and anti-aging effects. Background Technology

[0002] Ginseng and yam ( Dioscorea alata.L ) is a plant of the genus Dioscorea in the family Dioscoreaceae. It is a different species from yam, belonging to the same family and genus. It is suitable for living in mountain shrublands or forest edges at an altitude of 400-800 m. Its whole body is purple. At present, purple yam is mainly used for food. Purple yam has high edible and medicinal value. In addition to containing a large amount of anthocyanins, it also contains the following substances: (1) Dioscin / Diosgenin, a steroidal saponin compound in yam tuber, has anti-inflammatory, antioxidant, immunomodulatory, hypoglycemic and anti-tumor effects. (2) Dioscorea polysaccharide, a water-soluble polysaccharide, has immunomodulatory, antioxidant, anti-aging and hypoglycemic effects. (3) Flavonoids, polyphenols, have antioxidant, anti-inflammatory and anti-allergic effects. (4) Mucilage, a polysaccharide-protein complex, has moisturizing, intestinal lubrication and gastric mucosa protection effects. (5) Amino acids and trace elements, including essential amino acids such as arginine, and trace elements such as zinc, iron, and magnesium, which help with metabolism and immune regulation.

[0003] Lactic acid bacteria are a general term for a class of non-spore-forming Gram-positive bacteria that produce lactic acid. Previous reports have shown that they can regulate intestinal flora, enhance human immunity, and have good effects. Currently, researchers at home and abroad have used various commercial lactic acid bacteria to ferment plants or vegetables to improve their nutritional and sensory properties. A study reported (Zhou Xinyong et al., Analysis of Nutritional Components in Dioscorea opposita and its related species Dioscorea parasitica, Anhui Agricultural Sciences, 2010, No. 35) showed that lactic acid bacteria fermentation affects the nutritional components and sensory properties of yam juice. The results showed that yam juice fermented with a combination of L. plantarum 101 and L. paracasei HGD was more sensorily acceptable overall. Compared with unfermented yam juice, lactic acid bacteria fermentation reduced the pH from 6.12 to 3.88, decreased the total sugar content by 69.35%, and increased the contents of total phenols, soluble dietary fiber, total acid, and free amino acids by 98.00%, 53.15%, 493.51%, and 64.30%, respectively. Meanwhile, gas chromatography-mass spectrometry analysis showed that lactic acid bacteria fermentation significantly altered the types and contents of flavor compounds in yam juice. Acids increased by 195.02%, and esters increased by 172.73%. The increase in the types and contents of esters endowed the fermented yam juice with a rich aroma. Ketones and alcohols increased by 88.27% and 7.46%, respectively, while aldehydes decreased by 85.82%, improving the acceptability of the final fermented yam juice. Currently, there are no reports on the use of lactic acid bacteria to ferment purple yam. Furthermore, in reports on lactic acid bacteria fermentation of yam, the effects on components such as anthocyanins, diosgenin, arginine, and selenium have not been systematically studied. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing and applying a fermented product of Bifida Ferment Lysate and Dioscorea tuber that enhances moisturizing and anti-aging effects.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a *Bifidobacterium longum*, the nucleotide sequence of which is shown in SEQ ID NO: 1. The *Bifidobacterium longum* is *Bifidobacterium longum* HJ-005, which was deposited at the China Center for Type Culture Collection on July 23, 2025, with accession number CCTCC NO: M 20251676.

[0006] This invention has screened a new Bifidobacterium longum (Bifida ferment lysate), which, compared with other lactic acid bacteria, can increase the content of diosgenin, allantoin, vitamins and organic selenium in the fermentation products of fermented yam plants, thus exerting moisturizing and anti-aging effects.

[0007] Secondly, the present invention provides a fermentation product of yam plant, which is obtained by fermenting yam plant with Bifidobacterium longum.

[0008] Furthermore, the yam plant includes Dioscorea opposita and / or Dioscorea glutinosa.

[0009] Thirdly, the present invention provides a method for preparing the fermented product of the yam plant, characterized in that, after the yam plant is subjected to color protection and gelatinization, it is inoculated with the Bifidobacterium longum and fermented.

[0010] Furthermore, the yam plant was mixed with a citric acid solution and treated at 90-95℃ for 3-10 minutes to obtain a color-protected sample.

[0011] Furthermore, the citric acid solution has a mass concentration of 15% to 25%, preferably 20%.

[0012] Furthermore, the ratio of yam plant to citric acid solution is: yam plant : citric acid solution = 1 g : (1~3) mL, preferably yam plant : citric acid solution = 1 g : 2 mL.

[0013] Furthermore, protect the color at 90℃ for 5 minutes.

[0014] Further, the color-protecting sample was mixed with water, gelatinized at 90-95℃ for 20-40 min, saccharifying enzyme was added and saccharified at 55-65℃ for 2.5-3.5 h, homogenized, and yam plant matrix was obtained. Glucose was added, and then Bifidobacterium longum was inoculated and fermented.

[0015] Furthermore, the water volume is 2 to 5 times the volume of the color-protecting sample, preferably 3 times.

[0016] Furthermore, gelatinize at 95°C for 30 min, then add saccharifying enzyme and saccharify at 60°C for 3 h.

[0017] Furthermore, the final glucose concentration is 0.3% to 0.6%, preferably 0.5%.

[0018] Furthermore, the volume concentration of the inoculum of Bifidobacterium longum is 1% to 10%, preferably 5%.

[0019] Furthermore, the fermentation temperature is 36~37.5℃, preferably 37℃.

[0020] Furthermore, the fermentation time is 18-26 hours, with the preferred fermentation time for fermented yam being 22 hours and the preferred fermentation time for fermented purple ginseng sweet potato being 24 hours.

[0021] Fourthly, the present invention provides the application of the aforementioned Bifidobacterium longum and the aforementioned Dioscorea plant fermentation product in the preparation of cosmetics.

[0022] Furthermore, the cosmetic is a skin moisturizing and / or skin anti-aging cosmetic.

[0023] Furthermore, the skin anti-aging includes increasing the expression levels of skin elastin and / or collagen.

[0024] Fifthly, the present invention provides a cosmetic product containing the aforementioned yam plant fermentation product.

[0025] In a sixth aspect, the present invention provides a method for increasing the nutrient content in the fermentation products of yam plants, wherein the yam plants are fermented with the aforementioned Bifidobacterium longum; the nutrient components include at least one of diosgenin, allantoin, organic selenium, and arginine.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention utilizes Bifida Ferment Lysate (Bifidobacterium longum HJ-005) to ferment Dioscorea opposita and Dioscorea glutinosa. Through fermentation optimization and control, a Bifida Ferment Lysate ferment of Dioscorea opposita is prepared, simultaneously increasing the content of absorbable diosgenin, anthocyanins, arginine, and organic selenium. The effect is better than that of other lactic acid bacteria. It can significantly reduce the water loss and shrinkage of zebrafish embryos, thus having a moisturizing effect. It can also significantly promote the expression of type I collagen and Elna genes in zebrafish, promote the regeneration of collagen and elastin, and has anti-aging effects. It can be used as a new raw material for cosmetics. Attached Figure Description

[0027] Figure 1 Phylogenetic tree of Bifidobacterium longum HJ-005.

[0028] Figure 2 The results of Gram staining of Bifidobacterium longum HJ-005 are shown in the microscopic examination.

[0029] Figure 3 The results represent the moisturizing efficacy test results; where A is the model control group; B is the positive control group; C is the sample treatment group; and D is the blank control group.

[0030] Figure 4 The bar chart shows the results of the moisturizing efficacy test.

[0031] Figure 5 This is a bar chart showing the relative expression levels of type I collagen genes in zebrafish.

[0032] Figure 6 This is a bar chart showing the relative expression levels of the elastin gene in zebrafish. Detailed Implementation

[0033] To better illustrate the purpose, technical solution, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, other materials and reagents used in the embodiments are commercially available.

[0034] Example 1: Screening and Identification of Fermentation Strains 1. Isolation and Purification: Samples from the intestinal contents (feces) of healthy individuals and traditional fermented food (pickled vegetables) were collected. Modified MRS and M17 media were used, with separate culture conditions set for different bacterial strains, followed by serial dilutions and streaking. The cultures were incubated at 37°C, and single colonies were picked for repeated purification to obtain pure cultures. Specifically, *Lactobacillus*, *Bifidobacterium*, and *Leuconostoc* strains were cultured on modified MRS medium, with *Lactobacillus* and *Bifidobacterium* requiring anaerobic incubation for 48–72 h; *Lactococcus* and *Streptococcus* strains were cultured on M17 medium under aerobic or microaerobic conditions for 24–48 h.

[0035] The modified MRS medium (per liter) contains: 10.0 g peptone, 10.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 1.0 g Tween 80, 2.0 g K2HPO4·3H2O, 5.0 g sodium acetate trihydrate, 2.0 g triammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, and 0.5 g L-cysteine, adjusted to pH 6.2±0.1.

[0036] M17 medium (per liter) contains: 5.0 g peptone, 5.0 g soybean peptone, 2.5 g beef extract, 2.5 g yeast extract, 5.0 g glucose, 5.0 g lactose, 0.5 g ascorbic acid, 0.25 g MgSO4·7H2O, 2.5 g K2HPO4, and pH adjusted to 6.8±0.1.

[0037] 2. Identification: Gram staining, microscopic examination and basic physiological and biochemical tests were performed on the pure cultures obtained in step 1. The physiological and biochemical characteristics of different strains were different.

[0038] Lactobacillus, Bifidobacterium, Lactococcus, and Streptococcus are all catalase-negative. Lactobacillus ferments glucose to produce lactic acid without producing gas. Bifidobacterium ferments glucose to produce lactic acid and acetic acid (molar ratio 2:3). Lactococcus ferments glucose to produce lactic acid without producing gas. Streptococcus strains are hemolytic.

[0039] Following the method described in the literature (Xu Chao, Zhang Shaobing, Meng Jun, et al. Preliminary study on antioxidant activity and cholesterol degradation capacity of different lactic acid bacteria [J]. Food and Fermentation Industries, 2023, 49(02):152-158+165.DOI:10.13995 / j.cnki.11-1802 / ts.031305.), the antioxidant capacity of pure cultures with potential probiotic functions was compared, and strains with strong antioxidant capacity were obtained.

[0040] Genomic DNA was further extracted from each lactic acid bacteria strain, and PCR amplification was performed using universal primers for the 16S rRNA gene. The primer sequences were: upstream primer 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and downstream primer 1492R (5'-TACGGYTACCTTGTTACGACTT-3'). The amplification system (25 μL) consisted of: 2.5 μL of 10×PCR Buffer, 2 μL of dNTPs mixture (2.5 mmol / L), 0.5 μL each of upstream and downstream primers (10 μmol / L), 0.2 μL of Taq DNA polymerase (5 U / μL), 1 μL of template DNA, and sterile double-distilled water to a total amplification volume of 25 μL. The amplification program was as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1.5 min, for a total of 35 cycles; 72℃ final extension for 10 min, and incubation at 4℃. After sequencing the amplified products, the obtained sequences were compared with the NCBI database to classify and identify the selected strains.

[0041] Bifidobacterium longum was finally screened from the feces. Bifidobacterium longum Streptococcus thermophilus was screened from kimchi. Streptococcus thermophilus Lactobacillus plantarum ( Lactiplantibacillus plantarum ) and lactococci ( Lactococcus sp.).

[0042]

[0043] Its and Bifidobacterium longum subsp. longum The ATCC 15707 strain showed high homology; comparison results from the NCBI Blast database showed a similarity of over 95%, and the phylogenetic tree is as follows. Figure 1 As shown, the microscopic results of Gram staining are as follows: Figure 2 As shown. The obtained Bifidobacterium longum (also known as Bifida ferment lysate) was named Bifidobacterium longum (as shown). Bifidobacterium longum HJ-005 was deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M 20251676, at Wuhan University, China.

[0044] The obtained thermophilic streptococcus was named thermophilic streptococcus ( Streptococcus thermophilus HJA09, the obtained Lactobacillus plantarum was named Lactobacillus plantarum ( Lactiplantibacillus plantarum HJB73, the obtained lactococcus was named Lactococcus lactis ( Lactococcus sp.)HJG02.

[0045] Example 2: Changes in the active ingredients of purple ginseng, sweet potato, or yam after fermentation with Bifidobacterium longum HJ-005 I. Experimental Methods 1. The purple yam originated from Hunan Province, and the Chinese yam originated from Jiaozuo, Henan Province. Both are iron-stick Chinese yam, and the edible tubers were used for the experiment. Fresh purple yam or Chinese yam were washed, blanched, peeled, and sliced ​​into thin slices 2-3 mm thick. 50 g of the slices were accurately weighed and immersed in 100 mL of 20% (mass concentration) citric acid aqueous solution at 90℃ for color protection. After 5 min, the sample was removed to obtain the color-protected sample. Water (3 times the volume of the color-protected sample) was added, and the sample was pulverized in a tissue homogenizer. Then, it was gelatinized in a 95℃ water bath for 30 min. Saccharifying enzyme was added according to the fresh sample weight at a dosage of 150 U / g (enzyme activity is defined as: 1 U refers to the amount of enzyme that decomposes soluble starch to produce 1 mg of glucose in 1 h under pH 4.6 and 60℃ conditions). Saccharification was carried out at 60℃ for 3 h. After cooling, the sample was homogenized in a high-pressure homogenizer to obtain the purple yam matrix or Chinese yam matrix. Add 0.5% (final mass concentration) glucose to the purple ginseng and sweet potato substrate or yam substrate, inoculate with 5% (volume concentration), and grow to the logarithmic phase (strain OD). 600 A value of 8 indicates a colony count greater than 1 × 10⁻⁶. 8 The seed culture of Bifidobacterium longum HJ-005 (number / mL) was incubated at 37℃ for 24 h to obtain the fermentation broth of purple ginseng and sweet potato or yam.

[0046] The optimal fermentation time for yam was 22 hours, at which point the viable count of Bifidobacterium longum reached a peak of 1.2 × 10⁻⁶. 9With a CFU / mL concentration and a pH of 4.2, the fermented yam broth exhibits a pleasant yam aroma without excessive acidification or bitterness. The optimal fermentation time for purple ginseng sweet potato is 24 hours, with a peak viable count of 9.5 × 10⁻⁶ cells / mL. 8 With a concentration of CFU / mL, a pH of 4.1, and an anthocyanin retention rate of ≥85%, the fermented broth of purple ginseng sweet potato is naturally purplish-red, possessing both fruity and milky aromas, and exhibiting the highest retention rate of polyphenols.

[0047] 2. The contents of diosgenin, allantoin, organic selenium, and arginine in the raw materials of purple ginseng sweet potato or yam and the fermentation liquid of purple ginseng sweet potato or yam were tested respectively.

[0048] (1) Raw material pretreatment Take fresh purple ginseng sweet potato or yam slices (2-3 mm thick) after washing and peeling, place them in a vacuum drying oven at 45℃ and dry them to constant weight (moisture content ≤5%), pulverize them and pass them through a 60-mesh sieve to obtain purple ginseng sweet potato raw material powder or yam raw material powder for later use.

[0049] Accurately weigh 2.0 g of purple ginseng and yam raw material powder, place them separately in stoppered Erlenmeyer flasks, add 50 mL of 2 mol / L hydrochloric acid solution, shake well, reflux in an 85℃ water bath for 2 h, cool to room temperature, adjust the pH to neutral with sodium hydroxide solution, and transfer to a separatory funnel. Add 50 mL of petroleum ether (boiling range 60~90℃), shake and extract for 15 min, allow to stand and separate into layers, and collect the upper organic phase; repeat the extraction 3 times, and combine the organic phases. Dehydrate the combined organic phase through an anhydrous sodium sulfate column, collect the filtrate; concentrate to dryness using a rotary evaporator at 45℃, dissolve the residue in methanol and transfer to a 10 mL volumetric flask, dilute to the mark with methanol, and filter through a 0.45 μm organic phase filter membrane to obtain the sample solution to be tested before fermentation, for subsequent analysis.

[0050] (2) Pretreatment methods for fermentation broth (adapted to the complex substrate characteristics of the fermentation system) The fermentation broth contains impurities such as bacterial cells, polysaccharides, and proteins, which need to be specifically removed. The pretreatment steps are as follows: Accurately measure 10 mL of purple ginseng and sweet potato fermentation broth or yam fermentation broth, place it in a centrifuge tube, centrifuge at 8000 r / min for 15 min, discard the precipitate (to remove bacterial cells and undegraded starch granules), and collect the supernatant. Add 5% (volume concentration) trichloroacetic acid solution to the supernatant until the final concentration is 1% (volume concentration), shake well, and let stand for 30 min to precipitate proteins; centrifuge again at 8000 r / min for 15 min, collect the supernatant, and adjust the pH to neutral with sodium hydroxide solution.

[0051] Add 50 mL of 2 mol / L hydrochloric acid solution, shake well, and reflux in an 85℃ water bath for 2 h. Cool to room temperature, adjust the pH to neutral with sodium hydroxide solution, and transfer to a separatory funnel. Add 50 mL of petroleum ether (60~90℃), shake and extract for 15 min, allow to stand for separation, and collect the upper organic phase. Repeat the extraction three times and combine the organic phases. Dehydrate the combined organic phases through an anhydrous sodium sulfate column and collect the filtrate. Concentrate to dryness by rotary evaporation at 45℃. Dissolve the residue in methanol and transfer to a 10 mL volumetric flask, dilute to the mark with methanol, and filter through a 0.45 μm organic phase filter membrane to obtain the post-fermentation sample solution for subsequent analysis.

[0052] (3) The content of diosgenin in purple yam or yam before fermentation and the content of diosgenin in fermentation liquid after fermentation were detected according to the method in the literature (HPLC analysis of allantoin and diosgenin content in yam with different fertilizers [J]. Chinese Medicinal Herbs, 2014, 37(4):558-560).

[0053] Following the method described in the literature (Analysis of total polysaccharide and allantoin content in yam from different origins [J]. Global Chinese Medicine, 2016, 9(3):295-298), the allantoin content in purple ginseng sweet potato or yam before fermentation and the allantoin content in the fermentation liquid after fermentation were determined.

[0054] Following the method described in the literature (Research on the determination method of organic selenium in food, Shaanxi Agricultural Sciences, 2014, Vol. 2, pp. 35-37), the organic selenium content in purple ginseng sweet potato or yam before fermentation and the organic selenium content in the fermentation broth after fermentation were determined.

[0055] The arginine content in purple yam or yam before fermentation was determined according to the method described in the literature (Analysis and comprehensive evaluation of amino acid composition of different varieties of yam, Journal of Food Safety and Quality Testing, 2024, Vol. 15, No. 3, pp. 293-300).

[0056] II. Experimental Results Table 1. Changes in various active ingredients over time in the fermentation broth of purple ginseng, sweet potato, or yam fermented with Bifidobacterium longum HJ-005. Table 2. Changes in various active ingredients in the fermentation broth of purple ginseng, sweet potato, or yam fermented with Bifidobacterium longum HJ-005. As shown in Tables 1 and 2, fermentation of yam with *Bifidobacterium longum* HJ-005 promoted the production of diosgenin, allantoin, organic selenium, and arginine, reaching 1.1, 2.3, 1.7, and 2.9 times higher than the unfermented form, respectively. Fermentation of purple sweet potato with *Bifidobacterium longum* HJ-005 showed even more significant increases, reaching 10.1, 17.3, 132.1, and 18.3 times higher than the unfermented form, respectively. p <0.0001). The contents of diosgenin, allantoin, organic selenium, and arginine showed a trend of first increasing and then decreasing with fermentation time. After exceeding the optimal time, the contents of active ingredients showed a slow decreasing trend due to excessive acidification of the fermentation system and inhibition of strain activity.

[0057] Compared to yam, purple yam is more suitable for fermentation with Bifidobacterium longum HJ-005. Under the experimental conditions of this invention, Bifidobacterium longum HJ-005 enters the stable phase after 18-22 hours of fermentation. During yam fermentation, the maximum OD of Bifidobacterium longum HJ-005 is... 600 The value reached 5.7, while the maximum OD of Bifidobacterium longum HJ-005 during the fermentation of purple ginseng sweet potato was... 600 The value reached 9.4, an increase of nearly 64.9%. Under anaerobic fermentation conditions, *Bifidobacterium longum* HJ-005 secretes a series of enzymes (such as β-glucosidase and saponins) to decompose macromolecules in yam, including diosgenin, in order to grow and reproduce. More vigorous *Bifidobacterium longum* HJ-005 secretes more enzymes to promote the release and transformation of active substances in purple yam. Furthermore, the increased growth of *Bifidobacterium longum* HJ-005 also partially promotes the production of amino acids and organic selenium. In conclusion, compared to yam, purple yam is more suitable for the growth, transformation, and fermentation of *Bifidobacterium longum* HJ-005.

[0058] Example 3: Effects of Bifidobacterium longum HJ-005 and other lactic acid bacteria on the active ingredients of fermented purple ginseng sweet potato I. Experimental Methods Bifidobacterium longum HJ-005 was replaced with Streptococcus thermophilus HJA09, Lactobacillus plantarum HJB73 or Lactococcus lactis HJG02, and the purple ginseng sweet potato was fermented according to the method of Example 2. The changes of each active ingredient were then detected.

[0059] II. Experimental Results Table 3. Changes in various active ingredients in the fermentation broth of purple sweet potato fermented by different strains. As shown in Tables 2 and 3, all four lactic acid bacteria strains increased the content of various active ingredients in the fermentation broth of purple ginseng and sweet potato, but the degree of increase varied. Relatively speaking, the increase was most significant with Bifidobacterium HJ-005. p The value is <0.05, which may be related to the special physiological characteristics of Bifidobacterium longum, making it most suitable for fermentation of purple ginseng and sweet potato.

[0060] Example 4: Efficacy test of fermentation broth from purple ginseng sweet potato fermented with Bifidobacterium longum HJ-005 I. Experimental Methods 1. Moisturizing effect (zebrafish embryo water loss inhibition test) Twenty-four 3-day-old zebrafish embryos were exposed to fermentation broth samples of purple sweet potato fermented with Bifidobacterium longum HJ-005 (sample treatment group). At the same time, a model control group (15 g / L NaCl), a positive control group (glycerol, 0.5 g of glycerol dissolved in 1000 mL of water), and a blank control group (fish embryo culture medium) were set up. After 3 h of exposure, the fish embryos were photographed under a microscope to measure the tail area and perform statistical analysis.

[0061] Preparation of fish embryo culture medium: Weigh 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride and dissolve them in 10 L of water to prepare the medium with a pH of 6.5~8.5.

[0062] 2. Anti-aging effects (zebrafish elastin and type I collagen test) Thirty-six 6-day-old zebrafish were divided into three groups and exposed to fermentation broth samples of purple sweet potato fermented with Bifidobacterium longum HJ-005 (sample treatment group). A blank control group (fish embryo culture medium) was set up at the same time. After 24 h of exposure, RNA was extracted from the zebrafish, reverse transcribed into cDNA, and real-time PCR amplification was performed using β-actin as the housekeeping gene. The Ct value was used as the amplification result, and the relative expression levels of col1a1a, col1a1b, col1a2 and Elna were calculated and statistically analyzed.

[0063] PCR amplification system (20 μL): 10 μL SYBR Green Master Mix, 0.6 μL forward primer (10 μM), 0.6 μL reverse primer (10 μM), 2 μL cDNA template, 6.8 μL RNase-free water. Primer sequences are shown in Table 4.

[0064] PCR program: Pre-denaturation: 95℃ for 3 min; Cyclic reaction: 95℃ for 10 s, annealing temperature adjusted according to primer Tm value (60℃ for β-actin, col1a1a, and col1a2, 62℃ for col1a1b), 30 s, for a total of 40 cycles; Melting curve: 95℃ for 15 s, 60℃ for 1 min, 95℃ for 15 s (to verify primer specificity).

[0065] Table 4 II. Experimental Results 1. For example Figure 3 A, Figure 3 B Figure 3 C Figure 3 D and Figure 4 As shown, the moisturizing effect in the model control group was not significant (0% tail area reduction inhibition rate), the moisturizing effect in the positive control group was significant (30% tail area reduction inhibition rate), and the moisturizing effect in the sample treatment group was significant (66% tail area reduction inhibition rate).

[0066] 2. For example Figure 5 As shown, the sample treatment group promoted the expression of zebrafish col1a1a, col1a1b, and col1a2 genes by 64% ( p =0.00029), 40% p =0.0072) and 53% ( p =0.0013), which can significantly promote the expression of type I collagen gene in zebrafish, promote the regeneration of type I collagen, and has anti-aging effects.

[0067] like Figure 6 As shown, the sample treatment group promoted the expression of the Elna gene in zebrafish by 48% ( p =0.0020), can significantly promote the expression of the Elna gene in zebrafish, promote elastin regeneration, and has anti-aging effects.

[0068] This embodiment uses a zebrafish model to confirm that the fermentation broth of purple ginseng sweet potato fermented with Bifidobacterium longum HJ-005 has significant moisturizing and anti-aging effects, providing experimental evidence for its application in the cosmetics field.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A type of Bifidobacterium longum, characterized in that, The nucleotide sequence of the 16S rRNA of the aforementioned Bifidobacterium longum is shown in SEQ ID NO:

1.

2. A fermentation product of Dioscorea opposita, characterized in that, It is obtained by fermenting Dioscorea opposita with Bifidobacterium longum as described in claim 1.

3. The fermented product of Dioscorea opposita as described in claim 2, characterized in that, The yam plants mentioned include purple yam and / or Chinese yam.

4. The method for preparing the fermented product of Dioscorea opposita according to claim 2 or 3, characterized in that, After color preservation and gelatinization of the yam plant, it is inoculated with Bifidobacterium longum as described in claim 1 and fermented.

5. The preparation method according to claim 4, characterized in that, Fermentation time is 18-26 hours.

6. The use of the Bifidobacterium longum as described in claim 1, or the fermentation product of Dioscorea opposita as described in claim 2 or 3, in the preparation of cosmetics.

7. The application as described in claim 6, characterized in that, The cosmetics mentioned are skin moisturizing and / or skin anti-aging cosmetics.

8. The application as described in claim 7, characterized in that, The skin anti-aging treatment includes increasing the expression levels of elastin and / or collagen in the skin.

9. A cosmetic product, characterized in that, The cosmetic contains the yam plant fermentation product as described in claim 2 or 3.

10. A method for increasing the nutrient content in fermented products of Dioscorea opposita, characterized in that, Dioscorea plants are fermented with the Bifidobacterium longum as described in claim 1; the nutrients include at least one of diosgenin, allantoin, organic selenium, and arginine.

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