Pine pollen leavening with anti-wrinkle and anti-aging functions as well as preparation method and application of pine pollen leavening

Abstract

The invention provides a pine pollen fermentation product with anti-wrinkle and anti-aging functions as well as a preparation method and application thereof, and belongs to the technical field of cosmetics. The pine pollen extract is prepared by fermenting lactobacillus plantarum, the total polysaccharide content of the obtained pine pollen fermentation product is maximally increased by 77.3%, the FRAP total antioxidant capacity is maximally increased by 2.85 times compared with that of the unfermented pine pollen fermentation product, the anti-wrinkle effect of human skin cells can be effectively improved, and the pine pollen extract has a relatively strong proliferation effect on an ultraviolet damage model of human skin fibroblasts; the pine pollen extract has the advantages that the pine pollen extract is fermented, the elastin content is remarkably increased, the life of nematodes is effectively prolonged, lipofuscin deposition is reduced, and compared with unfermented pine pollen extract, the anti-aging capacity of the pine pollen can be effectively improved; the preparation method provided by the invention is low in price and simple in process, and the active ingredients and the efficacy of the traditional Chinese medicine composition are improved in a simplest and effective manner.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cosmetics, and particularly relates to a pine pollen ferment with anti-wrinkle and anti-aging functions, a preparation method thereof and application thereof. BACKGROUND

[0002] Pine pollen (English: POLLEN PINI) is also known as pine pollen and pine yellow, which refers to the dry pollen produced by the anthers of Pinus massoniana, Pinus tabulaeformis, Pinus koraiensis, Pinus armandii and Pinus sylvestris. It was first recorded in the Eastern Han Dynasty "Shennong's Herbal Classic", and is a fine powder of fresh yellow or light yellow, with a sweet taste and no toxicity. It is a traditional Chinese food. In addition to general nutrients such as protein, fat and carbohydrates, it also contains more than 200 nutrients and bioactive substances such as vitamins, trace elements, polyphenols, flavonoids, enzymes, fatty acids and coenzymes. It has broad development and application prospects. However, most of the active substances, such as insoluble bound polyphenols, are usually covalently complexed with structural components of the cell wall, such as cellulose, hemicellulose, lignin, protein, polysaccharide, pectin, etc., and are not easily released. Microbial fermentation produces enzymes to break covalent bonds and promote the release of active ingredients.

[0003] For example, Chinese patent CN115537344A discloses a process for improving the content and activity of antioxidant substances in pine pollen by Rhizopus oryzae fermentation, which specifically includes the following steps: configuration of pine pollen liquid culture medium, activation and inoculation of Rhizopus oryzae, and fermentation to obtain fermentation products. The method provided by the application uses Rhizopus oryzae fermentation to improve the content of free active substances such as polyphenols and flavonoids in pine pollen, and to improve the antioxidant activity. Through fermentation to produce enzymes, the active substances combined with macromolecules in the cells of pine pollen are released, the content of free polyphenols and flavonoids in broken pine pollen is increased, and the antioxidant capacity of broken pine pollen is improved.

[0004] For another example, Chinese patent CN114081854A discloses a pine pollen extract with barrier repair function, which is prepared by fermentation of Lactobacillus plantarum. The Lactobacillus plantarum has a preservation number of CCTCC NO: M20211332 and is preserved in the China Center for Type Culture Collection. The pine pollen extract provided by the application introduces Lactobacillus plantarum, which can effectively promote the transformation and decomposition of protein components in the pine pollen extract, effectively improve the barrier repair function of the pine pollen while ensuring safety, and can be applied to skin care products with keratin repair function. However, the preparation process of the application involves extraction and enzyme hydrolysis, and carbon dioxide gas is used for fermentation culture, which is complex and expensive, and is not easy to industrialize.

[0005] For example, Chinese patent CN114177121A discloses a method for preparing a pine pollen probiotic fermented cosmetic raw material. The preparation process comprises the following steps: breaking the cell wall and crushing the raw material; complex enzyme hydrolysis; sterilization treatment; inoculation of one or more mixed strains of Lactobacillus rhamnosus, Lactobacillus paracasei, Streptococcus thermophilus, Bacillus amyloliquefaciens, Bacillus subtilis, Lactobacillus plantarum, and yeast; fermentation treatment; and then secondary leaching treatment by separation and precipitation, and combination of the supernatant to obtain a pine pollen probiotic fermentation liquid. The process can extract active substances from pine pollen, increase the content of effective components, and produce a pine pollen probiotic fermented cosmetic with antioxidant function and the ability to inhibit melanin production, thereby achieving skin whitening.

[0006] However, the flavonoid content and polysaccharide content, especially the polysaccharide content, of the pine pollen extract obtained by the prior art cannot meet the requirements well. Therefore, it is necessary to develop a pine pollen fermentation product with anti-wrinkle and anti-aging functions. SUMMARY

[0007] Based on the deficiencies in the prior art, the present application aims to provide a pine pollen fermentation product with anti-wrinkle and anti-aging functions, a preparation method thereof, and applications thereof.

[0008] To achieve the above-mentioned purposes, the present application adopts the following technical solutions: On the one hand, the present application provides a pine pollen fermentation product with anti-wrinkle and anti-aging functions. The pine pollen extract is prepared by fermentation of Lactobacillus plantarum. The Lactobacillus plantarum has a preservation number of CICC 22195 and is purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0009] On the other hand, the present application also provides a preparation method of the above-mentioned pine pollen fermentation product, comprising the following steps: (1) adding pine pollen to a fermentation medium, adjusting the pH value, and sterilizing to obtain a sterilized culture medium; (2) inoculating the sterilized culture medium with bacterial liquid and culturing at 37°C for 24 h to obtain a fermentation liquid, which is ready for use; (3) centrifuging the fermentation liquid and taking the supernatant, which is the pine pollen fermentation product.

[0010] In the above step (1), the fermentation medium comprises 10 g / L of glucose, 3 g / L of potassium dihydrogen phosphate, and 1.5 g / L of magnesium sulfate heptahydrate.

[0011] In the above step (1), the addition amount of pine pollen is 0.2%-2%. Preferably, the addition amount of pine pollen is 1%.

[0012] In the above step (1), the pH value is 4-8. Preferably, the pH value is 6.

[0013] The sterilization temperature in step (1) is 110-121℃, preferably 115℃; and the sterilization time is 10-30min, preferably 20min.

[0014] The inoculation amount of the strain in step (2) is 1-10%; Preferably, the inoculation amount of the strain is 5%.

[0015] The viable cell count of the bacterial solution is 1×10 9 CFU / g-2×10 9 CFU / g.

[0016] The centrifugation speed in step (3) is 3000-5000r / min, and the time is 10-20min. Preferably, the centrifugation speed is 5000r / min, and the time is 10min.

[0017] In another aspect, the application also provides the use of the pine pollen fermentation product in the preparation of a skin care product, food or health food with anti-wrinkle and anti-aging functions.

[0018] A skin care product with anti-wrinkle and anti-aging functions, comprising the pine pollen fermentation product and a skin care product auxiliary.

[0019] The skin care product auxiliary is selected from one or more of emollients, emulsifiers, suspending agents, thickening agents, humectants, antioxidants and fragrances.

[0020] A food with anti-wrinkle and anti-aging functions, comprising the pine pollen fermentation product and a food auxiliary.

[0021] The food auxiliary is selected from one or more of acidity regulators, anti-caking agents, antioxidants, colorants, color protection agents, emulsifiers, flavor enhancers, nutritional enhancers, preservatives, stabilizers, sweeteners, thickening agents and food fragrances.

[0022] A health food with anti-wrinkle and anti-aging functions, comprising the pine pollen fermentation product and a health food auxiliary.

[0023] The health food auxiliary is selected from one or more of acidity regulators, anti-caking agents, antioxidants, colorants, color protection agents, emulsifiers, flavor enhancers, nutritional enhancers, preservatives, stabilizers, sweeteners, thickening agents and food fragrances.

[0024] Compared with the prior art, the application has the following advantages: (1) The preparation method of the pine pollen fermentation provided by the present application is to directly add Lactobacillus plantarum fermentation to pine pollen, which is low in price and simple in process, and uses the simplest and most effective way to improve the active ingredients and efficacy.

[0025] (2) The Lactobacillus plantarum provided by the present application can effectively decompose macromolecular substances in pine pollen, release active ingredients, and after fermentation by Lactobacillus plantarum, the total polysaccharide content is increased by 77.3%, and the total antioxidant capacity of FRAP is increased by 2.85 times at most compared with that before fermentation. (2) The pine pollen fermentation broth produced in the present application can effectively improve the anti-wrinkle effect of human skin cells, has a strong proliferation effect on the ultraviolet damage model of human skin fibroblasts, and has a very significant effect on increasing the content of elastin. (3) The pine pollen fermentation broth prepared in the present application can effectively improve the lifespan of nematodes and reduce the deposition of lipofuscin, and compared with unfermented pine pollen extract, it can effectively improve the anti-aging ability of pine pollen. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a cell safety concentration column chart of unfermented pine pollen sample; Figure 2 is a cell safety concentration column chart of pine pollen Lactobacillus plantarum fermentation broth sample; Figure 3 is the influence of unfermented pine pollen sample on the proliferation activity of HFF-1 cell anti-wrinkle model; In the figure, “##” represents a very significant difference from the blank group, p<0.01, and “**” represents a very significant difference compared with the model group, p<0.01.

[0027] Figure 4 is the influence of pine pollen Lactobacillus plantarum fermentation broth sample on the proliferation activity of HFF-1 cell anti-wrinkle model; In the figure, “##” represents a very significant difference from the blank group, p<0.01, and “**” represents a very significant difference compared with the model group, p<0.01.

[0028] Figure 5 is the influence of unfermented pine pollen sample on the elastin of HFF-1 cell anti-wrinkle model; In the figure, “##” represents a very significant difference from the blank group, p<0.01, and “**” represents a very significant difference compared with the model group, p<0.01.

[0029] Figure 6 is the influence of pine pollen Lactobacillus plantarum fermentation broth sample on the elastin of HFF-1 cell anti-wrinkle model; In the figure: "##" represents a significant difference from the blank group, p<0.01, "**" indicates a significant difference compared with the model group, p<0.01.

[0030] Figure 7 The figure is a schematic diagram of the effect of the unfermented pine pollen sample on the life of the nematode; Figure 8 The figure is a schematic diagram of the effect of the pine pollen plant lactobacillus fermentation liquid sample on the life of the nematode; Figure 9 The figure is a column chart of the average individual lipofuscin fluorescence intensity of each sample. DETAILED DESCRIPTION

[0031] The technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0033] The plant lactobacillus used in the following embodiments has the preservation number CICC 22195; the lactobacillus casei used has the preservation number CICC 23184; and the lactobacillus rhamnosus used has the preservation number CICC 23119, all of which are purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0034] Example 1: Pine pollen fermentation product with anti-wrinkle and anti-aging functions and preparation method thereof The preparation method is: (1) The fermentation medium used is: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pine pollen 1%, pH=6, 115°C high-temperature high-pressure sterilization for 20 min, to obtain the sterilized culture medium; (2) 5% of the plant lactobacillus seed liquid is added to the culture medium obtained in step (1), and cultured at 37°C for 24 h to obtain the fermentation liquid; (3) The fermentation liquid is centrifuged at 5000 r / min for 10 min in a centrifuge, and the supernatant is the pine pollen fermentation product.

[0035] Example 2: Pine pollen fermentation product with anti-wrinkle and anti-aging functions and preparation method thereof The preparation method is: (1) using fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pine pollen 0.5%, pH=5, 110℃ high temperature and high pressure sterilization 25 min, to obtain the sterilized medium; (2) adding 2.5% of Lactobacillus plantarum seed liquid to the medium obtained in step (1), and culturing at 37℃ for 24h to obtain a fermentation broth; (3) taking the fermentation broth to a centrifuge at 5000 r / min for 10 min, and the supernatant is pine pollen fermentation product.

[0036] Example 3: A pine pollen fermentation product with anti-wrinkle and anti-aging functions and a preparation method thereof The preparation method is: (1) using fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pine pollen 8%, pH=6, 120℃ high temperature and high pressure sterilization 10 min, to obtain the sterilized medium; (2) adding 8% of Lactobacillus plantarum seed liquid to the medium obtained in step (1), and culturing at 37℃ for 24h to obtain a fermentation broth; (3) taking the fermentation broth to a centrifuge at 5000 r / min for 10 min, and the supernatant is pine pollen fermentation product.

[0037] Comparative Example 1: A pine pollen fermentation product with anti-wrinkle and anti-aging functions and a preparation method thereof The preparation method is: (1) using fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pine pollen 1%, pH natural, 115℃ high temperature and high pressure sterilization 20 min, to obtain the sterilized medium; (2) adding 5% of Lactobacillus casei seed liquid to the medium obtained in step (1), and culturing at 37℃ for 24h to obtain a fermentation broth; (3) taking the fermentation broth to a centrifuge at 5000 r / min for 10 min, and the supernatant is pine pollen fermentation product.

[0038] Comparative Example 2: A pine pollen fermentation product with anti-wrinkle and anti-aging functions and a preparation method thereof The preparation method is: (1) using fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pine pollen 1%, pH natural, 115℃ high temperature and high pressure sterilization 20 min.

[0039] (2) Add 5% Lactobacillus rhamnosus seed liquid to the sterilized culture medium of (1), and culture at 37°C for 24 h for standby.

[0040] (3) Centrifuge the fermented broth at 5000 r / min for 10 min, and take the supernatant.

[0041] Comparative Example 3 The difference from Example 1 is that pine pollen not fermented by bacteria is used.

[0042] Comparative Example 4 The difference from Example 1 is that pine pollen is not added during fermentation, that is, the fermentation medium is used: glucose 10 g / L, potassium dihydrogen phosphate 3 g / L, magnesium sulfate heptahydrate 1.5 g / L, pH natural, 115°C high-temperature high-pressure sterilization for 20 min to obtain the sterilized culture medium; add 5% Lactobacillus plantarum seed liquid to the sterilized culture medium, and culture at 37°C for 24 h to obtain the fermented broth, centrifuge the fermented broth at 5000 r / min for 10 min, and take the supernatant, which is the blank fermentation product.

[0043] Comparative Example 5 A pine pollen extract is prepared according to the method of Example 2 in the prior art CN114081854A.

[0044] Effect detection Test Example 1: Determination of hydroxyl radical scavenging capacity: The ABBKINE hydroxyl radical detection kit is used for detection.

[0045] Test Example 2: Determination of FRAP total antioxidant capacity: The Biyun Tian FRAP total antioxidant capacity detection kit is used for detection.

[0046] Test Example 3: Determination of polysaccharide content: (1) Sample treatment: Take 2 ml of sample in a 15 mL centrifuge tube. Add 10 mL of 80% anhydrous ethanol, and use a vortex shaker to shake to mix evenly, and ultrasonic wash for 30 min. After washing, centrifuge at 3000 rpm for 10 min, and discard the supernatant. The insoluble material is washed with 10 mL of 80% ethanol solution and centrifuged. The above insoluble material is dissolved in 5 mL of water, and ultrasonic washing is performed for 30 min until the precipitate is completely dissolved. This solution is the sample determination solution.

[0047] (2) Preparation of standard curve: Take 0 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL of the standard glucose working solution respectively into a 10 mL test tube, and make up to 1.0 mL with distilled water. Add 1.0 mL of phenol solution to the test solution, then quickly add 5.0 mL of sulfuric acid, and stand for 10 min. Mix the reaction solution thoroughly using a vortex oscillator, then place the test tube in a 30 ℃ water bath for 20 min, and measure the absorbance at 490 nm. Prepare a standard curve with the mass concentration of glucose as the abscissa and the absorbance value as the ordinate.

[0048] (3) Determination method: Take 1 mL of the sample determination solution into a 10 mL test tube, and operate according to step (2) to determine the absorbance.

[0049] (4) Calculation: The content of polysaccharide in the sample is calculated according to formula (2) in terms of mass fraction ω, and the unit is mg / mL.

[0050] Formula (2) In the formula: m1: the content of sugar in the sample determination solution (μg) calculated from the standard curve; V1: the sample determination volume (mL); V2: the volume of the sample determination solution taken for absorbance determination (mL); m2: the sample amount (mL); 0.9: the correction coefficient for converting glucose into glucan.

[0051] Test Example 4: Determination of flavonoid content: The second method for determining total flavonoids in health foods was used for detection.

[0052] The detection results are shown in Table 1 below.

[0053] Table 1

[0054] According to the detection results in Table 1 above, the total polysaccharide and flavonoid contents in the pine pollen fermentation products obtained after fermentation by Lactobacillus plantarum in Examples 1-3 were all increased to different extents, and in particular, the polysaccharide content in the pine pollen fermentation product obtained in Example 1 increased more obviously, which was 1.45 times higher than that of unfermented pine pollen, and far exceeded the total polysaccharide and flavonoid contents in the pine pollen fermentation products after fermentation by other strains in Comparative Examples 1-2. The polysaccharide content in the pine pollen fermentation product obtained in Example 1 was 42.1% higher than that in Comparative Example 5, and the flavonoid content was 1.16 times higher than that in Comparative Example 3, and 80.5% higher than that in Comparative Example 5.

[0055] The antioxidant capacity of the pine pollen ferment after fermentation by Lactobacillus plantarum in Examples 1-3 was also significantly increased. The FRAP total antioxidant capacity of the pine pollen ferment obtained in Example 1 was 3.23 times higher than that of Comparative Example 3, 21.6% higher than that of Comparative Example 5, and the hydroxyl radical scavenging capacity was 20.3% higher than that of Comparative Example 3 and 42.2% higher than that of Comparative Example 5. Although the active ingredients and antioxidant capacity of the fermented pine pollen increased to different degrees, the pine pollen fermented by Lactobacillus plantarum was the most effective, greatly improving the active ingredients and having a very significant effect on antioxidant efficacy.

[0056] Test Example 5: Evaluation of Cell Anti-wrinkle Efficacy HFF-1 human fibroblasts were used for the experiment, and CCK-8 method was used for cell toxicity and cell proliferation detection, and enzyme-linked immunosorbent assay kit (enzyme-linked bio) was used for elastin content detection.

[0057] (1) Establishment of HFF-1 cell anti-wrinkle model HFF-1 cells with a growth density of 80%-90% were inoculated in a 96-well plate at a cell density of 5×10 3 / well (100 μL per well), and cultured for 24 h. Discard the old liquid, rinse the cells with PBS once, add 100 μL of PBS per well, and irradiate with UVB at 300 mJ / cm 2 Modeling, at least 6 replicate wells per dose group, UVB irradiation dose calculated according to formula (3): Irradiation dose (mJ / cm 2 ) = irradiation intensity (mW / cm 2 ) × irradiation time (s) formula (3) After irradiation, discard the PBS, and add 100 uL of culture medium per well, and continue to culture in the incubator. After 24 h of culture, the cell viability was detected by CCK-8 method; repeat the experiment 3 times.

[0058] (2) Sample processing The samples were dissolved and prepared with ultrapure water, and after filtration, the experiment was performed after gradient dilution with complete culture medium.

[0059] Table 2

[0060] (3) Determination of cell anti-wrinkle proliferation activity HFF-1 cells with a growth density of 80%-90% were inoculated in a 96-well plate at a cell density of 5×10 3 / well (100 μL per well), and cultured in a cell incubator.

[0061] After 24 h of culture, the cells were randomly divided into a blank control group, a UVB model group, and different concentrations of sample + UVB groups. The old liquid was discarded, the cells were rinsed once with PBS, 100 μL of drug-containing culture solution was added, the blank control group was covered with aluminum foil, and UVB irradiation was performed at 300 mJ / cm 2 After modeling, the cells were placed in a cell culture incubator and cultured for 24 h before determining the cell activity.

[0062] Before determination, the old liquid was discarded, the cells were rinsed twice with PBS, 100 μL of complete culture medium containing 10% CCK-8 solution was added to each well, and the absorbance at 450 nm was detected by a microplate reader after incubation at 37°C for 1.5, 2.0, 2.5, or 3 h (as needed for experimental exploration). The experiment was repeated three times. The relative activity of the cells (or the relative proliferation rate of the cells) was calculated according to formula (4): Cell relative activity = (A laboratory - A blank group) / (A control group - A blank group) x 100% The detection results are shown in Figures 3-4 .

[0063] As can be seen from Figures 3-4 , both pine pollen before and after fermentation have a significant anti-wrinkle effect, but unfermented pine pollen only has a very significant effect at a concentration of 5% and 1.25%, with a maximum increase of 7.9% compared with the model group. After fermentation by Lactobacillus plantarum, all concentrations have a very significant effect, and the maximum increase is 28.07% compared with the model group. Therefore, Lactobacillus plantarum fermented pine pollen can effectively improve its anti-wrinkle effect and has a significant effect.

[0064] (4) Determination of the content of elastin in the cell anti-wrinkle model HFF-1 cells with a growth density of 80%-90% were inoculated at a cell density of 5 x 10 3 / well (100 μL per well) in a 24-well plate and placed in a cell culture incubator for culture. The remaining operation methods were the same as above.

[0065] When determining, the supernatant was taken and determined according to the human elastin (ELN) detection kit.

[0066] The detection results are shown in Figures 5-6 .

[0067] As can be seen from Figures 5-6It can be seen that the unfermented pine pollen has a very significant effect on the cell elastin of the anti-wrinkle model at a concentration of 10%, and compared with the model group, the elastin content is increased by 10.26%; after fermentation by Lactobacillus plantarum, all concentrations have the effect of increasing elastin, and at 10% it reaches the highest, which is increased by 18.35% compared with the model group. It can be seen that Lactobacillus plantarum fermented pine pollen can increase the effect of increasing elastin content at a smaller concentration, and the effect is more significant than before fermentation.

[0068] Test Example 6: Evaluation of nematode anti-aging ability (1) Sample information Table 3

[0069] (2) Sample processing Each sample was diluted to 5 mg / mL with appropriate ultrapure water, sterilized by membrane, and mixed with OP50 bacterial solution to a final concentration of 50 μg / mL.

[0070] 6.1 Evaluation of nematode lifespan (1) Sample grouping ① Blank control group (CK): 30 nematodes were cultured on NGM medium coated with OP50 bacterial solution.

[0071] ② Each sample group: 30 nematodes were diluted and mixed with OP50 bacterial solution, and then coated on NGM medium to culture nematodes, with a sample concentration of 50 μg / mL.

[0072] (2) Experimental operation ① Randomly select synchronized L4 stage nematodes and divide them into 5 groups according to the description in 6.1 (1), of which 3 sample groups and 1 positive group are given bacterial solution containing corresponding drugs for culture; according to the synchronization time of 0 days, change the plate every 2 days, and track the survival status of nematodes.

[0073] ② When the nematodes do not react to the worm picker by repeated gentle touching, the nematodes are recorded as dead. When the nematodes die by crawling on the wall or die in the worm bag, they are recorded as lost. Pick worms every day until all die, and the results are statistically analyzed and plotted using Prism software.

[0074] The detection results are shown in Table 4 and Figures 7-8 .

[0075] Table 4 Life extension rate of each sample group

[0076] According to the detection results, it can be seen that the samples before and after fermentation can prolong the life of nematodes to a certain extent, and Lactobacillus plantarum itself also has a certain effect on prolonging the life of nematodes. The nematode life extension rate of unfermented pine pollen is 4.47%, after fermentation by Lactobacillus plantarum, the strain and pine pollen produce the effect of bidirectional fermentation, and the highest life extension rate can reach 15.47%, which is 2.46 times higher than that of comparative example 3, and 86.16% higher than that of comparative example 5, and is significantly higher than that of other strains, which has a strong anti-aging effect.

[0077] 6.2 Nematode lipofuscin deposition evaluation (1) Sample grouping ① Blank control group: 15 nematodes were cultured on NGM medium coated with OP50 bacterial solution.

[0078] ② Each sample group: 15 nematodes were cultured on NGM medium after each sample was appropriately diluted and mixed with OP50 bacterial solution, and the sample concentration was 50 μg / mL.

[0079] (2) Experimental operation ① Randomly select synchronized L4 stage nematodes, divide them into 5 groups according to the description in 6.1, and give the corresponding drug-containing bacterial solution to the 3 sample groups and 1 positive group for culture; according to the time of 0 days of synchronization, change the plate every 2 days, and feed for 10 days, then pick out the nematodes, rinse them with M9 for 3 times, centrifuge, and remove the supernatant; ② Slide preparation: prepare a glass slide containing 5% agarose gel, 10 μl 5mM L-methazol in the center of the agar, and then pick the nematodes into the agar for anesthesia, and gently cover the cover glass for observation and photography.

[0080] ③ Photography: observe the autofluorescence of lipofuscin under an inverted fluorescence microscope and take pictures.

[0081] ④ Capture the pictures and analyze the autofluorescence content of lipofuscin using Image J.

[0082] SPSS 23.0, Origin 9.0 and Excel 2016 software were used for data analysis, all data were expressed as mean ± standard deviation (x ± s), independent sample T test was used for comparison, and p<0.05 was considered statistically significant.

[0083] The detection results are shown in Table 5 and Figure 9 .

[0084] Table 5 Nematode lipofuscin deposition reduction rate after treatment of each sample group

[0085] ​According to the detection results, the deposition of lipofuscin in nematodes can be reduced to a large extent before and after fermentation, among which the effect of sample Lactobacillus plantarum fermentation is the most outstanding, and the reduction rate is as high as 48.18%, which is 48.66% higher than that of Comparative Example 3 and 1.08 times higher than that of Comparative Example 5. The effect is very significant. Therefore, Lactobacillus plantarum fermented pine pollen has a better effect of reducing the deposition of nematode lipofuscin, further highlighting its anti-aging effect.

[0086] In summary: After fermentation by Lactobacillus plantarum, the active ingredients and key effects are improved. Among them, the polysaccharide content increases more obviously, the polysaccharide content increases by 1.45 times at most, and the flavonoid content increases by 1.16 times at most; the antioxidant capacity also increases significantly, and the total antioxidant capacity of FRAP is increased by 3.23 times at most compared with that before fermentation; the cell anti-wrinkle proliferation activity is increased by 2.55 times at most compared with that before fermentation; and the effect of nematode lipofuscin deposition is increased by 48.66% at most. Therefore, Lactobacillus plantarum fermented pine pollen has a significant effect of improving the content of active ingredients and anti-wrinkle and anti-aging effect of pine pollen.

Claims

1. A pine pollen ferment with anti-wrinkle and anti-aging functions, characterized in that: The pine pollen extract is obtained by fermentation with Lactobacillus plantarum; the preparation method includes the following steps: (1) Add pine pollen to the fermentation medium, adjust the pH value, and then sterilize to obtain the sterilized medium. (2) Inoculate the sterilized culture medium with bacterial solution and incubate at 37°C for 24 hours to obtain fermentation broth for later use; (3) After centrifuging the fermentation liquid, take the supernatant, which is the pine pollen fermentation product.

2. The pine pollen fermentation product according to claim 1, characterized in that: The amount of pine pollen added in step (1) is 0.2%-2%, preferably 1%.

3. The pine pollen fermentation product according to claim 1, characterized in that: The pH value mentioned in step (1) is 4-8, preferably 6.

4. The pine pollen fermentation product according to claim 1, characterized in that: The sterilization temperature in step (1) is 110℃-121℃, and the sterilization time is 10min-30min.

5. The pine pollen fermentation product according to claim 1, characterized in that: The inoculation amount of the strain mentioned in step (2) is 1-10%, preferably 5%.

6. The pine pollen fermentation product according to claim 1, characterized in that: The viable count of the bacterial solution mentioned in step (2) is 1×10⁻⁶. 9 CFU / g-2×10 9 CFU / g.

7. The pine pollen fermentation product according to claim 1, characterized in that: The centrifugation speed in step (3) is 3000-5000 r / min, and the time is 10-20 min.

8. The use of the pine pollen ferment as described in any one of claims 1-7 in the preparation of skin care products, food or health food with anti-wrinkle and anti-aging functions.

9. A skincare product with anti-wrinkle and anti-aging functions, characterized in that: This includes the pine pollen fermentation product and its excipients as described in any one of claims 1-7.

10. The skincare product according to claim 9, characterized in that: The excipients are selected from one or more of emollients, emulsifiers, suspending agents, thickeners, moisturizers, antioxidants, and fragrances.

11. A food product with anti-wrinkle and anti-aging functions, characterized in that: This includes the pine pollen fermentation product and its food products as described in any one of claims 1-7.

12. The food product according to claim 11, characterized in that: The excipients are selected from one or more of the following: acidity regulators, anticaking agents, antioxidants, colorants, color protectants, emulsifiers, flavor enhancers, nutritional fortifiers, preservatives, stabilizers, sweeteners, thickeners, and food flavorings.

13. A health food product with anti-wrinkle and anti-aging functions, characterized in that: The ingredients include the pine pollen fermentation product and its health food products as described in any one of claims 1-7.

14. The health food according to claim 13, characterized in that: The excipients are selected from one or more of the following: acidity regulators, anticaking agents, antioxidants, colorants, color protectants, emulsifiers, flavor enhancers, nutritional fortifiers, preservatives, stabilizers, sweeteners, thickeners, and food flavorings.

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