Method for the synergistic conversion of pomegranate polyphenols by lactic acid bacteria and bifidobacterium infantis and the resulting fermented product composition

Abstract

The present application belongs to the technical field of biological fermentation engineering, and provides a method for synergistically converting pomegranate polyphenols by lactic acid bacteria and infant bifidobacterium and a fermented product composition prepared by the method, the method comprising: inoculating lactic acid bacteria and infant bifidobacterium into a pomegranate extract solution, performing primary fermentation to obtain a fermentation product; readjusting the pH value of the fermentation product, and then inoculating Gordan anaerobes to perform secondary fermentation to obtain a pomegranate fermentation product composition containing urolithin A.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation engineering technology, and relates to a method for the synergistic transformation of pomegranate polyphenols by lactic acid bacteria and Bifidobacterium infantis, and the resulting fermentation product composition. Background Technology

[0002] Pomegranate fruit is rich in punicalin, a compound that is the most abundant and most active ellagitannin polyphenol in pomegranates, with a molecular weight of approximately 1084 Da and extremely strong antioxidant potential. However, due to its large molecular weight, punicalin has a very low skin absorption rate and is difficult to be directly absorbed by the gastrointestinal tract after oral administration, resulting in severely insufficient bioavailability.

[0003] Urolithin A is the core metabolite of punicalin under the influence of gut microbiota, with the chemical structure 3,8-dihydroxydibenzo[b,d]pyran-6-one. Studies have shown that urolithin A can promote the clearance of damaged mitochondria in aging cells, inhibit the abnormal accumulation of reactive oxygen species (ROS), and significantly promote the synthesis of type I collagen by activating the mitophagy pathway, thus demonstrating outstanding efficacy in delaying skin aging. However, some individuals lack specific strains in their gut microbiota capable of converting ellagic acid into urolithin A through dehydroxylation, and this conversion ability significantly declines with age and metabolic diseases.

[0004] Currently, the acquisition of urolithin A faces the following technical bottlenecks: (1) Chemical synthesis method: Urolithin A is prepared by chemical dehydroxylation of ellagic acid, but the product obtained is a mixture of racemic compounds and has poor transdermal absorption, which poses a safety hazard and cannot meet the requirements of high purity and high safety of cosmetic raw materials.

[0005] (2) Biotransformation by a single strain: Existing technologies mostly use anaerobic bacteria of the genus *Gordonella* to directly convert ellagic acid to produce urolithin A (e.g., *Streptococcus thermophilus* FUA329, conversion rate 82%, patent CN115747098A; *Lactobacillus plantarum* CCTCC AB2013128, patent CN202411214357). However, these strains lack potent tannin hydrolases and cannot directly degrade bound tannins, resulting in extremely low urolithin A yields when using pomegranate extract (rich in punicin rather than free ellagic acid) as a substrate.

[0006] (3) Simple co-culture: Lactic acid bacteria and anaerobic bacteria are simultaneously inoculated into the substrate for mixed fermentation. However, due to the acid production by lactic acid bacteria, the pH of the environment drops rapidly, and the dehydroxylation enzyme system of anaerobic bacteria is inactivated under acidic conditions, which severely limits the conversion rate of urolithin A. Experimental data show that the conversion rate of urolithin A in traditional three-strain mixed fermentation is only about 18%, which is far lower than expected.

[0007] (4) Targeted regulation of gut microbiota: Improve gut microbiota structure in people with type B / O by supplementing with specific probiotics (patent CN202310583571), but this scheme is for oral health care purposes and cannot directly obtain standardized raw materials for external skin use.

[0008] In summary, the existing technology lacks a complete technical solution for efficiently producing a urolithin A fermentation product composition through biotransformation using pomegranate whole fruit extract (rich in macromolecular tannins) as a substrate, which can be directly applied to topical anti-aging skin preparations. Summary of the Invention

[0009] To address the shortcomings of existing technologies, the present invention aims to provide a method for the synergistic transformation of pomegranate polyphenols by lactic acid bacteria and Bifidobacterium infantis, and the resulting fermentation product composition.

[0010] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for the co-transformation of pomegranate polyphenols by lactic acid bacteria and Bifidobacterium infantis, the method comprising: Lactic acid bacteria and Bifidobacterium infantis were inoculated into a pomegranate extract solution for primary fermentation to obtain a fermentation product. The pH of the fermentation product was adjusted, and then anaerobic bacteria of the genus *Gordonella* were inoculated for secondary fermentation to obtain a pomegranate fermentation product composition containing urolithin A.

[0011] This invention uses pomegranate whole fruit extract (containing more than 40% punicalin) as a substrate, and sequentially performs primary and secondary fermentation. During the primary fermentation stage, lactic acid bacteria (preferably *Lactobacillus plantarum*, accession number CCTCC M 208150, Chinese patent document CN101748083A) and *Bifidobacterium infantis* (accession number CGMCC No. 32401, Chinese patent document CN122208461A) are simultaneously inoculated. The lactic acid bacteria are responsible for hydrolyzing large molecular weight tannins, cleaving punicalin (molecular weight approximately 1084 Da) into ellagic acid and oligoellagic tannin fragments, reducing the molecular weight of the substrate and expanding the accessible substrate library. *Bifidobacterium infantis* is the key innovative strain of this invention. The *Bifidobacterium infantis* specific strains screened by the screening method provided by this invention have the following unique functions: (1) This specific strain has a strong survival ability in extreme environments. Punicanthin is the most abundant polyphenol compound in pomegranate extract, and it has a certain inhibitory effect on microorganisms. The higher the concentration, the more obvious the inhibitory effect. In the fermentation process, the starting substrate of the primary fermentation contains a high concentration of punicanthin. If the inoculated strain cannot tolerate the high tannin environment, its growth and metabolic activities will be severely inhibited, and it will not be able to perform the pre-modification function normally. The specific strain of Bifidobacterium infantis screened in this invention has a tolerance concentration of punicanthin of more than 5 mg / mL, and its survival rate in the high tannin environment exceeds 92%. In contrast, the survival rate of the commonly used Bifidobacterium bifidum under the same conditions is only about 48%, and the difference between the two is obvious. High tolerance means that this specific strain can quickly adapt to the substrate environment in the early stage of fermentation, maintain normal cell activity and metabolic function, and provide sufficient cell quantity and enzyme activity reserves for subsequent pre-modification reactions.

[0012] (2) Ellagic acid intermediates produced after hydrolysis by lactic acid bacteria have multiple hydroxyl groups in their molecular structure. The arrangement and spatial configuration of these hydroxyl groups directly affect the recognition and binding efficiency of subsequent Gordon's anaerobic dehydroxylases. Ordinary Bifidobacterium strains lack enzyme systems for targeted modification of ellagic acid intermediates. Therefore, the intermediates obtained after treatment by lactic acid bacteria and ordinary Bifidobacterium strains still retain their original molecular configuration and have low affinity for dehydroxylases. However, the specific strains screened in this invention have unique phenolic hydroxyl modification enzyme systems that can hydroxylate or esterify ellagic acid intermediates, changing the hydroxyl distribution in their molecular structure. The modified intermediates have changed molecular configuration, making them more compatible with the binding sites of Gordon's anaerobic dehydroxylases, thereby improving the efficiency and selectivity of the dehydroxylation reaction.

[0013] (3) During fermentation, Bifidobacterium infantis decomposes the substrate and grows while secreting various organic acids into the fermentation broth, mainly L-lactic acid and acetic acid. These organic acids are not only products of normal bacterial metabolism, but also play an important auxiliary role in the final fermentation product composition. The specific strain screened in this invention has a significantly stronger organic acid secretion capacity than ordinary strains, and the total organic acid content produced by its fermentation reaches 12.4 mmol / L, which is about 134% higher than that of ordinary strains. The increase in organic acid content has two effects. On the one hand, an appropriate amount of organic acid can regulate the pH environment of the fermentation system, providing a clear signal for subsequent pH adjustment operations. On the other hand, in the final fermentation product composition, L-lactic acid and acetic acid have a certain cuticle softening effect, which can improve the transdermal absorption efficiency of active ingredients to a certain extent and form a natural permeation-enhancing matrix. This permeation-enhancing component produced by the strain's own metabolism avoids the need for artificial addition of chemical permeation enhancers in the later stage, ensuring the natural properties and safety of the product.

[0014] (4) During the later stages of fermentation, some Bifidobacterium infantis cells undergo autolysis, releasing internal active substances after the cell walls and cell membranes rupture. β-glucan is an important component of the Bifidobacterium cell wall and is released into the fermentation broth during cell autolysis. The specific strains screened in this invention release approximately 58% more β-glucan after cell autolysis than ordinary strains. β-glucan is an active substance with moisturizing and skin barrier repair functions, which can promote the proliferation and differentiation of keratinocytes, enhance the skin barrier function, and reduce moisture loss. In the fermentation product composition, β-glucan, urolithin A, organic acids, and other components form a multi-component synergistic system. The barrier repair function of β-glucan and the anti-aging function of urolithin A work together to give the fermentation product composition a more comprehensive effect in skin care. The natural release of β-glucan by the strain through autolysis eliminates the need for additional extraction and addition steps, simplifying the production process while ensuring the natural activity of β-glucan.

[0015] The second stage involves inoculating the fermentation system with *Goldenella* anaerobic bacteria. The role of *Goldenella* in the secondary fermentation is to dehydroxylate the intermediate product pre-modified by *Bifidobacterium infantis*, generating urolithin A. When this strain is introduced into the fermentation system, the fermentation broth has already undergone pH adjustment, with the pH value adjusted to the range of 6.5–7.0, providing suitable environmental conditions for the activity of the dehydroxylation enzyme system. *Goldenella* anaerobic bacteria are strict anaerobes; their growth and metabolic activities require anaerobic conditions. Therefore, the secondary fermentation process must be completed in a strictly anaerobic environment. During the secondary fermentation, *Goldenella* anaerobic bacteria utilize their unique dehydroxylation enzyme system to recognize and act on the pre-modified ellagic acid intermediate product, selectively removing hydroxyl groups at specific positions, transforming the molecular structure of the intermediate product into the chemical structure of urolithin A. The dehydroxylation reaction is the final step in the urolithin A biosynthetic pathway. The efficiency of the dehydroxylation reaction is affected by various factors, the most important of which are environmental pH and oxygen concentration. If the pH adjustment is insufficient or timed improperly, residual organic acids will gradually inhibit dehydroxylase activity, leading to a decrease in the conversion rate. If anaerobic conditions are not strictly controlled, oxygen entering the fermentation system will directly inhibit the activity of the dehydroxylase system, and may even cause enzyme inactivation. As fermentation time increases, intermediate products are gradually consumed, the concentration of urolithin A continuously increases, and the composition of other metabolites in the fermentation broth also continuously changes. When the substrate is completely consumed, the conversion reaction stops, the secondary fermentation ends, and the obtained fermentation product is a fermentation product composition containing urolithin A and other fermentation metabolites.

[0016] As a preferred embodiment of the present invention, the lactic acid bacteria is *Lactobacillus plantarum*.

[0017] The Bifidobacterium infantis strain is a specific strain with accession number CGMCC No. 32401.

[0018] As a preferred technical solution of the present invention, the temperature of the primary fermentation is 35℃~40℃, for example, it can be 35℃, 35.5℃, 36℃, 36.5℃, 37℃, 37.5℃, 38℃, 38.5℃, 39℃, 39.5℃ or 40℃, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0019] In some optional instances, the duration of the primary fermentation is 20 to 28 hours, for example, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, or 28 hours, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0020] Primary fermentation plays a core role in substrate pretreatment, and its function is mainly reflected in two aspects: first, degrading macromolecular pomegranate polyphenols into small molecule intermediates; and second, chemically modifying these intermediates for subsequent transformation.

[0021] The addition of lactic acid bacteria primarily addresses the challenge of directly utilizing large-molecule tannins. The most abundant active ingredient in pomegranate extract is punicalin, a high-molecular-weight ellagic tannin compound. Due to its complex molecular structure and high molecular weight, it presents significant obstacles to both skin absorption and microbial metabolism. Lactic acid bacteria secrete tannin-hydrolyzing enzymes that cleave the large molecular structure of punicalin, generating ellagic acid and smaller oligomeric ellagic tannin fragments. This hydrolysis reduces the substrate's molecular weight, transforming the originally tightly packed, difficult-to-access, and difficult-to-utilize large-molecule polyphenols into smaller molecules that are more easily recognized and converted by subsequent bacterial strains. Without the hydrolytic action of lactic acid bacteria, subsequent anaerobic bacteria would face insufficient substrate accessibility, severely impacting conversion efficiency.

[0022] Bifidobacterium infantis plays a pre-modification role. Although the molecular weight of the ellagic acid intermediates produced after hydrolysis by lactic acid bacteria has decreased, their chemical structure is not yet optimally suited to the action of dehydroxylases from *Goldenella* anaerobic bacteria. *Bifidobacterium infantis* possesses a unique phenolic hydroxyl-modifying enzyme system, capable of hydroxylating or esterifying these intermediates to generate structurally specific intermediate metabolites. The modified intermediates undergo changes in molecular conformation, increasing their affinity for dehydroxylases and making them more readily recognized and transformed by subsequent anaerobic bacteria.

[0023] During primary fermentation, in addition to the substrate conversion reactions mentioned above, the chemical composition of the fermentation system also changes. Lactic acid bacteria and Bifidobacterium infantis secrete various metabolites during their growth and metabolism, the most important of which are organic acids such as L-lactic acid and acetic acid. As fermentation time increases, organic acids accumulate, causing the pH of the fermentation broth to gradually decrease. Moderate acidification helps inhibit the growth of other microorganisms and maintain the purity of the fermentation system; however, excessively low pH can inhibit the dehydroxylation enzymes of subsequently introduced Gordon's bacteria anaerobic bacteria. Therefore, the pH needs to be adjusted after primary fermentation. Furthermore, both bacteria release secondary metabolites such as β-glucan, extracellular polysaccharides, and various amino acids during metabolism. The presence of these substances enriches the chemical composition of the final fermentation product, endowing the product with additional functions beyond urolithin A.

[0024] As a preferred technical solution of the present invention, the temperature of the secondary fermentation is 30℃~40℃, for example, it can be 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0025] In some optional instances, the total duration of the primary fermentation and the secondary fermentation is 72 hours.

[0026] The core function of secondary fermentation is to convert the intermediate products produced by primary fermentation into the target product urolithin A. The Gordonella anaerobic bacteria introduced at this stage are strict anaerobic bacteria with dehydroxylase activity. The dehydroxylase system of Gordonella anaerobic bacteria can recognize and act on polyphenol intermediate products that have been hydrolyzed by lactic acid bacteria and pre-modified by Bifidobacterium infantis. These intermediate products contain multiple hydroxyl groups in their molecular structure. Gordonella anaerobic bacteria can selectively remove hydroxyl groups at specific positions, converting ellagic acid into urolithin A.

[0027] During the secondary fermentation process, as the metabolic activity of *Gordonella* anaerobic bacteria continues, intermediate products are gradually consumed, and the concentration of urolithin A continuously increases. Simultaneously, residual organic acids and other metabolites in the fermentation system also continuously affect the activity of *Gordonella* anaerobic bacteria. If pH adjustment is timely and sufficient, the dehydroxylase system of *Gordonella* anaerobic bacteria can maintain high activity for a longer period, allowing urolithin A accumulation to continue until the substrate is completely consumed. Conversely, if pH adjustment is insufficient or at an inappropriate time, residual organic acids will gradually inhibit dehydroxylase activity, leading to premature termination of the conversion reaction, and affecting the final yield and conversion rate of urolithin A. The fermentation product obtained after the secondary fermentation is the fermentation product composition, which contains urolithin A, organic acids, β-glucan, and other metabolites produced during fermentation, and can be directly used for subsequent product development and applications.

[0028] As a preferred technical solution of the present invention, the fermentation environment of the primary fermentation and the secondary fermentation is an anaerobic environment.

[0029] As a preferred technical solution of the present invention, an alkaline solution is added dropwise to the primary fermentation product to adjust its pH value back to 6.5~7.0, for example, it can be 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0, but it is not limited to the listed values. Other unlisted values ​​within this range are also applicable.

[0030] The reason for adjusting the pH of the fermentation product is that the dehydroxylase system of the subsequently introduced Gordon's anaerobic bacteria is sensitive to acidic environments. Gordon's anaerobic bacteria are responsible for converting the pre-modified intermediate product into urolithin A. This conversion process depends on the catalytic activity of their dehydroxylase system, which exhibits higher activity under neutral to slightly alkaline conditions and is inhibited under acidic conditions. If Gordon's anaerobic bacteria are directly introduced into the unadjusted acidic fermentation broth, the dehydroxylase system cannot effectively perform its catalytic function, and the conversion efficiency of urolithin A will be significantly reduced.

[0031] As a preferred embodiment of the present invention, the Bifidobacterium infantis is obtained by screening using the following method: (a) Isolation of Bifidobacterium strains from infant gut samples; (b) The isolated Bifidobacterium strains were cultured in a medium with a pungent glycoside concentration of 5 mg / mL for 24 h, and strains with a survival rate of ≥60% were selected as the initial screening strains. (c) The primary screening strains were cultured for 24 h in a high-concentration medium with a pungent glycoside concentration of 7 mg / mL, and for 2 h in a medium containing bile salts at pH 3; strains with a survival rate of ≥60% in both the high-concentration medium and the bile salt medium were selected as candidate strains. (d) The lactic acid bacteria and the candidate strain were inoculated into a pomegranate extract solution for primary fermentation to obtain a primary fermentation product; the pH of the primary fermentation product was adjusted, and then anaerobic bacteria of the genus *Gordonella* were inoculated for secondary fermentation to obtain a secondary fermentation product; the conversion rate of urolithin A in the secondary fermentation product was detected, and strains with a urolithin A conversion rate ≥80% were screened as the *Bifidobacterium infantis*.

[0032] In this invention, Bifidobacterium strains are first isolated from samples derived from the infant gut, which is a natural habitat for Bifidobacterium, especially in breastfed infants where the number and variety of Bifidobacterium are abundant. Isolating strains from this source yields a large number of Bifidobacterium strains with potential functional diversity, providing ample candidates for subsequent screening.

[0033] Subsequently, the isolated strains were cultured in a medium with a pungent glycoside concentration of 5 mg / mL, and strains with a survival rate of not less than 60% were selected as primary screening strains. This step establishes the first tolerance screening criterion. Pomegranate extract contains high concentrations of pungent glycoside, a polyphenolic compound that inhibits many microorganisms. Only strains that can maintain a high survival rate in this high-polyphenol environment are likely to function normally in subsequent fermentation processes. By screening out strains sensitive to pungent glycoside, the candidate pool is narrowed down.

[0034] Two tolerance tests were then conducted: first, the strains were cultured for 24 hours in a high-concentration medium with 7 mg / mL punicalin; second, they were cultured for 2 hours in a medium containing bile salts at pH 3. Strains exhibiting a survival rate of at least 60% under both conditions were selected as candidate strains. This step further refined the screening criteria, examining the strains' tolerance from two dimensions. The high-concentration punicalin test assessed the strains' survival ability at higher substrate concentrations, which is related to the stability of the process in the face of substrate concentration fluctuations. The acid-bile salt test simulated the gastrointestinal environment, examining the strains' survival ability in an acidic environment and in the presence of bile salts, which is directly related to whether the strains can maintain their activity in oral applications.

[0035] Finally, validation was conducted in a real fermentation scenario. Lactic acid bacteria and candidate strains were inoculated into a pomegranate extract solution for primary fermentation, followed by pH adjustment, and then secondary fermentation was performed using Gordon's bacteria (anaerobic bacteria). The conversion rate of urolithin A in the secondary fermentation product was measured, and strains with a conversion rate of not less than 80% were selected as the final fermentation strains. The previous screening steps focused on the strains' tolerance indicators, but these indicators do not directly reflect the strains' performance in a complete two-stage fermentation process. By placing candidate strains in a real fermentation process and using the final conversion rate of urolithin A as the evaluation criterion, the actual performance of the candidate strains can be directly measured. This ensures that the finally selected strains not only have good tolerance but also play an effective pre-modification role in synergistic work with lactic acid bacteria and Gordon's bacteria, achieving a high urolithin A conversion rate.

[0036] In a second aspect, the present invention provides a fermentation product composition prepared by the method described in the first aspect, characterized in that the fermentation product composition contains urolithin A, organic acids and β-glucan.

[0037] In some optional examples, the urolithin A content in the fermentation product composition is 300-500 mg / L, for example, it can be 300 mg / L, 320 mg / L, 340 mg / L, 360 mg / L, 380 mg / L, 400 mg / L, 420 mg / L, 440 mg / L, 460 mg / L, 480 mg / L or 500 mg / L, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0038] In some optional instances, the total organic acid content in the fermentation product composition is ≥10 mmol / L, for example, it may be 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, 17 mmol / L, 18 mmol / L, 19 mmol / L or 20 mmol / L, but is not limited to the listed values, other unlisted values ​​within this range are also applicable.

[0039] In some alternative examples, the fermentation product composition contains β-glucan at a concentration ≥0.5 mg / mL, such as 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, or 1.5 mg / mL, but is not limited to the listed values; other unlisted values ​​within this range are also applicable.

[0040] The fermentation product composition provided by this invention can be used to prepare topical anti-aging agents for the skin, functional foods, or dietary supplements.

[0041] In the field of topical anti-aging preparations, the most prominent advantage of fermentation product compositions lies in their naturally formed multi-component synergistic penetration-enhancing system. Although traditional urolithin A monomers possess the core functions of activating mitochondrial autophagy and promoting collagen synthesis, their transdermal absorption rate is extremely low. In in vitro porcine skin experiments, the cumulative transdermal penetration of pure urolithin A over 24 hours was only 11.4 μg / cm³. 2 Furthermore, most of these substances remain in the stratum corneum and struggle to reach the dermis to exert their effects. However, the fermented product composition contains high concentrations of L-lactic acid and acetic acid produced by the fermentation of Bifidobacterium infantis. These organic acids can temporarily open the lipid barrier channels of the stratum corneum. Simultaneously, the network structure formed by β-glucan and extracellular polysaccharides can act as a drug carrier, promoting the delivery of urolithin A to deeper tissues. This results in the fermented product composition obtained in this invention having a dermal retention rate 4.45 times that of the pure urolithin A group, and a 6.5-fold increase in the drug distribution ratio between the dermis and stratum corneum. By generating penetration-enhancing components through fermentation itself, cosmetic manufacturers can achieve highly efficient delivery of active ingredients without adding additional chemical penetration enhancers. Moreover, all penetration-enhancing components are natural fermentation products, offering far superior safety and gentleness compared to artificially synthesized penetration enhancers.

[0042] In the field of functional foods and dietary supplements, the advantage of fermented product compositions lies in their complete spectrum of gut-friendly metabolites. The specific strain of *Bifidobacterium infantis* produced up to 15.6 mmol / L of total organic acids during fermentation, with L-lactic acid and acetic acid reaching 8.24 mmol / L and 4.81 mmol / L, respectively. It also synthesized 628 μg / mL of β-glucan and 301 μmol / L of γ-aminobutyric acid (GABA). These components themselves have clear gut health benefits: organic acids can lower intestinal pH and inhibit the growth of harmful bacteria; β-glucan can act as a prebiotic to promote the proliferation of *Bifidobacterium* and *Lactobacillus*; and GABA has soothing and anti-inflammatory effects on intestinal smooth muscle. More importantly, the urolithin A in the fermented product composition is generated through natural fermentation, rather than a chemically synthesized racemic mixture, resulting in higher bioavailability and safety. For individuals whose gut microbiota lacks anaerobic strains of the genus *Gordonella* and who are unable to convert pomegranate polyphenols into urolithin A, directly ingesting the fermentation product composition is equivalent to skipping the complex intestinal metabolic process and obtaining metabolic end products with anti-aging activity in one step.

[0043] The fermentation product composition actually has an intrinsic connection between these two seemingly different application areas. Whether applied topically or taken orally, its core advantage stems from the multiple active ingredients generated by the same fermentation process. In the skin, organic acids and β-glucan play a role in promoting penetration and repairing the skin barrier; in the gut, the same components play a role in regulating the gut microbiota and repairing the mucous membrane. The fact that a single product achieves dual efficacy in different areas gives the fermentation product composition obtained in this invention extremely high industrialization value. Cosmetic companies can use it to develop anti-aging serums that do not require separation and purification, while health supplement companies can use it to develop functional foods targeting gut health and overall anti-aging. The raw material sources for both product lines are exactly the same, greatly reducing the complexity of supply chain management and production costs.

[0044] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) In the prior art, the bioconversion of urolithin A using pomegranate whole fruit extract as a substrate has always faced difficulties. The main reason is that punicalin has a large molecular weight and complex structure, and single strains or simple mixed fermentation cannot effectively utilize this type of large molecular polyphenol. The prior art usually can only use pre-hydrolyzed small molecular ellagic acid as a substrate, or use chemical synthesis methods, but these methods have problems such as high cost, poor safety or low conversion rate. The present invention uses a two-stage fermentation process to enable lactic acid bacteria, Bifidobacterium infantis and Gordon's bacteria to play different roles. Lactic acid bacteria are responsible for hydrolyzing the large molecular punicalin into small molecular intermediates, Bifidobacterium infantis pre-modifies the intermediates, and Gordon's bacteria finally complete the dehydroxylation reaction to generate urolithin A. This makes the pomegranate whole fruit extract, which was originally difficult to utilize, a highly efficient bioconversion substrate, and the conversion rate of urolithin A is increased from 18.2% in the prior art to 82.6%, which is about 4.5 times higher.

[0045] (2) In this invention, Bifidobacterium infantis not only plays a pre-modification role of polyphenols, but its metabolic activities also produce a variety of secondary metabolites. During fermentation, Bifidobacterium infantis secretes substances such as L-lactic acid, acetic acid, and β-glucan while decomposing the substrate. L-lactic acid and acetic acid have a certain stratum corneum softening effect, which can improve the transdermal absorption efficiency of active ingredients to a certain extent; β-glucan has moisturizing and barrier repair functions. The fermentation product composition containing these secondary metabolites has a transdermal absorption efficiency that is 3.4 times higher than that of pure urolithin A monomer. This indicates that the secondary metabolites in the fermentation product composition not only do not affect the absorption of urolithin A, but also promote its distribution and retention in skin tissue through synergistic effects. The secondary metabolites generated by the fermentation process itself eliminate the need for the later addition of penetration enhancers or moisturizing ingredients, ensuring the natural properties and safety of the product.

[0046] (3) During the primary fermentation process, the metabolic activities of lactic acid bacteria and Bifidobacterium infantis lead to a large accumulation of organic acids, and the pH value of the fermentation broth continues to decrease. The acidic environment has little effect on the normal metabolism of lactic acid bacteria and Bifidobacterium infantis, but it has a significant inhibitory effect on the dehydroxylase system of anaerobic bacteria of the genus *Gordonella*. Therefore, it is necessary to adjust the pH value of the fermentation product after the primary fermentation is completed to ensure the full progress of the primary fermentation and to avoid damage to the dehydroxylase system by lactic acid.

[0047] (4) The fermentation product obtained after fermentation is a complex mixture, containing not only the target product urolithin A, but also L-lactic acid, acetic acid, β-glucan, γ-aminobutyric acid, extracellular polysaccharides, and incompletely converted intermediates. In traditional production processes, obtaining high-purity urolithin A monomer requires a series of purification steps such as extraction, concentration, and chromatographic separation. These steps increase production costs, reduce overall yield, and introduce safety issues such as residual organic solvents. The fermentation product composition prepared by this invention can be directly used as a raw material for cosmetics and topical anti-aging skin preparations without the need for separate separation and purification of urolithin A. This is because other components in the fermentation product composition have skin care functions, and these components do not interfere with urolithin A; on the contrary, they can synergistically enhance each other's effects. Directly using the fermentation product as the raw material for the final product greatly simplifies the production process, reduces equipment investment and energy consumption, shortens the production cycle, and retains all the active ingredients produced during fermentation, making the final product more functional.

[0048] (5) The screening method for Bifidobacterium infantis provided by this invention includes four core steps: strain isolation, initial screening for tolerance, secondary screening under dual stress, and verification of fermentation function. Each step has clear screening criteria and quantifiable evaluation indicators. The operation process is standardized and easy to replicate and implement under different laboratory and production conditions. For subsequent strain improvement work, researchers can use this screening framework to obtain strain variants with better performance by changing the screening pressure intensity, adjusting the evaluation indicator threshold, or introducing new screening dimensions. For example, strains with stronger tolerance can be screened by increasing the concentration of pungent glycosides or extending the culture time, and strains with better pre-modification ability can be screened by adjusting the timing of pH adjustment or secondary fermentation conditions. In addition, the evaluation system established by this screening method can also be extended to the screening of other functional strains, providing a methodological reference for the development of similar microbial fermentation processes. Attached Figure Description

[0049] Figure 1 This is a schematic diagram of the fermentation process provided in Embodiment 1 of the present invention; Figure 2 A comparative graph showing the effects of different fermentation modes on the conversion rate of urolithin A; Figure 3 A comparison of the survival rates of infantile Bifidobacterium specific strain (Strain B) and general Bifidobacterium bifidum in a high-tannin environment; Figure 4 This is a comparison of the cumulative transdermal permeation of the fermentation product composition prepared in this invention and pure urolithin A reference standard in an isolated pig skin model over 24 hours. Figure 5This is a comparison of the 24-hour dermal retention of the fermentation product composition prepared in this invention and pure urolithin A reference standard in an isolated pig skin model. Figure 6 A comparison of the LC3-II / I ratios in different treatment groups in a UVA-induced photoaging model of human primary fibroblasts (HDF). Figure 7 A comparison of ROS levels in different treatment groups in a UVA-induced photoaging model of human primary fibroblasts (HDF). Figure 8 This is a comparison of type I collagen secretion levels in different treatment groups in a UVA-induced photoaging model of human primary fibroblasts (HDF). Detailed Implementation

[0050] The technical solutions of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings. The embodiments described herein are specific implementations of the present invention, used to illustrate the concept of the present invention; these descriptions are explanatory and exemplary, and should not be construed as limiting the implementation methods or the scope of protection of the present invention. In addition to the embodiments described herein, those skilled in the art can employ other obvious technical solutions based on the content disclosed in the claims and specification of this application. These technical solutions include those that make any obvious substitutions and modifications to the embodiments described herein.

[0051] Example 1: Comparison of Urolithin A Conversion Rates Using Different Fermentation Processes 1.1 Matrix Preparation Accurately weigh natural pomegranate extract powder containing 40% (w / w) punicin (purchased from Shaanxi Angsheng Biotechnology Co., Ltd., batch number AS-PE-20230912), prepare an aqueous solution with a total polyphenol concentration of 10 mg / mL (calculated as gallic acid equivalent, Folin-Ciocalteu method) with distilled water, and autoclave at 121℃ for 15 minutes for later use.

[0052] 1.2 Activation and Counting of Microbial Strains Lactobacillus plantarum (CCTCC M 208150): After anaerobic culture in MRS liquid medium (Difco, USA) at 37°C for 18 hours, the viable count was (9.63±0.41)×10⁻⁶ using the 10-fold serial dilution plate count method (MRS + 0.05% L-cysteine, anaerobic culture for 48 hours). 8 CFU / mL; Bifidobacterium infantis (CGMCC No. 32401): After anaerobic culture at 37°C for 24 hours in modified MRS medium containing 0.05% L-cysteine, the viable count was (8.17±0.58)×10⁻⁶. 8CFU / mL; Gordonibacter sp. (Gord-B3), anaerobes of the genus *Gordonibacter*, was cultured anaerobically at 37°C for 72 hours in PY liquid medium containing 0.5 g / L L-cysteine. The viable count was (4.08 ± 0.33) × 10⁻⁶. 6 CFU / mL; Counter-evidence control strain: Bifidobacterium breve ATCC 15700, viable count (7.24±0.49)×10⁻⁶ 8 CFU / mL; Bifidobacterium bifidum ATCC 29521, viable count (8.83±0.62)×10⁻⁶ 8 CFU / mL; Bifidobacterium longum subsp. infantis ATCC15697, viable count (7.91±0.55)×10⁻⁶ 8 CFU / mL.

[0053] 1.3 Group Design (n=3 per group, independent inoculation / fermentation / sampling): Group 1 (Simultaneous Mixed Fermentation): Three strains (Lactobacillus plantarum, Bifidobacterium infantis, and Gordoniae anaerobic bacteria) were simultaneously inoculated into the substrate at 2% (v / v) at 0h, and anaerobic fermentation was carried out at 37℃ for 72h without pH adjustment. Group 2 (The fermentation process provided by this invention, such as...) Figure 1 As shown): At 0h, inoculate with *Lactobacillus plantarum* and *Bifidobacterium infantis* (2% v / v each); after culturing for 24h, adjust the pH of the primary fermentation product to 6.8 ± 0.1 with sterile 1M NaOH solution; then inoculate with Gord-B3 (2% v / v) and continue fermentation for 72h; Group 3 (without Bifidobacterium infantis): Lactobacillus plantarum (2% v / v) was inoculated at 0h; after culturing for 24h, the pH of the primary fermentation product was adjusted back to 6.8±0.1 with sterile 1M NaOH solution; then Gord-B3 (2% v / v) was inoculated and fermentation continued for 72h; Group 4 (without Lactobacillus plantarum): Bifidobacterium infantis (2% v / v) was inoculated at 0h, and after culturing for 24h, the pH of the primary fermentation product was adjusted back to 6.8±0.1 with sterile 1M NaOH solution; then Gord-B3 (2% v / v) was inoculated and fermentation continued for 72h. Group 5 (pH delayed by 30h): The operation is the same as Group 2, but the pH adjustment is delayed to 30h; that is: at 0h, inoculate with Lactobacillus plantarum and Bifidobacterium infantis (2% v / v each); after culturing for 24h, inoculate with Gord-B3 (2% v / v) and continue fermentation to 72h; at 30h, adjust the pH value to 6.8±0.1 (at this time, it is already in the Gord-B3 fermentation stage); Group 6 (Gord-B3 only): Gord-B3 (2% v / v) was directly added at 0h and cultured for 72h without pH adjustment.

[0054] 1.4 Detection Methods At 0h, 6h, 12h, 18h, 24h, 30h, 36h, 48h, 60h, and 72h, 2mL samples were aseptically collected from each group, centrifuged at 12000r / min for 10min (4℃), and the supernatant was filtered through a 0.22μm filter membrane. The concentration of urolithin A was determined by HPLC. HPLC conditions: InertSustain C18 column (250mm×4.6mm, 5μm, GL Sciences); mobile phase methanol:0.1% formic acid water = 40:60 (v / v); flow rate 1.0mL / min; detection wavelength 305nm; column temperature 30℃; each sample was measured twice in parallel, and the mean value was taken.

[0055] 1.5 The conversion rate of urolithin A over 72 hours was measured (mean ± variance, n=3). The results are shown in Table 1. Table 1 ; Statistical analysis (Student's t-test, two-tailed): Group 2 vs. Group 1, t=57.4, **P<0.001; Group 2 vs. Group 3, t=88.1, **P<0.001; Group 2 vs. Group 4, t=71.3, **P<0.001; Group 2 vs. Group 5, t=38.9, **P<0.001; Group 2 vs. Group 6, t=102.7, **P<0.001; Group 3 vs. Group 4, t=13.1, **P<0.01.

[0056] Figure 2 This is a comparative graph showing the effects of different fermentation methods on the conversion rate of urolithin A, combined with... Figure 2 As shown in Table 1, ① Group 2 was significantly better than Group 1, with a conversion rate increase of about 4.5 times; ② Both Bifidobacterium infantis and Lactobacillus infantis are indispensable; ③ The timing of pH adjustment is crucial. When the conversion rate was delayed to 30 hours (Group 5), it dropped to 30.5%, indicating that lactic acid had partially inhibited the dehydroxylase; ④ The efficiency of direct conversion by a single bacterium (Group 6) was extremely low (5.29%), confirming that large tannin molecules cannot be directly utilized by Gord-B3.

[0057] 1.6 Time-course study (Group 1 vs. Group 2, urolithin A concentration mg / L, mean ± variance, n=3), the results are shown in Table 2: Table 2 ; As shown in Table 2, the urolithin A production in the two groups was similar at 24 h (53.1 mg / L vs 49.3 mg / L), with no significant difference (t=1.21, P>0.05). At 30 h (6 h after pH adjustment), group 2 showed a clear inflection point for rapid accumulation of urolithin A (an increase of 133.4 mg / L from 24 to 30 h), while group 1 only increased by 16.3 mg / L during the same period (t=23.8, P>0.05). ** P<0.001); from 36 to 72 h, urolithiasis A in group 2 continued to increase linearly, while group 1 tended to plateau (only 12.9 mg / L increased after 36 h), indicating that the activity of Gord-B3 dehydroxylase in mixed fermentation was completely inhibited by lactate.

[0058] Example 2: Multidimensional screening and systematic evaluation of specific strains of Bifidobacterium infantis 2.1 Strain source and initial screening Fecal samples from healthy infants (8 full-term newborns with informed consent from their parents) were serially diluted 10-fold and inoculated into modified MRS Bifidobacterium selective medium (containing 0.05% L-cysteine, 50 μg / mL amphotericin B, and 12.5 μg / mL polymyxin B). The samples were anaerobic at 37°C for 72 h. Typical Bifidobacterium colonies were picked, and 17 candidate strains were obtained after identification by full-length 16S rRNA gene sequencing (primers 27F / 1492R). Four standard strains (ATCC 29521, ATCC 15700, ATCC15697, and ATCC 15703) were simultaneously introduced as controls.

[0059] 2.2 First round of screening: Tolerance test of pungent glycoside 5 mg / mL (37℃, 24h, survival rate %, mean ± variance, n=3), the test results are shown in Table 3: Table 3 ; Figure 3 This chart compares the survival rates of a specific strain of Bifidobacterium infantis (Strain B) and the general Bifidobacterium bifidum in a high-tannin environment. Figure 3 As shown in Table 3, only Strain B and Strain D among the 17 candidate strains had a survival rate exceeding 60%, while all four standard strains failed. The successful strains proceeded to the second round of screening.

[0060] 2.3 Second round of screening: High concentration tolerance and acid bile salt tolerance test (mean ± variance, n=3), the test results are shown in Table 4: Table 4 ; As can be seen from the test data provided in Table 4, the survival rate of Strain D dropped sharply to 52.3% at a high concentration of 7 mg / mL, and the survival rate of acid bile salts was significantly lower than that of Strain B. Based on the comprehensive evaluation, it was not included in the final screening.

[0061] 2.4 Third round (final screening) – Fermentation function verification (10 mg / mL pungent glycoside, 72 h, mean ± variance, n=3), the test results are shown in Table 5: Table 5 ; As can be seen from the test data provided in Table 5, the urolithin A conversion rate of the Strain B group was 92.9% higher than that of ATCC 29521 (t=17.3, ** P<0.001), an improvement of 145.9% compared to ATCC 15700 (t=19.6, ** P<0.001). The contents of three key intermediate metabolites in the fermentation supernatant of Strain B were significantly higher than those of the standard strain, indicating that it has stronger polyphenol pre-modification activity. Strain B was formally named "Bifidobacterium infantis-specific strain B", with accession number CGMCC No.32401.

[0062] Example 3: Systematic analysis of secondary metabolites of Bifidobacterium infantis Strain B and control Bifidobacterium were inoculated into fermentation medium containing 5 mg / mL pungent glycoside and cultured anaerobicly at 37°C for 72 hours. The fermentation supernatant was collected for metabolite analysis.

[0063] 3.1 Quantitative detection of organic acids (GC-MS method, 5 strains, 5 mg / mL punicein, anaerobic at 37℃ for 72 h, mean ± variance, n=3), the results are shown in Table 6: Table 6 ; As shown in Table 6, the total organic acids in the Strain B group (15.6 ± 1.83 mmol / L) increased by 129.8% compared to ATCC 29521 (t = 7.24). ** P<0.01), an improvement of 175.4% compared to ATCC 15700 (t=8.13, ** P<0.01). L-lactic acid accounts for 52.8%, which is the material basis for its natural permeation-enhancing function.

[0064] 3.2 Quantitative detection of β-glucan (phenol-sulfuric acid method + ELISA, mean ± variance, n=3), the results are shown in Table 7: Table 7 ; As can be seen from the test data provided in Table 7, the β-glucan content in the Strain B group (628±52.4 μg / mL) was increased by 60.6% compared with ATCC29521 (t=5.87, ** P<0.01).

[0065] 3.3 Quantitative analysis of free amino acids (Hitachi L-8900 amino acid analyzer, content ≥5μmol / L, mean ± variance, n=3), the results are shown in Table 8: Table 8 ; As can be seen from the test data provided in Table 8, the total free amino acid content of Strain B (1459±158 μmol / L) increased by 89.2% compared with ATCC 29521 (771±109 μmol / L) (t=6.18, ** P<0.01. The GABA content (301±34 μmol / L) was significantly higher than that of ATCC 29521 (138±26 μmol / L), indicating that it has skin-soothing and anti-inflammatory effects.

[0066] Example 4: Substrate Concentration Gradient Conversion Efficiency and Industrial Feasibility Verification Punicin was set at different concentrations (simulating industrial-grade pomegranate extract) and the fermentation process was carried out according to Example 1: lactic acid bacteria and Strain B were introduced at hour 0 (primary fermentation), and after the pH was adjusted to 6.8±0.1 at hour 24, Gordon's anaerobic bacteria were introduced (secondary fermentation).

[0067] Five groups with different concentrations of punicalin (2, 5, 10, 15, 20 mg / mL), each with a fermentation process (n=3), were tested. The results are shown in Table 9. Table 9 ; The test data provided in Table 9 show that: ① When the substrate concentration is ≤10 mg / mL, the conversion rate of urolithin A is stable at 78.6%~83.4%, which is the highest efficiency; ② When the lactic acid accumulation (11.8 and 14.3 mmol / L) in the 15 and 20 mg / mL groups exceeds the critical threshold, the conversion rate drops to 69.7% and 72.9% respectively; ③ Although the conversion rate decreases at high concentrations, the absolute yield of urolithin A still increases (527.1 and 734.8 mg / L), indicating that the process is still industrially feasible for high-concentration substrates; ④ Considering the overall benefits (yield × conversion rate), the recommended substrate concentration for industrial application is 8~12 mg / mL.

[0068] Example 5: Fine-tuning of pH callback time window and study on lactate inhibition threshold Nine time points were used for the pullback (16h~32h), and a control with no pullback was used. Other conditions were the same as in Group 1 of Example 1 for the fermentation process. The final conversion rate of urolithin A was measured at 10 mg / mL substrate, n=3. The results are shown in Table 10. Table 10 ; The test data provided in Table 10 show that: ① the optimal pullback time window is 22-24h, with the highest conversion rate (84.1%±2.18%) at 24h; ② the lactate inhibition threshold is approximately 15mmol / L, and even if pH pullback is successful after exceeding this value, dehydroxylase activity cannot be fully recovered; ③ when the pullback time is earlier than 20h, substrate hydrolysis and pre-modification are not fully completed, resulting in a significantly lower conversion rate; ④ the recommended optimal pullback time is in the range of 22-26h, with the best point being 24h.

[0069] Example 6: Multidimensional performance comparison of Bifidobacterium infantis with other Bifidobacterium strains. The test results are shown in Table 11: Table 11 ; Statistical analysis (compared with Strain B, **P<0.01): Strain B was significantly superior to all five control strains in all five performance indicators (survival rate, organic acid, β-glucan, urolithin A conversion rate, and EPS). The H2O2 production of Strain B (12.4±3.1 μmol / L) was significantly lower than that of all control strains. This low H2O2 characteristic helps protect the anaerobic dehydroxylase system of Gord-B3 from oxidative damage, which may be one of the mechanisms by which Strain B exerts a synergistic effect during fermentation. Comprehensive evaluation shows that the *Bifidobacterium infantis* specific strain Strain B has irreplaceable advantages in terms of tolerance, metabolite diversity, contribution to urolithin A conversion, and enzyme protection function.

[0070] Example 7: Comparative experiment on the transdermal absorption of multi-component fermentation product composition obtained by the patent in vitro by pig skin 7.1 Franz diffusion pool system: Ex vivo abdominal skin of Bama miniature pigs (keratin layer thickness approximately 15.3 μm, structurally intact confirmed by HE staining), effective diffusion area 1.77 cm². 2 The receiving cell contained 12 mL of 1% Tween-80 PBS (pH=7.4), and was magnetically stirred at 32±0.5℃ and 300r / min.

[0071] 7.2 Experimental Grouping (n=6 per group, 3 pigskins, 2 pools per pigskin): Group A: Pure urolithiasis A (purchased from Sigma-Aldrich, ≥98%, 500 μg / mL, containing 1% DMSO); Group B: Urolithin A + Lactic acid mixture (Urolithin A 500 μg / mL + L-lactic acid 4 mmol / L); Group C: Urolithin A + β-glucan mixture (urolithin A 500 μg / mL + β-1,3-glucan 500 μg / mL); Group D: A mixture of three components: urolithiasis A, lactic acid, and β-glucan (all three components are mixed simultaneously). Group E: Deacidification fermentation product composition (activated carbon adsorption for deacidification, retention of urolithin A and β-glucan); Group F: The whole fermentation product composition obtained in Example 1 of this invention.

[0072] 7.3 Cumulative transdermal permeation Q (μg / cm³) at each time point 2 (mean ± variance, n=6), the detection results are shown in Table 12: Table 12 ; Figure 4 This is a comparison chart of the cumulative transdermal permeation of the fermentation product composition prepared in this invention (Group F) and pure urolithin A reference standard (Group A) in an isolated porcine skin model over 24 hours. Figure 4 As can be seen from the test data provided in Table 12, group F (38.9±3.42μg / cm) 2 The level was significantly higher than that in group D (22.1±2.74 μg / cm). 2 (t=7.86, **P<0.001), indicating that the osmotic effect is not a simple superposition of urolithin A + lactic acid + β-glucan, but rather the result of the combined action of a multi-component synergistic network (extracellular polysaccharide network structure, lipid intermediate metabolites, short-chain fatty acid esterification products, etc.) naturally formed during fermentation.

[0073] Group E vs. Group C comparison (t=2.34, *P<0.05): Deacidification fermentation product composition (14.6±2.04μg / cm) 2 It is superior to the mixture of urolithin A and β-glucan (13.9±1.92μg / cm³). 2 This indicates that the contribution of the polysaccharide network structure to the permeation-enhancing effect of urolithin A is independent of organic acids.

[0074] 7.4 Skin layer distribution data (24h, mean ± variance, n=6), the results are shown in Table 13: Table 13 ; Figure 5This is a comparison chart of the 24-hour dermal retention of the fermentation product composition prepared in this invention (Group F) and pure urolithin A reference standard (Group A) in an isolated porcine skin model. Figure 5 As can be seen from the test data provided in Table 13, group F (14.6±2.31μg / cm) 2 () is group A (3.28±1.09μg / cm) 2 The value was 4.45 times that of group D (t=8.24, **P<0.001), which is 7.13±1.54μg / cm. 2 2.05 times that of t=6.17, **P<0.001.

[0075] The dermal / keratin ratio increased from 0.040 in group A to 0.260 in group F (a 6.5-fold increase), fully demonstrating that the whole fermentation product composition not only increased the total transdermal amount of urolithin A, but also significantly improved the targeted retention in the dermal layer. This is a key pharmacokinetic parameter for achieving anti-aging effects on the skin.

[0076] Total skin retention was actually the lowest in group F (70.8 μg / cm). 2 This indicates that the penetration-enhancing components mainly promote the active transport of urolithin A to the dermis, rather than its ineffective accumulation in the stratum corneum.

[0077] Example 8: Multi-dimensional verification of anti-aging efficacy (UVA-induced photoaging model of human primary fibroblasts) 8.1 Cell Model: HDF (ATCC PCS-201-012, passages 3-6), DMEM high glucose + 10% FBS, cultured at 37℃ and 5% CO2. Cells were synchronized for 12 hours without serum after reaching 80% confluence. UVA irradiation: 365nm, 1.52mW / cm². 2 Dosage 5 J / cm 2 The irradiation time is approximately 55 minutes. During irradiation, retain 1 mL of PBS solution. Immediately after irradiation, replace the culture medium containing the test substance.

[0078] 8.2 Experimental Grouping (n=6 per group, 3 independent experimental batches × 2 technical replicates): ①CON (blank control): Normal serum-containing culture medium, without UVA, and without any treatment; ②DMSO (UVA control): Serum-free culture medium containing 0.1% DMSO was added after UVA. ③Urolithin AL (pure urolithin A 5μg / mL); ④ Urolithin AH (pure urolithin A 10μg / mL); ⑤PM-L (a fermentation product composition with 5 μg / mL urolithin A; actual urolithin A 5.0 μg / mL, total organic acids approximately 0.62 mmol / L, β-glucan approximately 31.4 μg / mL); ⑥PM-H (a fermentation product composition containing 10 μg / mL of urolithin A; actual urolithin A 10.0 μg / mL, total organic acids approximately 1.24 mmol / L, and β-glucan approximately 62.8 μg / mL); ⑦SA (equal concentration organic acid control: L-lactic acid 1.24 mmol / L + acetic acid 0.62 mmol / L); ⑧BG (equal concentration β-glucan control: 62.8 μg / mL).

[0079] 8.3 Detection indicators: ①LC3-II / I ratio (Western Blot, ImageJ quantification, β-actin normalization); ②ROS level (DCFH-DA method, Ex 488nm / Em 525nm, with CON as 100%) ③Type I collagen (ELISA, Boster SEA057Hu); ④MMP-1 activity (gelatin zymography); ⑤ Total antioxidant capacity of T-AOC (ABTS method); ⑥ MTT cell viability (excluding cytotoxicity); ⑦ P62 / SQSTM1 expression (Western Blot, supplementary autophagy flux marker).

[0080] 8.4 MTT results: The survival rates of all treatment groups were in the range of 92% to 103%, with no significant difference from the CON group (P>0.05), excluding interference from cytotoxicity.

[0081] 8.5 Core anti-aging indicators (mean ± variance, n=6; compared with the DMSO group, *P<0.05, **P<0.01; compared with the same concentration of urolithiasis A group, #P<0.05, ##P<0.01), the test results are shown in Table 14: Table 14

[0082] 8.6 Results Analysis Figure 6 , Figure 7 and Figure 8 The figures show a comparison of the LC3-II / I ratio, ROS level, and type I collagen secretion in different treatment groups in a UVA-induced human primary fibroblast (HDF) photoaging model. Figure 6 , Figure 7 , Figure 8 As can be seen from the test data provided in Table 14: (1) UVA damage effect: The DMSO group showed typical photoaging phenotypes compared with the CON group: LC3-II / I decreased by 30.6% (P<0.001), ROS increased by 181.3% (P<0.001), type I collagen decreased by 71.9% (P<0.001), MMP-1 increased by 251% (P<0.001), and T-AOC decreased by 71.6% (P<0.001).

[0083] (2) Dose effect of pure urolithin A: Urolithin AL and urolithin AH were significantly better than the DMSO group in all 5 indicators (P<0.01), showing a clear dose-effect relationship, which confirmed that urolithin A monomer has anti-aging activity of activating mitochondrial autophagy and inhibiting ECM degradation.

[0084] (3) Synergistic effect of fermentation product composition: PM-L was significantly better than the same concentration of urolithin AL group in LC3-II / I (1.82 vs 1.08, P<0.01), ROS (134.2% vs 201.7%, P<0.01), type I collagen (157.8 vs 106.9 ng / mL, P<0.01) and MMP-1 (1.59 vs 2.68, P<0.01), and the type I collagen of PM-L (157.8 ng / mL) was even close to that of urolithin AH group (146.2 ng / mL, P>0.05), which reflects the synergistic effect of permeation-enhancing factors in greatly improving the intracellular delivery efficiency of urolithin A. The PM-H group showed the most comprehensive effects: LC3-II / I reached 2.79 (vs. urolithiasis AH 1.74, an increase of 60.3%), ROS decreased to 109.3% (close to the CON group level), type I collagen recovered to 200.7 ng / mL (89.8% of the CON group), and T-AOC recovered to 17.2 μmol / g protein (93.5% of the CON group).

[0085] (4) Independent effects of organic acids and β-glucan: There were no significant differences between the SA group and the DMSO group in any of the indicators (P>0.05), excluding the direct pharmacological effects of organic acids and β-glucan on autophagy or collagen synthesis, confirming that their anti-aging contribution is entirely achieved through the mechanism of "permeation-promoting delivery of urolithin A". The expression trend of P62 / SQSTM1 was consistent with that of LC3-II / I: CON 0.87±0.12, DMSO 1.34±0.18 (P<0.01, p62 accumulation indicates that autophagic flux is inhibited), PM-H 0.73±0.09 (P<0.01 vs DMSO), further supporting the conclusion that the fermentation product composition promotes intact autophagic flux.

[0086] Example 9: Formulation, stability and human efficacy evaluation of topical anti-aging agents 9.1 The specific components of the formulation (urolithiasis A content 412±14 mg / L, fermentation product composition pH=5.82) are shown in Table 15: Table 15 ; Essence preparation process (A phase + B phase mixing): A phase (fermentation product composition + butylene glycol + glycerin) is stirred at low speed for 30 min at 25℃; B phase (purified water + sodium hyaluronate) is stirred and dissolved; A phase is slowly added to B phase and stirred at 300 r / min for 30 min; phenoxyethanol is added and stirred for 10 min; pH is adjusted to 5.8~6.2; filtered through a 0.22μm sterile filter membrane and dispensed in the dark.

[0087] Cream preparation process (emulsification method): Phase A is heated to 75°C and stirred to dissolve; Phase B (squalane + cetearyl alcohol) is heated to 75°C and melted; Phase B is slowly added to Phase A and emulsified by high-speed shearing (10000r / min, 5min); after cooling to 35°C, fermentation product composition and phenoxyethanol are added; pH is adjusted to 5.8~6.2, filtered and dispensed.

[0088] 9.2 Stability test (F2 essence, 4 storage conditions, 0 days, 7 days, 14 days, 30 days, 60 days and 90 days to detect urolithiasis A retention rate (%) and pH change, mean ± variance, n=3).

[0089] The results of the urolithiasis A retention rate (%) are shown in Table 16: Table 16 ; The results of the pH value change detection are shown in Table 17: Table 17 ; The test data provided in Tables 16 and 17 show that: ① After 90 days of refrigeration at 4℃, the retention rate of urolithin A was 96.9%, and the pH remained stable, which is the recommended storage condition; ② After 90 days at room temperature of 25℃, the retention rate was 91.8%, and the pH only decreased by 0.16, which basically meets the shelf life requirements of commercially available cosmetics (shelf life of 24 months); ③ 40℃ and light have significant effects on urolithin A and pH, so it is necessary to store it in the dark and at low temperature; ④ Under all conditions for 90 days, the microbiological indicators all meet the requirements of the "Cosmetic Safety Technical Specifications" (2015 edition).

[0090] 9.3 Human efficacy evaluation (randomized, double-blind, placebo-controlled, F2 serum, 46 volunteers, 8 weeks): The experimental group consisted of 23 participants, and the control group consisted of 23 participants. The experimental group received 0.5 mL of F2 essence twice daily, morning and evening, while the control group received a blank matrix (without the fermentation product composition). Detection points were: week 0 (baseline), week 2, week 4, and week 8. Repeated measures ANOVA with Bonferroni correction was used.

[0091] The results of the main indicators (mean ± variance, *P<0.05, **P<0.01 compared with the control group at the same time point; #P<0.05, ##P<0.01 compared with baseline) are shown in Table 18: Table 18 ; The scores of the subjects' self-report questionnaire (5-point scale, 1 = no improvement, 5 = significant improvement, mean ± variance) are shown in Table 19: Table 19 ; Safety: During the 8-week use period, no local skin irritation reactions (erythema, itching, stinging, desquamation) were reported in either group. Dermatologist DVGS assessment: 0 / 23 subjects in the experimental group experienced ≥Grade 1 irritation reactions (1 subject in the control group experienced mild, transient stinging, not product-related); the overall safety assessment was "no irritation". Overall effects after 8 weeks: Skin elasticity R2 value increased by 18.6%, skin moisture content increased by 32.5%, fine line volume decreased by 29.1%, skin radiance L* value increased by 11.5%, TEWL decreased by 26.6%, and erythema index a* value decreased by 16.7%. Significant improvements in all indicators began in week 2, and the improvement further increased by week 8, indicating that continued use provides better long-term anti-aging effects.

[0092] Based on the above test results, it can be seen that the fermentation process provided by this invention uses pomegranate whole fruit extract (containing more than 40% pungent glycosides) as a substrate, which is then subjected to hydrolysis of macromolecular tannins by lactic acid bacteria, pre-modification of the polyphenol substrate by Bifidobacterium infantis (preservation number CGMCCNo.32401), and protection of the dehydroxylase system by precise pH adjustment (22~26h, optimal 24h, adjusted to 6.5~7.0), and then deep conversion by Gordonella anaerobic bacteria to generate urolithiasis A.

[0093] Using 10 mg / mL punicalin as a substrate, the process achieved a urolithin A conversion rate of 84.1 ± 2.18% (n=3) after 72 hours. This is approximately 4.5 times higher than the traditional simultaneous mixed fermentation (18.5 ± 0.92%, n=3), approximately 6.3 times higher than the control group without Bifidobacterium infantis (13.3 ± 0.83%, n=3), approximately 35.4% higher than the control group with pH adjustment delayed to 30 hours (62.1 ± 3.78%, n=3), and approximately 14.9 times higher than the control group with direct conversion of Gord-B3 alone (5.29 ± 0.47%, n=3).

[0094] The survival rate of the *Bifidobacterium infantis* specific strain Strain B in a high-tannin environment (5 mg / mL pungentin) reached 92.4 ± 2.9% (n=3), which was approximately 91% higher than that of the general strain ATCC 29521 (48.3 ± 4.1%, n=3). The resulting fermentation product composition contained urolithin A 394.3 ± 10.2 mg / L (n=3), total organic acids 15.6 ± 1.83 mmol / L (n=3), β-glucan 628 ± 52.4 μg / mL (n=3), GABA 301 ± 34 μmol / L (n=3), and EPS 218 ± 28.4 mg / L (n=3).

[0095] In the in vitro porcine skin experiment, the cumulative transdermal permeability of the whole fermentation product composition group (Group F) reached 38.9 ± 3.42 μg / cm³ over 24 hours. 2 (n=6), which is the pure urolithiasis A group (11.4±1.64μg / cm). 2 The concentration of the substance was 3.41 times that of n=6; the dermal retention amount reached 14.6±2.31 μg / cm³. 2 (n=6), which is the pure urolithiasis A group (3.28±1.09μg / cm). 2 The dermis / keratin ratio increased from 0.040 to 0.260 (a 6.5-fold increase).

[0096] In the UVA-induced photoaging model of human primary fibroblasts, the high-concentration group (PM-H) of the fermentation product composition with an isolithin A concentration (10 μg / mL) had an LC3-II / I ratio of 2.79±0.31 (n=6), a ROS decrease of 109.3±10.8% (n=6), and a type I collagen secretion of 200.7±26.3 ng / mL (n=6), all of which were significantly better than the isolithin A pure group (LC3-II / I 1.74±0.21, ROS 164.4±17.2%, type I collagen 146.2±24.6 ng / mL, P<0.01).

[0097] Eight-week human efficacy evaluation (n=23, F2 serum): skin elasticity R2 value increased by 18.6%, skin moisture content increased by 32.5%, fine line volume decreased by 29.1%, skin radiance L* value increased by 11.5%, TEWL decreased by 26.6%, overall subject satisfaction score was 4.19±0.78 (out of 5), and safety assessment was "no irritation". This fermented product composition can be directly used in the production of topical anti-aging preparations such as serums and creams, and has broad application prospects in the cosmetics and functional food fields.

[0098] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for the synergistic transformation of pomegranate polyphenols by lactic acid bacteria and Bifidobacterium infantis, characterized in that, The method includes: Lactic acid bacteria and Bifidobacterium infantis were inoculated into a pomegranate extract solution for primary fermentation to obtain a fermentation product. The pH of the fermentation product was adjusted, and then anaerobic bacteria of the genus *Gordonella* were inoculated for secondary fermentation to obtain a pomegranate fermentation product composition containing urolithin A.

2. The method according to claim 1, characterized in that, The lactic acid bacteria is *Lactobacillus plantarum*. The Bifidobacterium infantis strain is a specific strain with accession number CGMCC No. 32401.

3. The method according to claim 1, characterized in that, The temperature for the primary fermentation is 35℃~40℃; The duration of the primary fermentation is 20-28 hours.

4. The method according to claim 1, characterized in that, The temperature for the secondary fermentation is 30℃~40℃; The total duration of the primary fermentation and the secondary fermentation is 72 hours.

5. The method according to claim 1, characterized in that, The primary and secondary fermentations are carried out in an anaerobic environment.

6. The method according to claim 1, characterized in that, An alkaline solution was added dropwise to the primary fermentation product to adjust its pH value to 6.5-7.

0.

7. The method according to claim 1, characterized in that, The infantile bifidobacteria were obtained by screening using the following method: (a) Isolation of Bifidobacterium strains from infant gut samples; (b) The isolated Bifidobacterium strains were cultured in a medium with a pungent glycoside concentration of 5 mg / mL for 24 h, and strains with a survival rate of ≥60% were selected as the initial screening strains. (c) The primary screening strains were cultured for 24 h in a high-concentration medium with a pungent glycoside concentration of 7 mg / mL, and for 2 h in a medium containing bile salts at pH 3; strains with a survival rate of ≥60% in both the high-concentration medium and the bile salt medium were selected as candidate strains. (d) The lactic acid bacteria and the candidate strain were inoculated into a pomegranate extract solution for primary fermentation to obtain a primary fermentation product; the pH of the primary fermentation product was adjusted, and then anaerobic bacteria of the genus *Gordonella* were inoculated for secondary fermentation to obtain a secondary fermentation product; the conversion rate of urolithin A in the secondary fermentation product was detected, and strains with a urolithin A conversion rate ≥80% were screened as the *Bifidobacterium infantis*.

8. A fermentation product composition prepared by the method according to any one of claims 1 to 7, characterized in that, The fermentation product composition contains urolithin A, organic acids and β-glucan; The content of urolithin A in the fermentation product composition is 300~500 mg / L; The total organic acid content in the fermentation product composition is ≥10 mmol / L; The fermentation product composition contains ≥0.5 mg / mL of β-glucan.

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