Mucinophilic akkermansia superakk 001 with cosmetic effects and application thereof

Abstract

The application provides a mucinophilic Akkermansia muciniphila SuperAKK 001 with a cosmetic effect and an application thereof, and belongs to the technical field of microbial agents.The mucinophilic Akkermansia muciniphila SuperAKK 001 with the cosmetic effect provided by the application has a preservation number of CGMCC NO.47119.The mucinophilic Akkermansia muciniphila provided by the application has the physiological characteristics of extremely acid tolerance and a higher gastrointestinal survival rate, and has a stronger function of resisting UVB-induced skin photoaging of ultraviolet rays.The mucinophilic Akkermansia muciniphila, its bacterial powder, its postbiotic powder and microcapsule preparation provided by the application realize the multiple cosmetic effects of inhibiting skin photoaging, repairing photo-damage, repairing skin barrier and a wound, tightening and lightening wrinkles, whitening and moisturizing, break through many defects of the prior art, and have important industrialization value and clinical application significance.

Description

Technical Field

[0001] This invention belongs to the field of microbial inoculant technology, specifically relating to a Kermania superakinophilus strain with cosmetic effects, SuperAKK 001, and its applications. Background Technology

[0002] Skin aging is a complex degenerative skin disease caused by both endogenous aging and exogenous environmental stimuli, with ultraviolet radiation being the core cause of exogenous skin aging (photoaging). Long-term ultraviolet radiation induces skin cells to produce a large number of reactive oxygen free radicals, triggering severe oxidative stress reactions, activating key pathways such as matrix metalloproteinases, and continuously degrading core structural proteins in the dermis, such as collagen and elastin. Simultaneously, it causes DNA damage to skin cells, mitochondrial dysfunction, and epidermal barrier metabolic disorders, ultimately leading to a series of skin problems such as dryness, pigmentation, dull skin tone, wrinkles, sagging skin, and barrier damage, severely affecting the appearance and physiological health of the skin. In addition, daily external stimuli, skin trauma, and inflammatory stress can also exacerbate skin barrier damage, slow wound healing, and accelerated skin aging, making them major causes of sub-healthy skin and premature aging in the general population.

[0003] Currently, there are numerous technologies and products on the market targeting photoaging repair and skin care. The mainstream intervention methods are mainly divided into three categories: topical skincare, medical aesthetic intervention, and oral nutritional supplements. However, all of these have significant technical defects and application limitations, making it difficult to achieve safe, long-lasting, and multi-dimensional comprehensive beauty and skin care effects. Topical skincare products primarily focus on surface moisturizing, anti-oxidation, and concealing dullness. Their effective ingredients can only act on the epidermis and cannot penetrate the dermis to repair deep cell damage and collagen loss caused by ultraviolet radiation. Their effects on repairing existing wrinkles, skin laxity, and photoaging damage are weak, and their single-effect nature prevents the simultaneous achievement of multiple effects such as wound repair, barrier reconstruction, whitening, and firming. Furthermore, most topical products contain chemical whitening agents, preservatives, fragrances, and other additives, which can easily cause skin sensitivity and inflammation with long-term use, limiting their suitability for certain groups.

[0004] While medical aesthetic interventions such as laser repair, hyaluronic acid injections, and collagen fillers offer significant short-term tightening and anti-wrinkle effects, they suffer from drawbacks including invasiveness, high risks, short-lasting effects, and high costs. Multiple treatments are often required, and they cannot fundamentally improve skin cell metabolism and photoaging damage mechanisms. Post-treatment side effects such as redness, sensitivity, and hyperpigmentation are common, limiting their applicability and target audience, making them unsuitable for routine, daily skincare. Conventional oral beauty products, primarily composed of vitamins, collagen, hyaluronic acid, and plant extracts, only provide single antioxidant or basic moisturizing effects. They lack specificity and have dispersed efficacy, failing to specifically inhibit UV-induced oxidative stress and collagen degradation. Their effects on photoaging damage repair, skin barrier reconstruction, wound healing, and skin tightening and anti-aging are very limited, exhibiting technical shortcomings such as single efficacy, slow onset of action, unclear mechanisms of action, and poor overall skincare results. For example, existing hydrolyzed collagen peptides offer more immediate effects and are often used in combination with vitamin C and hyaluronic acid, allowing for greater flexibility in formulation design. However, these raw materials have large molecular weights and low absorption and utilization rates in the human body. Conventional powder forms are easily broken down by stomach acid, often requiring an additional enteric coating process, which increases production and raw material costs. Long-term use alone can only improve surface skin dehydration, failing to address skin laxity and pigmentation caused by photoaging in the dermis. Furthermore, some users experience protein intolerance, leading to acne, bloating, and other discomfort. For example, existing plant polyphenol antioxidants, such as astaxanthin, proanthocyanidins, and scutellaria baicalensis polyphenols, can eliminate singlet oxygen and peroxide free radicals generated by ultraviolet radiation, increasing the minimum erythema threshold and providing internal protection against ultraviolet radiation, alleviating skin redness and melanin deposition caused by UV radiation. However, these substances have short biological half-lives and limited time in the body; moreover, most polyphenols exhibit significant differences in hydrophilicity / lipophilicity, and are easily metabolized and broken down by the liver and kidneys after oral ingestion. The proportion of effective components acting on the skin is extremely low, requiring higher dosages to achieve results. Furthermore, these products rely solely on a single antioxidant mechanism and cannot address the root cause of photoaging in the skin. Another example is pharmacological whitening and anti-aging oral products: Glutathione, L-cysteine, oral nicotinamide, tranexamic acid, and other pharmacologically active oral ingredients can target and inhibit tyrosinase activity, blocking the melanin transport process induced by UVB radiation, resulting in significant improvements in sunspots and dull skin tone, with rapid whitening effects. However, these ingredients possess medicinal properties, and their application scenarios and safety are strictly limited. Long-term high-dose use can increase the metabolic burden on the liver and kidneys; domestic regulations also strictly limit their addition in health foods, making large-scale high-concentration production impossible. Excessive nicotinamide intake can also cause skin redness and gastrointestinal discomfort.At the same time, these products only focus on pigment regulation and do not have the ability to repair the skin or promote collagen regeneration, so they cannot improve the collagen loss and skin sagging caused by ultraviolet rays.

[0005] While there are existing reports on the use of probiotic preparations for cosmetic purposes, significant technical bottlenecks exist: First, most methods rely solely on live bacteria for indirect intestinal regulation. Live bacteria suffer from poor stability, require stringent storage conditions, and are easily inactivated by stomach acid and bile upon oral administration, resulting in a survival rate of less than 10%. Very few strains successfully reach the intestines and colonize at high concentrations, leading to low bioavailability and ultimately significant attenuation of product efficacy, resulting in weak and unstable cosmetic effects. Second, existing formulations are limited in dosage form and lack stability-enhancing formulations, failing to address industry pain points such as easy inactivation of active ingredients, low bioavailability, and poor targeting, making it difficult to achieve long-lasting, stable, and multi-dimensional oral cosmetic and health-preserving effects. Furthermore, the stringent storage conditions and rapid activity decay of ordinary live bacteria powders are also core challenges that urgently need to be addressed in the industry.

[0006] In summary, the current skin care industry generally faces technical challenges such as single product efficacy, weak targeting, treating symptoms but not the root cause, difficulty in balancing safety and stability, and inability to simultaneously achieve multiple synergistic effects such as photoaging inhibition and repair, barrier repair, anti-wrinkle and firming, and whitening and moisturizing. There is a market gap for exclusive, stable, and efficient bacterial raw materials, post-biotic products, and compound preparations. Summary of the Invention

[0007] In view of this, one of the objectives of the present invention is to provide a *Ackermania* strain SuperAKK 001 with cosmetic effects, which has extremely acid-resistant physiological characteristics and a higher gastrointestinal survival rate, thereby having a more significant function in resisting UVB-induced photoaging of the skin.

[0008] The second objective of this invention is to provide a bacterial powder and postbiotic powder with cosmetic effects, which breaks through the technical bottlenecks of existing oral cosmetic raw materials with single efficacy and poor stability of probiotic live bacteria preparations. Postbiotic products are free from dependence on live bacteria colonization, have high storage stability and good oral safety, open up a new path of beauty from the inside out based on gut-epidermal axis regulation, enrich oral cosmetic functional raw materials, and have broad prospects for industrialization in food and dietary supplements.

[0009] The third objective of this invention is to provide a microcapsule formulation with cosmetic effects, which uses acid-resistant microcapsule coating to resist the erosion of strong stomach acid and intestinal bile, and significantly improve the survival rate of live bacteria in the body and the ability to colonize the intestine; the post-biotic is a sterilized product, with no risk of live bacteria inactivation, and the coating is mainly used to achieve sustained release of the ingredients into the intestine, prolong the duration of action of active substances in the intestine, and further improve oral absorption efficiency.

[0010] The fourth objective of this invention is to provide the application of the above-mentioned products in the preparation of oral cosmetic preparations.

[0011] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a *Ackermania viride* SuperAKK 001 with cosmetic effects, wherein the *Ackermania viride* SuperAKK 001 is classified as *Ackermania viride*. Akkermansia muciniphila It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCCNO.47119.

[0012] The present invention also provides a bacterial powder with cosmetic effects, wherein the bacterial powder contains the above-mentioned Akkermansia muciniphila SuperAKK 001.

[0013] Preferably, when the bacterial powder is freeze-dried bacterial powder, the freeze-dried bacterial powder further contains a cryoprotectant, which is composed of trehalose and glycerol; the concentration of the bacterial powder suspension is 1.0 × 10⁻⁶. 9 The volume-to-weight ratio of the bacterial powder suspension, trehalose, and glycerol, based on CFU / mL, is 100 mL: 10 g: 1 g. The bacterial powder suspension is obtained by mixing bacterial cells with skim milk.

[0014] The present invention also provides an epigenetic powder with cosmetic effects, which is prepared by anaerobic culture, inactivation, concentration and drying of the above-mentioned Akkermansia muciniphila SuperAKK 001.

[0015] Preferably, the culture medium used for anaerobic culture is BHI liquid culture medium, which contains, per 1L, 38g of brain heart extract powder, 5g of yeast extract, 5g of sodium chloride, 2g of porcine gastric mucin, 0.5g of L-cysteine ​​hydrochloride, and the balance being water; the pH of the BHI liquid culture medium is 6.5; the concentration is to concentrate the bacterial solution obtained from anaerobic culture to 1 / 5 of the original volume; the drying includes freeze-drying, which includes the following steps: pre-freezing at -80℃ for 4h, then drying at -40℃ under vacuum <10Pa for 18h, and then drying at 26℃ for 6h.

[0016] The present invention also provides a microcapsule preparation with cosmetic effects, which is obtained by coating the above-mentioned bacterial powder or the above-mentioned post-biotic powder as the core material; the coating solution used for coating is composed of 8%~12% ethyl cellulose, 0.5%~1.0% magnesium stearate, 75%~85% anhydrous ethanol and the balance water in a mass ratio.

[0017] Preferably, the viscosity of the ethyl cellulose is 10-20 mPa·s; the weight gain of the coating is 15%-25% based on the core material mass; during coating, the inlet air temperature is 40-50℃, the coating solution temperature is 30-35℃, the atomization pressure is 1.5-2.5 bar, the coating solution spray rate is 2-5 mL / min, and the ventilation volume is 80-120 m³ / min. 3 / h.

[0018] The present invention also provides the application of the above-mentioned Akkermansia melanophila SuperAKK 001, the above-mentioned bacterial powder, the above-mentioned post-biotic powder, or the above-mentioned microcapsule preparation in the preparation of oral cosmetic preparations.

[0019] Preferably, the cosmetic treatment includes inhibiting photoaging of the skin, repairing photoaging of the skin, promoting the repair of skin wounds and barriers, firming the skin, improving wrinkles, whitening and / or moisturizing.

[0020] Preferably, the cosmetic procedure includes improving skin matrix loss and dermal thinning caused by photoaging, alleviating UV-induced skin oxidative stress damage, inhibiting collagen loss in skin tissue, improving skin tissue oxidative aging, promoting collagen fiber recovery and remodeling, improving dry skin, promoting skin cell regeneration, promoting the migration and repair of photodamaged skin cells, and / or blocking fibroblast apoptosis.

[0021] The beneficial effects of this invention are: The Akkermansia melanophila SuperAKK 001 provided by this invention has extremely acid-resistant physiological characteristics and a higher gastrointestinal survival rate, and has a stronger function in resisting UVB-induced photoaging of the skin.

[0022] The bioactive probiotic powder and postbiotic powder provided by this invention overcome the technical bottlenecks of existing oral beauty ingredients, which suffer from limited efficacy and poor stability of live probiotic preparations. Postbiotic products eliminate reliance on live bacteria colonization, exhibit high storage stability and good oral safety, and open up a new pathway for beauty from the inside out based on gut-epidermal axis regulation. This enriches the range of oral beauty functional ingredients and has broad prospects for industrialization in the food and dietary supplement sectors.

[0023] The microencapsulation formulation provided by this invention utilizes microencapsulation technology and acid-resistant microencapsulation, which can resist the erosion of strong stomach acid and intestinal bile, significantly improving the survival rate of live bacteria in vivo and their intestinal colonization ability, exhibiting significant advantages of stability and high efficiency. The post-biotic is a sterilized product, eliminating the risk of live bacteria inactivation. The coating is mainly used to achieve sustained intestinal release of the ingredients, prolonging the duration of action of active substances in the intestine, and further improving oral absorption efficiency. This overcomes the industry shortcomings of existing oral beauty products, which have single-target effects and cannot simultaneously address endogenous intestinal regulation and skin repair. Moreover, the microencapsulation formulation provided by this invention avoids the negative effects caused by drug use, has no side effects, and maintains long-term effectiveness.

[0024] In summary, the *Ackermania mucilaginosa* SuperAKK 001, its bacterial powder, post-biotic powder, and microcapsule formulation provided by this invention, through specific strain cultivation, exclusive post-biotic preparation process, and optimized dosage form design, achieve multiple cosmetic effects such as inhibiting skin photoaging, repairing photodamage, repairing skin barrier and wounds, firming and reducing wrinkles, and whitening and moisturizing. It overcomes many defects of existing technologies and has important industrialization value and clinical application significance.

[0025] Information on the preservation of biological materials This invention relates to *Ackermania*, a bacterophilic organism. Akkermansia muciniphila SuperAKK 001 was deposited on July 21, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China, with accession number CGMCC NO.47119. Attached Figure Description

[0026] Figure 1 HE staining results of mouse skin tissue in each group (×200, scale bar=50μm), from left to right are the results of blank control group, UVB model group, SuperAKK 001 live bacteria microcapsule preparation group and SuperAKK 001 postbiotic microcapsule preparation group.

[0027] Figure 2 The results of Masson trichrome staining of mouse skin tissue in each group (×200, scale bar=50μm) are shown from left to right, representing the results of the blank control group, the UVB model group, the SuperAKK 001 live bacteria microcapsule preparation group, and the SuperAKK 001 postbiotic microcapsule preparation group.

[0028] Figure 3 Immunohistochemical staining results of COL1A1 in mouse skin tissue of each group (×200, scale bar=50μm), from left to right are the results of blank control group, UVB model group, SuperAKK 001 live bacteria microcapsule preparation group and SuperAKK 001 postbiotic microcapsule preparation group. Detailed Implementation

[0029] This invention provides a *Ackermania viride* SuperAKK 001 with cosmetic effects, wherein the *Ackermania viride* SuperAKK 001 is classified as *Ackermania viride*. Akkermansia muciniphila It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47119.

[0030] The Akkermansia melanophila SuperAKK 001 provided by this invention was isolated, screened and purified from intestinal samples of athletes in Xishuangbanna, Yunnan. It has extremely acid-resistant physiological characteristics and a higher gastrointestinal survival rate. Compared with existing bacteria, it has a stronger function in resisting UVB-induced photoaging of the skin.

[0031] The present invention also provides a bacterial powder with cosmetic effects, wherein the bacterial powder contains the above-mentioned Akkermansia muciniphila SuperAKK 001.

[0032] In this invention, the bacterial powder refers to live bacterial powder. When the bacterial powder is freeze-dried, it preferably also contains a cryoprotectant, which is preferably composed of trehalose and glycerol; the concentration of the bacterial powder suspension is 1.0 × 10⁻⁶. 9 The preferred volume-to-weight ratio of the bacterial powder suspension, trehalose, and glycerol, based on CFU / mL, is 100 mL:10 g:1 g. The bacterial powder suspension is obtained by mixing bacterial cells with skim milk. In this invention, the skim milk preferably contains 15% by mass. When preparing the freeze-dried bacterial powder, the preferred freeze-drying method is to first pre-freeze at -80°C for 4 hours, then dry at -40°C under vacuum <10 Pa for 20 hours, followed by drying at 20°C~25°C for 4 hours. After freeze-drying, the powder is immediately sealed with nitrogen and stored at -20°C.

[0033] The present invention also provides an epigenetic powder with cosmetic effects, which is prepared by anaerobic culture, inactivation, concentration and drying of the above-mentioned Akkermansia muciniphila SuperAKK 001.

[0034] In this invention, the culture medium used for anaerobic culture is preferably BHI (biologically modified hygroscopic protein) liquid culture medium. The BHI liquid culture medium, per 1L, preferably contains 38g of brain heart extract powder, 5g of yeast extract, 5g of sodium chloride, 2g of porcine gastric mucin, 0.5g of L-cysteine ​​hydrochloride, and the balance being water; the pH of the BHI liquid culture medium is 6.5. This invention does not have specific limitations on the source of each raw material in the BHI liquid culture medium; commercially available products in the art can be used. In this invention, the inactivation method is preferably pasteurization; the concentration method is preferably concentrating the bacterial solution obtained from anaerobic culture to 1 / 5 of the original volume, increasing the solid content by 5 times; the concentration method is preferably centrifugation or ultrafiltration concentration; the drying method preferably includes freeze-drying, which preferably includes the following steps: pre-freezing at -80℃ for 4 hours, then drying at -40℃ under vacuum <10Pa for 18 hours, and then drying at 26℃ for 6 hours.

[0035] The present invention also provides a microcapsule preparation with cosmetic effects, which is obtained by coating the above-mentioned bacterial powder or the above-mentioned post-biotic powder as the core material; the coating solution used for coating is composed of 8%~12% ethyl cellulose, 0.5%~1.0% magnesium stearate, 75%~85% anhydrous ethanol and the balance water in a mass ratio.

[0036] In this invention, the microencapsulation formulation is divided into bacterial powder microencapsulation formulation and metabiotic powder microencapsulation formulation. This invention uses acid-resistant microencapsulation coating on Akkermansia myxophilus live bacterial powder, which can resist the erosion of strong gastric acid and intestinal bile, and significantly improve the survival rate and intestinal colonization ability of live bacteria in vivo. This invention also uses acid-resistant microencapsulation coating on Akkermansia myxophilus metabiotics. The metabiotics are sterilized products, so there is no risk of live bacteria inactivation. The coating is mainly used to achieve intestinal sustained release of the ingredients, prolong the duration of action of active substances in the intestine, and further improve oral absorption efficiency.

[0037] In this invention, the mass percentage of ethyl cellulose in the coating solution is preferably 9% to 11%, more preferably 10%; the viscosity of the ethyl cellulose is preferably 10 to 20 mPa·s, more preferably 12 to 18 mPa·s; the mass percentage of magnesium stearate is preferably 0.6% to 0.9%, more preferably 0.7% to 0.8%; and the mass percentage of anhydrous ethanol is preferably 76% to 84%, more preferably 78% to 82%. In this invention, based on the core material mass, the weight gain of the coating is preferably 15%~25%, more preferably 18%~22%; during coating, a bottom-spray fluidized bed coating device is preferably used; the inlet air temperature is preferably 40~50℃, more preferably 42~48℃; the temperature of the coating solution is preferably 30~35℃, more preferably 31~34℃; the atomization pressure is preferably 1.5~2.5 bar, more preferably 1.8~2.2 bar; the spray rate of the coating solution is preferably 2~5 mL / min, more preferably 3~4 mL / min; and the ventilation volume is preferably 80~120 m³ / min. 3 / h, more preferably 90~110m 3 / h. In this invention, after coating, preferably all materials continue to undergo fluidized bed drying in a fluidized bed. The drying temperature is preferably 35~40℃, more preferably 37~39℃, and the processing time is preferably 30~60min, more preferably 40~50min, until the final moisture content of the material is controlled below 6%. The dried material is preferably sieved through a 60~80 mesh screen to remove lumps and large particles; then it is individually packaged in aluminum foil bags, preferably 2g per bag, and the finished product is stored in a light-proof, sealed environment.

[0038] The present invention also provides the application of the above-mentioned Akkermansia melanophila SuperAKK 001, the above-mentioned bacterial powder, the above-mentioned post-biotic powder, or the above-mentioned microcapsule preparation in the preparation of oral cosmetic preparations.

[0039] This invention does not specifically limit the dosage form of oral cosmetic preparations; they can be solid or liquid formulations. The *Ackermania mutans* SuperAKK 001, bacterial powder, post-biotic powder, or microcapsule formulations provided by this invention can improve photoaging through multiple pathways. In this invention, the preferred cosmetic effects include inhibiting skin photoaging, repairing skin photoaging, promoting skin wound and barrier repair, firming skin, improving wrinkles, whitening, and / or moisturizing. More preferably, these effects include improving skin matrix loss and dermal thinning caused by photoaging, alleviating UV-induced skin oxidative stress damage, inhibiting collagen loss in skin tissue, improving skin tissue oxidative aging, promoting collagen fiber recovery and remodeling, improving dry skin, promoting skin cell regeneration, promoting the migration and repair of photodamaged skin cells, and / or blocking fibroblast apoptosis.

[0040] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0041] Unless otherwise specified, the following embodiments are all conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] All experimental data obtained in the following examples are expressed as ±S. Statistical software (SPSS 26.0) was used to analyze the data. When the comparisons among multiple groups met the requirements of normal distribution and homogeneity of variance, one-way ANOVA was used. P < 0.05 was considered statistically significant.

[0044] Example 1 Isolation, identification and microencapsulation of strains 1. Strains screening and purification (1) Sampling: Take 1g of intestinal sample from an athlete in Xishuangbanna, Yunnan. After aseptic anaerobic collection, transport it to the laboratory anaerobic incubator for sample preparation in the cold chain. (2) Sample preparation: ① Take 1g of sample and add it to a sterile conical flask containing 9mL of sterile PBS buffer. Shake and mix at 180r / min for 35min in an ice bath environment to prepare the stock solution suspension for later use. ② Prepare diluted solutions using a tenfold serial dilution method, with dilution gradients of 10:10. -1 10 -2 10 -3 10-4 10 -5 10 -6 10 -7 The entire process must be carried out on ice in the dark; be prepared. (3) Preparation of BHI selective solid plate culture medium containing 0.2% porcine mucin Culture medium components and preparation method: Brain Heart Extract (BHI) 38g, yeast extract 5g, sodium chloride 5g, porcine gastric mucoprotein 2g, L-cysteine ​​hydrochloride 0.5g, agar 15g, distilled water to a final volume of 1000mL; After mixing the raw materials, adjust the pH to 6.5, heat and stir until the powder is completely dissolved, and sterilize with high-pressure steam at 121℃ and 0.1MPa for 20 minutes. After sterilization, pour the mixture into plates in an anaerobic clean bench, let it solidify at room temperature, and then store it at 4℃ for later use.

[0045] (4) Culture: 100 μL of each gradient dilution was spread on BHI plates containing mucin and cultured in a strictly anaerobic environment at 37°C for 5 days.

[0046] (5) Selecting colonies: Target colony selection criteria: colony diameter 0.8~1.5mm, round and raised, with smooth and neat edges, milky white and translucent, with obvious mucin decomposition zone (the culture medium around the colony is sunken and transparent).

[0047] (6) Separation and purification Suspected single colonies were picked from the initial screening plates based on morphological characteristics and transferred to the same formulation of mucin BHI solid medium using the streak plate method. The culture was anaerobic at 37°C for 5 days and purified by streak plate for 3 generations. After purification, pure single colonies were picked, inoculated with 20% sterile glycerol bacterial solution, and stored in an ultra-low temperature freezer at -80°C as candidate strains.

[0048] 2. Identification and Preservation The isolated and purified single colony (SuperAKK 001) was sent for identification by Jinan Tianyi Biotechnology Co., Ltd. The primers used in the identification process were as follows: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO.1), 1492R: 5'-CTACGGCTACCTTGTTACGA-3' (SEQ ID NO.2).

[0049] The identification result is: This strain is Akkermansia muciniphila The identified strain was named *Ackermania myxophilus* SuperAKK 001 and deposited with the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC NO. 47119. This invention relates to *Ackermania myxophilus* (…). Akkermansia muciniphilaThe 16S rDNA sequence of SuperAKK 001 is shown in SEQ ID NO.3.

[0050] Example 2 Preparation of live bacteria powder of Akkermansia melanophila SuperAKK 001 The *Ackermania superakok* strain SuperAKK 001 obtained in Example 1 and stored at -80℃ was activated on a mucin BHI solid medium plate (the specific composition is the same as step (3) in Part 1 of Example 1). One activated single colony was transferred to 100 mL of mucin BHI liquid medium and cultured anaerobically at 37℃ for 72 h to obtain a seed culture. The seed culture was inoculated into mucin BHI liquid medium (the difference from mucin BHI solid medium is that no agar is added, but everything else is the same as mucin BHI solid medium) at an inoculation rate of 1% (v / v). Then, it was cultured anaerobically at 37℃ for 72 h to obtain a viable count of 1.0 × 10⁻⁶. 9 Bacterial suspension with cfu / mL.

[0051] After centrifuging the bacterial culture, the bacterial cells were collected, washed with sterile physiological saline, and resuspended in 15% (w / w) reconstituted skim milk to obtain a suspension; the concentration of the suspension was adjusted to 1.0 × 10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension. The bacterial suspension was mixed with a cryoprotectant composed of trehalose and glycerol at a volume-to-weight ratio of 100 mL:10 g:1 g. The mixture was then freeze-dried as follows: pre-frozen at -80°C for 4 hours, followed by a first drying at -40°C under vacuum <10 Pa for 20 hours, and then a second drying at 23°C for 4 hours. After the second drying, the mixture was immediately sealed with nitrogen and stored at -20°C to obtain bacterial powder. In this example, the prepared bacterial powder contained 1.0 × 10⁻⁶ cells. 10 CFU / g (10 billion / g).

[0052] Example 3 Preparation of post-biotic powder from *Ackermania superakok* 001 The *Ackermania superakok* strain SuperAKK 001 obtained in Example 1 and stored at -80℃ was activated on a mucin BHI solid medium plate (the specific composition is the same as step (3) in Part 1 of Example 1). One activated single colony was transferred to 100 mL of mucin BHI liquid medium and cultured anaerobically at 37℃ for 72 h to obtain a seed culture. The seed culture was inoculated into mucin BHI liquid medium (the difference from mucin BHI solid medium is that no agar is added, but everything else is the same as mucin BHI solid medium) at an inoculation rate of 1% (v / v). Then, it was cultured anaerobically at 37℃ for 72 h to obtain a viable count of 1.0 × 10⁻⁶. 9 Bacterial suspension with cfu / mL.

[0053] After pasteurizing the bacterial solution (containing bacterial cells and their metabolites), it was concentrated to 1 / 5 of its original volume by ultrafiltration (increasing the solid content by 5 times), and then freeze-dried as follows: pre-freezing at -80℃ for 4 hours, followed by the first drying at -40℃ and vacuum <10Pa for 18 hours, and then the second drying at 26℃ for 6 hours to obtain post-biotic powder.

[0054] Example 4 The microencapsulation preparation of Akkermansia mesophila SuperAKK 001 live bacterial powder (obtained in Example 2) is as follows: (1) Preparation of coating solution The coating system was prepared according to the following mass ratios: 8% ethyl cellulose with a viscosity of 10 mPa·s was added, 0.5% magnesium stearate was added, 75% anhydrous ethanol was added, and the remaining components were made up with purified water.

[0055] Preparation process: First, add ethyl cellulose into anhydrous ethanol system in portions and stir until completely dissolved. Then, add magnesium stearate and mix well. Finally, slowly add purified water and stir continuously for 30 minutes. After mixing, filter through a 100-mesh sieve and set aside the filtrate for later use.

[0056] (2) Wurster bottom spray fluidized bed coating operation Bottom-spray type fluidized bed coating equipment was selected, and the process parameters were set as follows: inlet air temperature controlled at 40℃, coating solution temperature maintained at 30℃; atomization pressure set at 1.5 bar, coating solution spray rate at 2 mL / min, and equipment ventilation volume at 80 m³ / min. 3 / h; Based on the quality of the live bacteria powder (as core material) obtained in Example 2, the overall coating weight gain was controlled to be within 15%.

[0057] (3) Post-drying process After coating, the material continued to undergo fluidized bed drying at 35°C for 30 minutes. The final moisture content of the material was measured to be less than 6%.

[0058] (4) Finished product sorting and packaging The dried material is sieved through a 60-mesh screen to remove lumps and large particles; then it is individually packaged in aluminum foil bags, with each bag containing 2g. The finished product is stored in a dark, sealed environment.

[0059] Example 5 The microencapsulation preparation of Akkermansia mesophila SuperAKK 001 live bacterial powder (obtained in Example 2) is as follows: (1) Preparation of coating solution The coating system was prepared according to the following mass proportions: 12% of ethyl cellulose with a viscosity of 20 mPa·s was added, 1.0% of magnesium stearate was added, 85% of anhydrous ethanol was added, and the remaining components were made up with purified water.

[0060] Preparation process: First, add ethyl cellulose into anhydrous ethanol system in portions and stir until completely dissolved. Then, add magnesium stearate and mix well. Finally, slowly add purified water and stir continuously for 30 minutes. After mixing, filter through a 100-mesh sieve and set aside the filtrate for later use.

[0061] (2) Wurster bottom spray fluidized bed coating operation A bottom-spray type fluidized bed coating system was selected, with the following process parameters set: inlet air temperature controlled at 50℃, coating solution body temperature maintained at 35℃; atomization pressure set at 2.5 bar, coating solution spray rate at 5 mL / min, and equipment ventilation volume at 120 m³ / min. 3 / h; Based on the quality of the live bacteria powder (as core material) obtained in Example 2, the overall coating weight gain range was controlled to be 25%.

[0062] (3) Post-drying process After coating, the material continued to undergo fluidized bed drying at 40°C for 60 minutes. The final moisture content of the material was measured to be less than 6%.

[0063] (4) Finished product sorting and packaging The dried material is sieved through an 80-mesh screen to remove lumps and large particles; then it is individually packaged in aluminum foil bags, with each bag containing 2g. The finished product is stored in a dark, sealed environment.

[0064] Example 6 The microencapsulation preparation of Akkermansia mesophila SuperAKK 001 live bacterial powder (obtained in Example 2) is as follows: (1) Preparation of coating solution The coating system was prepared according to the following mass ratios: 10% of ethyl cellulose with a viscosity of 15 mPa·s was added, 0.8% of magnesium stearate was added, 80% of anhydrous ethanol was added, and the remaining components were made up with purified water.

[0065] Preparation process: First, add ethyl cellulose into anhydrous ethanol system in portions and stir until completely dissolved. Then, add magnesium stearate and mix well. Finally, slowly add purified water and stir continuously for 30 minutes. After mixing, filter through a 100-mesh sieve and set aside the filtrate for later use.

[0066] (2) Wurster bottom spray fluidized bed coating operation A bottom-spray type fluidized bed coating system was selected, with the following process parameters set: inlet air temperature controlled at 45℃, coating solution body temperature maintained at 32.5℃; atomization pressure set at 2.0 bar, coating solution spray rate at 4 mL / min, and equipment ventilation volume at 100 m³ / min. 3 / h; Based on the quality of the live bacteria powder (as core material) obtained in Example 2, the overall coating weight gain was controlled to be within 20%.

[0067] (3) Post-drying process After coating, the material continued to undergo fluidized bed drying at 38°C for 45 minutes. The final moisture content of the material was measured to be less than 6%.

[0068] (4) Finished product sorting and packaging The dried material is sieved through a 70-mesh screen to remove lumps and large particles; then it is individually packaged in aluminum foil bags, with each bag containing 2g. The finished product is stored in a dark, sealed environment.

[0069] Example 7 Microencapsulation formulation of *Ackermania superakok* 001 post-vitamin powder (obtained in Example 3) The preparation method is the same as in Example 4, except that the live bacteria powder obtained in Example 2 in step (2) of Example 4 is replaced with the post-vitamin powder of Akkermansia melanophila SuperAKK 001 obtained in Example 3.

[0070] Example 8 Microencapsulation formulation of *Ackermania superakok* 001 post-vitamin powder (obtained in Example 3) The preparation method is the same as in Example 5, except that the live bacteria powder obtained in Example 2 in step (2) of Example 5 is replaced with the post-vitamin powder of Akkermansia melanophila SuperAKK 001 obtained in Example 3.

[0071] Example 9 Microencapsulation formulation of *Ackermania superakok* 001 post-vitamin powder (obtained in Example 3) The preparation method is the same as in Example 6, except that the live bacteria powder obtained in Example 2 in step (2) of Example 6 is replaced with the post-vitamin powder of Akkermansia melanophila SuperAKK 001 obtained in Example 3.

[0072] Experimental Example 1 The microcapsule formulations obtained in Examples 4 to 9 were subjected to quality evaluation as shown in Table 1 below, and the results are shown in Table 1.

[0073] Table 1. Microcapsule quality evaluation results

[0074] Experimental Example 2 Single-factor controlled experiment on key parameters of coating solution To verify the irreplaceability of the coating solution formulation of this invention, using the SuperAKK 001 live bacteria powder obtained in Example 2 as the core material, and following the Wurster bottom-spray fluidized bed coating process described in Example 6 (material body temperature 32.5℃, coating weight gain 20%), single-factor investigations were conducted on four key parameters: ethyl cellulose viscosity, ethyl cellulose addition ratio, magnesium stearate addition ratio, and anhydrous ethanol content. The survival rate, encapsulation efficiency, and live bacteria count of the microcapsule product in simulated gastric fluid at pH 3.0 at 37℃ for 2 hours were used as the main evaluation indicators, with each experiment repeated in triplicate.

[0075] (1) Effect of ethyl cellulose (EC) viscosity on the acid resistance of microcapsule formulations With a fixed addition ratio of 10% ethyl cellulose, 1.0% magnesium stearate, and 80% anhydrous ethanol, coating solutions with viscosities of 5, 10, 15, 20, and 30 mPa·s were prepared, and coating was carried out using the same process. The results are shown in Table 2.

[0076] Table 2. Effect of ethyl cellulose viscosity on the acid resistance of microcapsules

[0077] Note: Survival rate was calculated based on the number of viable bacteria before coating; for comparisons between groups within the EC viscosity range of 10-20 mPa·s and groups outside the range, P < 0.05.

[0078] Table 2 shows that when the viscosity of ethyl cellulose is below 10 mPa·s, the film-forming strength is insufficient, and the coated film ruptures rapidly in simulated gastric fluid, with a survival rate of only 45.2%. When the viscosity is above 20 mPa·s, the excessively high solution viscosity leads to coarse atomized droplets, particle agglomeration, and a significant decrease in coating uniformity. The survival rate is significantly better in the viscosity range of 10–20 mPa·s than outside this range (P < 0.05), with 15 mPa·s being the optimal range.

[0079] (2) Effect of ethyl cellulose (EC) addition ratio on acid resistance of microcapsule formulations With a fixed ethyl cellulose viscosity of 15 mPa·s, a magnesium stearate addition ratio of 1.0%, and anhydrous ethanol content of 80%, the ethyl cellulose addition ratios were set to 4%, 6%, 8%, 10%, 12%, and 16%, respectively, and the coating was carried out using the same process. The results are shown in Table 3.

[0080] Table 3. Effect of Ethyl cellulose addition ratio on the acid resistance of microcapsules

[0081] Note: When comparing groups with EC addition ratios of 8% to 12% with groups outside the range, P < 0.05.

[0082] Table 3 shows that when the ethyl cellulose content is below 8%, the coating film thickness is insufficient, and gastric juice penetration leads to the inactivation of a large number of viable bacteria, with a survival rate of only 38.5%~62.1%. When the content is above 12%, the solution viscosity increases sharply, resulting in uneven atomization, particle agglomeration, and a significant decrease in both encapsulation efficiency and survival rate. All indicators within the 8%~12% range are significantly better than those outside the range (P<0.05), with 10% being the optimal value.

[0083] (3) Effect of magnesium stearate addition ratio on acid resistance of microcapsule formulations With a fixed ethyl cellulose viscosity of 15 mPa·s, an addition ratio of 10%, and an anhydrous ethanol content of 80%, magnesium stearate was added at ratios of 0%, 0.3%, 0.5%, 1.0%, and 1.5%, respectively, and coated using the same process. The results are shown in Table 4.

[0084] Table 4. Effect of magnesium stearate addition ratio on the acid resistance of microcapsule formulations.

[0085] Note: When comparing groups with magnesium stearate content within the range of 0.5% to 1.0% with groups outside the range, P < 0.05.

[0086] Table 4 shows that when magnesium stearate, as both an anti-adhesion agent and a pore-forming agent, is added at a concentration below 0.5%, the particles severely agglomerate, resulting in uneven coating and a survival rate of only 58.6%–72.8%. At concentrations above 1.0%, the excessive hydrophobicity leads to decreased wettability and slow dissolution of the microcapsules in artificial intestinal fluid, along with the appearance of white precipitates on the surface, reducing the survival rate to 68.2%. A concentration within the range of 0.5%–1.0% balances anti-adhesion and dissolution performance, resulting in a significantly better survival rate than outside this range (P < 0.05).

[0087] (4) Effect of anhydrous ethanol ratio on the acid resistance of microcapsule formulations With a fixed ethyl cellulose viscosity of 15 mPa·s, an addition ratio of 10%, and a magnesium stearate addition ratio of 1.0%, the proportion of anhydrous ethanol was set to 65%, 70%, 75%, 80%, 85%, and 95% (the remainder being purified water), and coating was carried out using the same process. The results are shown in Table 5.

[0088] Table 5. Effect of anhydrous ethanol content on the acid resistance of microcapsule formulations.

[0089] Note: When comparing groups with ethanol content of 75% to 85% with groups outside the range, P < 0.05.

[0090] As shown in Table 5, when the ethanol content is below 75%, the solvent boiling point is too high, the drying rate is insufficient, and residual moisture causes particle clumping and bacterial powder to become damp and inactive, resulting in a survival rate of only 48.6%~65.3%. When the ethanol content is above 85%, the solvent evaporates too quickly, the coating film solidifies rapidly, producing a large number of micropores, and gastric juice permeates through the pores, reducing the survival rate to 55.8%. The solvent evaporation rate and film formation rate are best matched within the range of 75%~85%, and the survival rate is significantly better than outside this range (P<0.05).

[0091] As can be seen from Tables 2 to 5, the four component parameters of the coating solution of the present invention—ethyl cellulose viscosity of 10~20 mPa·s, addition ratio of 8%~12%, magnesium stearate of 0.5%~1.0%, and anhydrous ethanol of 75%~85%—form a precise ratio window with mutual synergy. Any deviation from any parameter will lead to a significant decrease in the acid resistance of the microcapsules (P<0.05), confirming that this ratio is irreplaceable.

[0092] Experimental Example 3 Single-factor control experiment of key parameters of fluidized bed process To verify the irreplaceability of the fluidized bed coating process parameters of this invention, under the preferred coating solution formulation obtained in Experimental Example 2, single-factor investigations were conducted on two key process parameters: material temperature and coating weight gain. In addition to acid resistance survival rate, encapsulation efficiency, and viable cell count, the evaluation index included the dissolution rate of artificial intestinal fluid (pH 6.8, containing 0.3% bile salts) at 37°C for 45 minutes to evaluate the enteric release characteristics of the microcapsule formulation.

[0093] (1) Effect of material temperature on the acid resistance of microcapsule formulations With a fixed optimal coating solution formulation (ethyl cellulose viscosity 15 mPa·s, addition ratio 10%, magnesium stearate 1.0%, anhydrous ethanol 80%) and a coating weight gain of 20%, the material body temperature was controlled at 20, 25, 30, 32.5, 35, and 40℃ by adjusting the inlet air temperature, while keeping other process parameters unchanged. The results are shown in Table 6.

[0094] Table 6. Effect of material bulk temperature on the acid resistance of microcapsule formulations.

[0095] Note: When comparing groups with material temperatures within the range of 30~35℃ with groups outside the range, P < 0.05.

[0096] Table 6 shows that when the material's bulk temperature is below 30℃, the ethanol evaporation rate is lower than the coating solution spraying rate, resulting in solvent residue on the particle surface and causing agglomeration. The coating film is discontinuous, with a survival rate of only 48.5%–62.3%. Above 35℃, although the film formation rate is fast, *Ackermania pseudomallei* is a strict anaerobic bacterium and is heat-sensitive; at 40℃, the viable cell count drops from 7.2 × 10⁻⁶. 9 Reduced to 5.5×10 9 The cfu / g concentration decreased, and the survival rate dropped to 55.8%. The temperature range of 30~35℃ balances solvent evaporation efficiency and the heat tolerance limit of the bacteria, which is the special temperature window of this invention for AKK bacteria. Within this range, all indicators are significantly better than outside the range (P<0.05).

[0097] (2) Effect of coating weight gain on acid resistance and enteric release of microcapsule formulations With the optimal coating solution formulation and material temperature fixed at 32.5℃, the coating weight gain was controlled to be 5%, 10%, 15%, 20%, 25%, and 30% by adjusting the coating solution spraying time, while keeping other process parameters unchanged. The results are shown in Table 7.

[0098] Table 7. Effects of coating weight gain on acid resistance and enteric release of microcapsule formulations.

[0099] Note: For groups with a coating weight gain of 15%–25%, P < 0.05 compared to groups outside this range. The intestinal fluid dissolution rate at 45 min refers to the proportion of live bacteria released from the microcapsules after incubation at 37°C for 45 min in artificial intestinal fluid (pH 6.8, containing 0.3% bile salts).

[0100] Table 7 shows that when the coating weight gain is less than 15%, the coating thickness is insufficient to provide a complete acid-resistant barrier, and the survival rate is only 42.3%~68.5%. After the weight gain reaches 25%, the acid-resistant survival rate tends to plateau (80.2%~81.5%), but the intestinal fluid dissolution rate drops sharply from 82.1% to 55.3%~75.8% at 45 min, indicating that excessively thick coating severely hinders the release and colonization of live bacteria in the intestine. The optimal balance between acid resistance protection and enteric release is achieved within the range of 15%~25%, with 20% being the optimal value. Within this range, all indicators are significantly better than outside the range (P<0.05).

[0101] Based on Tables 6 and 7, the material body temperature of 30-35℃ and the coating weight gain of 15%-25% specified in this invention are key process parameter windows locked through systematic optimization, taking into account the strictly anaerobic and heat-sensitive physiological characteristics of Akkermansia myxophilus. When any parameter deviates from the specified range, the acid-resistant survival rate of the microcapsule product decreases by 28.9%-53.2% (P<0.05), or the enteric release rate decreases to 55.3%, confirming that the above process parameters are irreplaceable.

[0102] The microcapsule formulation prepared by the formula and process of this invention has a moisture content controlled below 6%. Combined with the aluminum foil sealing and light-proof storage method, it effectively improves the product's stability at room temperature and solves the industry problem of easy activity decay of traditional AKK powder.

[0103] Test Example 4 Evaluation of intestinal colonization and intestinal barrier function of the SuperAKK 001 microcapsule formulation obtained in Example 6 1. Dynamic detection of fecal viable bacteria count Healthy, SPF-grade male ICR mice, weighing (20±2) g and 6 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After purchase, they underwent a one-week acclimatization period. The rearing conditions were: ample food and water, temperature 25℃, and a 12-hour light / 12-hour dark cycle.

[0104] After mice had adapted to the environment for one week, mice with abnormal conditions were removed and randomly divided into three groups of 10 mice each: blank control group, SuperAKK 001 uncoated live bacteria group, and SuperAKK 001 live bacteria microcapsule preparation group. Mice in the SuperAKK 001 live bacteria microcapsule group were administered a uniform suspension of SuperAKK 001 live bacteria microcapsule preparations prepared with sterile physiological saline (5 million CFU / day per mouse) by gavage. Mice in the SuperAKK 001 uncoated live bacteria group were administered SuperAKK 001 live bacteria preparations dissolved in physiological saline by gavage (5 million CFU / day per mouse). The control group was given an equal volume of physiological saline. The mice were administered the mixture by gavage for 2 consecutive weeks. During the experiment, the mice ate and drank normally. Fresh fecal samples were collected from each group of mice at 0h, 6h, 12h, 24h, 48h, and 72h after gavage. The fecal samples were accurately weighed, and 9 times the volume of sterile PBS was added. The mixture was vortexed to thoroughly mix and prepare a 10% fecal homogenate.

[0105] A series of diluents were prepared using a tenfold serial dilution method. 100 μL of each diluent was spread onto BHI selective plates containing 0.2% mucin. After strict anaerobic incubation at 37°C for 72 h, the number of colonies was counted. The results were expressed as the number of viable AKK bacteria per gram of feces (CFU / g).

[0106] The results are shown in Table 8. No bacteria were detected in the blank control group. There was a significant difference in bacterial count between the uncoated SuperAKK 001 live bacteria group and the SuperAKK 001 live bacteria microcapsule preparation group (P < 0.05), indicating that microcapsule coating significantly improved the survival rate of AKK bacteria after passing through the stomach. After 24 hours, the fecal live bacteria count in the uncoated SuperAKK 001 live bacteria group had decreased to 10. 4 Below CFU / g, it is basically undetectable after 48 hours; however, the SuperAKK 001 live bacteria microcapsule preparation group still showed a detectable level of 3.6 × 10⁻⁶ after 72 hours. 4 CFU / g of live bacteria. Microencapsulation increased the number of live bacteria in feces by approximately 770 times over 24 hours, demonstrating that the coating technology effectively improves the in vivo survival time of the strains.

[0107] Table 8 Results of dynamic detection of fecal viable bacteria count

[0108] Note: ND indicates not detected; # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the SuperAKK 001 uncoated live bacteria group (P < 0.05). 2. Detection of colonization distribution in different segments of the intestine After 14 days of continuous gavage, five mice from each group were randomly selected and sacrificed. Under aseptic conditions, the contents of the duodenum, jejunum, ileum, and colon were collected. The abundance of SuperAKK 001 bacteria in each intestinal segment was detected by qPCR. Quantitative detection was performed using AKK-specific primers (forward: 5'-CAGCACGTGAAGGTGGGGAC-3' (SEQ ID NO.4), reverse: 5'-CCTTGCGGTTGGCTTCAGAT-3' (SEQ ID NO.5)). The results are expressed as the 16S rRNA gene copy number of AKK bacteria per gram of contents. The results are shown in Table 9.

[0109] Table 9. Detection of colonization distribution in different segments of the intestine.

[0110] Note: ND indicates not detected (below the detection limit); # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the SuperAKK 001 uncoated live bacteria group (P < 0.05). As shown in Table 9, SuperAKK 001 primarily colonizes the colon, which is consistent with the physiological characteristic of AKK bacteria preferring a mucus-rich environment in the large intestine. The colonic colonization rate of the SuperAKK 001 live bacteria microcapsule preparation group reached 9.5 × 10⁻⁶. 8 The copy number / g was approximately 1100 times that of the uncoated live bacteria group of SuperAKK 001, demonstrating that microencapsulation technology significantly enhances the intestinal colonization ability of the strain.

[0111] 3. Intestinal barrier function testing Immunohistochemistry of tight junction proteins: Mid-colon tissue from mice in each group was collected, fixed in 4% paraformaldehyde, routinely embedded in paraffin, and sectioned. Immunohistochemistry was used to detect the expression of tight junction proteins ZO-1 and Occludin. Image-Pro Plus software was used to analyze the average optical density (AOD) of positive staining, and the AOD value was used to represent the relative protein expression level.

[0112] Serum endotoxin (LPS) detection: Blood was collected from the orbital region of mice, serum was separated, and the LPS content in the serum was detected using the Limulus Amebocyte Lysate (LAL) endpoint colorimetric method, strictly following the kit instructions. The results are shown in Table 10.

[0113] Table 10 Results of intestinal barrier function testing

[0114] Note: # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the SuperAKK 001 uncoated live bacteria group (P < 0.05). As shown in Table 10, there was a significant difference between the uncoated SuperAKK 001 live bacteria group and the SuperAKK 001 live bacteria microcapsule preparation group (P < 0.05), indicating that the microcapsule-coated SuperAKK 001 can significantly upregulate the expression of colonic tight junction proteins ZO-1 and Occludin, while significantly reducing serum LPS levels. This demonstrates that SuperAKK 001 effectively improves intestinal barrier function, reduces endotoxin entry into the blood, helps alleviate systemic low-grade inflammation, and provides intestinal-level mechanistic support for the improvement of skin photoaging.

[0115] Experimental Example 5 In vitro cell efficacy verification experiment Experimental cells: Human skin fibroblasts (HSF) (SV40 immortalized human skin fibroblasts), purchased from a reputable cell collection center in China, with complete STR identification reports and cell traceability certificates, suitable for standardized cell efficacy verification experiments. Cells were routinely cultured in DMEM high-glucose medium containing 10% fetal bovine serum at 37°C and 5% CO2.

[0116] Experimental groups: blank control group, UVB model group, SuperAKK 001 live bacteria microcapsule preparation group (prepared in Example 6), and SuperAKK 001 postbiotic microcapsule preparation group (prepared in Example 9). Except for the blank group, the other groups were irradiated with UVB (irradiation dose 30 mJ / cm) to construct a skin photodamage cell model. The SuperAKK 001 live bacteria microcapsule preparation group and the SuperAKK 001 postbiotic microcapsule preparation group were treated with SuperAKK 001 live bacteria microcapsule preparation and postbiotic microcapsule preparation 24 hours in advance. The amount added to both groups was 100 μg / mL, based on the mass of the preparation.

[0117] 1. Fibroblast proliferation viability assay (CCK-8 assay) Skin fibroblasts are the core cells for the synthesis of dermal collagen and elastic fibers, and their cell proliferation activity directly reflects the cell's ability to survive, grow, and repair.

[0118] Experimental Methods: Human immortalized skin fibroblasts (HSFs) in the logarithmic growth phase were selected and digested with trypsin. After centrifugation and resuspending, the cell suspension concentration was adjusted. Cells were seeded into 96-well plates at appropriate densities, with a total culture medium volume of 200 μL per well. The plates were incubated statically at 37°C and 5% CO2 until complete cell adhesion. After cell adhesion, except for the blank control group, all other groups received 30 mJ / cm² treatment. 2A skin photodamage cell model was constructed by irradiating the cells with UVB radiation. The SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 postbiotic microcapsule group were treated with SuperAKK 001 live bacteria microcapsule preparation and SuperAKK 001 postbiotic microcapsule preparation culture medium, respectively. The blank control group and the UVB model group were treated with equal volumes of complete culture medium. Each group had six replicates. After 24 h of continuous culture, 10 μL of CCK-8 assay reagent was added to each well in the dark, and the cells were incubated for another 2 h in the dark. The absorbance (OD value) at 450 nm was measured using a microplate reader to calculate cell proliferation activity.

[0119] Cell viability calculation formula: Cell viability (%) = (OD value of experimental group - OD value of blank background) / (OD value of blank control group - OD value of blank background) × 100%. The results are shown in Table 11.

[0120] Table 11 Results of Fibroblast Proliferation Viability Detection

[0121] Note: # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the model group (P < 0.05). As shown in Table 11, the cell proliferation activity in the UVB model group was significantly reduced, with cell viability only 52.36% of that in the blank control group, showing a significant difference (P < 0.05). Compared with the UVB model group, the cell proliferation activity in the SuperAKK 001 live bacteria microcapsule preparation group increased to 92.97%, and the cell proliferation activity in the SuperAKK 001 postbiotic microcapsule preparation group increased to 93.18%, showing a significant difference compared with the UVB model group (P < 0.05), but no significant difference compared with the blank control group (P > 0.05). This demonstrates that the SuperAKK 001 microcapsule preparation and the postbiotic microcapsule preparation can effectively reverse the inhibition of fibroblast proliferation caused by UVB damage and activate the skin cell regeneration capacity.

[0122] 2. Fibroblast migration and repair assay (scratch assay) Experimental Methods: HSF skin fibroblasts in the logarithmic growth phase were collected, digested, resuspended, and their concentration adjusted before being seeded into 6-well culture plates. Cells were cultured under standard conditions until they filled the wells and formed a dense monolayer. Using a 200 μL sterile pipette tip, uniform vertical scratches were made on the surface of the culture plate, ensuring consistent scratch width across all experimental groups. After scratching, the cells were gently washed three times with sterile PBS buffer to remove floating cells and debris, eliminating experimental interference. Experimental grouping, modeling, and intervention methods were the same as in the CCK-8 experiment described above. The blank control group and UVB model group received equal volumes of complete culture medium, with three replicates per group. After scratching, the cells were incubated at 37℃ with 5% CO2 for 24 hours. The scratch area width was recorded under a microscope at 0h and 24h. ImageJ software was used to calculate the scratch healing area and the 24h scratch healing rate. The formula for calculating the scratch healing rate is: Scratch healing rate (%) = (0h scratch width - 24h scratch width) / 0h scratch width × 100%.

[0123] The experimental results are shown in Table 12. As shown in Table 12, after 24 hours of scratch culture, the scratch healing rate of the UVB model group was only 21.31%, and the cell migration ability was severely impaired, which was significantly different from the blank control group (P < 0.05). Compared with the UVB model group, the scratch healing rate of the SuperAKK 001 live bacteria microcapsule preparation group was 86.17%, and the scratch healing rate of the SuperAKK 001 postbiotic microcapsule preparation group was 85.68%, which were significantly different from the UVB model group (P < 0.05) but not significantly different from the blank control group (P > 0.05). This demonstrates that the SuperAKK 001 microcapsule preparation and the postbiotic microcapsule preparation can significantly promote the migration and repair of photodamaged skin cells and help repair skin wounds and barriers.

[0124] Table 12 Results of Fibroblast Migration Repair Assay (Scratch Assay)

[0125] Note: # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the model group (P < 0.05). 3. Detection of gene and protein expression related to collagen and hyaluronic acid synthesis (qPCR and Western blot methods) COL1A1 is the gene encoding the α1 chain of type I collagen. Its translation product, COL1A1 protein, is the most important subunit of type I collagen in the dermis, determining the skin's dermal tension, firmness, and structural integrity. HAS2 is the most important rate-limiting enzyme in dermal fibroblasts, catalyzing the polymerization of glucuronic acid and N-acetylglucosamine to produce high molecular weight hyaluronic acid (HA), thus determining the total amount of HA synthesized in the dermis.

[0126] Western blot protein expression detection experimental method Cells from each group were collected and lysed thoroughly on ice for 30 min with RIPA lysis buffer containing protease inhibitors. The cells were then centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected to obtain total protein. Protein concentration was determined using a BCA protein quantification kit, and the loading amount was standardized for each group. After protein separation by SDS-PAGE gel electrophoresis, the proteins were wet-transferred to PVDF membranes and blocked with 5% skim milk at room temperature for 1 h. Primary antibodies COL1A1, HAS2, and the internal control GAPDH were added, and the membranes were incubated overnight at 4°C. The following day, the membranes were washed three times with TBST for 10 min each time, followed by incubation with the corresponding secondary antibody at room temperature for 1 h, and then washed again with TBST. Imaging was performed using an ECL chemiluminescence kit, and the band gray values ​​were quantitatively analyzed using ImageJ software to calculate the relative expression level of the target protein.

[0127] The experimental results are shown in Table 13. The expression levels of COL1A1 and HAS2 genes and proteins in the UVB model group were significantly downregulated, showing a significant difference compared to the blank control group (P < 0.05). After intervention with SuperAKK 001 microcapsule live bacteria and post-biotic microcapsule preparations, the expression levels of various skin matrix synthesis-related indicators were significantly upregulated in a dose-dependent manner. The SuperAKK 001 live bacteria microcapsule preparation group and the SuperAKK 001 post-biotic microcapsule preparation group showed significant differences compared to the UVB model group (P < 0.05), but no significant differences compared to the blank control group (P > 0.05). This demonstrates that SuperAKK 001 microcapsule preparations and post-biotic microcapsule preparations can effectively activate the skin's autologous collagen and hyaluronic acid synthesis pathways, improving the skin matrix loss and dermal thinning caused by photoaging.

[0128] Table 13 Results of gene and protein expression detection related to collagen and hyaluronic acid synthesis

[0129] Note: # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the model group (P < 0.05). 4. Detection of intracellular reactive oxygen species (ROS) content (DCFH-DA fluorescence method) ROS is a collective term for oxygen-containing reactive substances in cells, such as superoxide anion, hydrogen peroxide, and hydroxyl radicals, and is a core marker of oxidative stress. Normal levels of ROS participate in cell signaling regulation; excessive ROS attack cell membranes, DNA, and proteins, inducing lipid peroxidation, fibroblast senescence and apoptosis, inhibiting collagen and hyaluronic acid synthesis, and activating collagen degradation pathways, thus being an initiating factor of photoaging of the skin.

[0130] Experimental Methods: Intracellular ROS accumulation levels were detected using the DCFH-DA fluorescent probe method. Logarithmically growing HSF human skin fibroblasts were seeded at a concentration of 1 × 10⁶ cells per well in 24-well cell culture plates and cultured overnight for adhesion. Experimental grouping, UVB modeling, and SuperAKK 001 microcapsule formulation intervention protocols were the same as described in the previous cell experiments, with three replicates per group. After the intervention period for each group, the original culture medium was removed from the wells. The DCFH-DA fluorescent probe was diluted to 10 μmol / L using serum-free DMEM medium and added to the wells, incubated at 37°C in the dark for 20 min. After incubation, the probe solution was aspirated, and the cells were washed three times with pre-cooled sterile PBS to thoroughly remove any undeclared free probe. The fluorescence intensity of each group was observed using an inverted fluorescence microscope, and the average fluorescence intensity was quantitatively detected using flow cytometry. The control group was used as a reference, and the fluorescence value of the model group was set as 100%. The relative ROS content of each group was calculated.

[0131] The results are shown in Table 14. After UVB irradiation, the UVB model group showed a significant accumulation of ROS and a marked increase in fluorescence intensity, which was significantly different from the blank control group (P < 0.05). The SuperAKK 001 live bacteria microcapsule preparation group and the SuperAKK 001 postbiotic microcapsule preparation group showed significant differences compared with the UVB model group (P < 0.05), but no significant differences compared with the blank control group (P > 0.05). This demonstrates that the SuperAKK 001 microcapsule preparation and the postbiotic microcapsule preparation effectively alleviate UVB-induced skin oxidative stress damage and block the oxidative stress-mediated skin photoaging cascade damage response.

[0132] Table 14 Results of intracellular reactive oxygen species (ROS) content detection

[0133] Note: # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the model group (P < 0.05). 5. Apoptosis detection (Annexin V-FITC / PI double staining flow cytometry) Apoptosis, or programmed cell death, is induced by UVB, high concentrations of ROS, and oxidative stress, leading to a large number of apoptotic cells in the skin. Surviving fibroblasts are the core carriers for the synthesis of collagen and hyaluronic acid. An increased apoptosis rate directly leads to a sharp decline in the number of dermal cells and a decrease in the ability to synthesize extracellular matrix, ultimately resulting in photoaging characteristics such as dermal atrophy, loose skin, and wrinkles.

[0134] Experimental Methods: The apoptosis level of each group was detected by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit. Logarithmically growing HSF human skin fibroblasts were harvested, and after adjusting the cell concentration, 2 × 10⁶ cells per well were seeded into 6-well cell culture plates and cultured overnight for adhesion. The experimental grouping, UVB modeling method, and SuperAKK 001 microcapsule intervention protocol were the same as described in the in vitro cell experiments, with 3 replicates per group. After 24 h of intervention, the cell culture medium was discarded, and the cells were gently washed twice with pre-cooled PBS. Trypsin was added to digest and collect the cells. The cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was collected. The cells were resuspended in 1 × Binding Buffer to adjust the cell concentration to 1 × 10⁶ cells / mL. 100 μL of the cell suspension was transferred to flow cytometry tubes, and 5 μL of Annexin V-FITC and 5 μL of PI staining solution were added in the dark. The mixture was gently mixed and stained at room temperature in the dark for 15 min. After staining, add 400 μL of 1×Binding Buffer, mix gently, and use flow cytometry within 1 hour to analyze the proportion of early apoptotic, late apoptotic, and total apoptotic cells, and calculate the total apoptosis rate of each group.

[0135] The experimental results are shown in Table 15. The apoptosis rate of cells in the UVB model group was as high as 37.62%, indicating that UVB ultraviolet radiation could significantly induce abnormal apoptosis of fibroblasts, which was significantly different from the blank control group (P < 0.05). The apoptosis rates of cells in the SuperAKK 001 live bacteria microcapsule preparation group and the SuperAKK 001 postbiotic microcapsule preparation group decreased to 5.14% and 5.12%, respectively, which were significantly different from the UVB model group (P < 0.05) but not significantly different from the blank control group (P > 0.05). This demonstrates that the SuperAKK 001 microcapsule preparation and the postbiotic microcapsule preparation can effectively block the UVB-induced fibroblast apoptosis pathway, maintain the stability of dermal fibroblast numbers, and inhibit the photoaging process.

[0136] Table 15 Results of apoptosis detection

[0137] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0138] Experimental Example 6 Effects of SuperAKK 001 live bacteria microcapsule formulation (prepared in Example 6) and postbiotic microcapsule formulation (prepared in Example 9) on improving UVB-induced dry skin in mice. 1. Mouse model construction Fifty healthy SPF-grade ICR mice, female, weighing (20±2) g and 6 weeks old, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. After purchase, they underwent a one-week acclimatization period. The rearing conditions were: ample food and water, temperature 25℃, and a 12-hour light / 12-hour dark cycle.

[0139] After one week of acclimatization, mice exhibiting abnormal behavior were excluded, and the mice were randomly divided into four groups of ten mice each: a blank control group, a UVB model group, a SuperAKK 001 live bacteria microcapsule group, and a SuperAKK 001 postbiotic microcapsule group. Except for the blank control group, a 2cm × 3cm area was marked on the back of each mouse in the other groups, and the hair was shaved. The mice were then given a daily dose of 40 mJ / cm². 2 Mice were exposed to UVB radiation once daily for 8 weeks to establish a mouse model of UVB photoaging of the skin. The control group of mice did not receive UVB exposure and were fed normally.

[0140] Mice in the SuperAKK 001 live bacteria microcapsule group were administered a uniform suspension of SuperAKK 001 live bacteria microcapsule preparations prepared with sterile physiological saline by gavage (addition amount: 10 mg / kg body weight / day per mouse). Mice in the SuperAKK 001 postbiotic microcapsule preparation group were administered a uniform suspension of SuperAKK 001 postbiotic microcapsule preparations prepared with sterile physiological saline by gavage (addition amount: 10 mg / kg body weight / day per mouse). The gavage volume and preparation concentration were the same for both groups. The blank group and UVB model group were given the same amount of physiological saline. The mice were administered the preparations by gavage for 8 consecutive weeks. During the experiment, the mice ate and drank normally, and their body weight, skin condition, and eating and activity were recorded weekly.

[0141] 2. Observation of mouse condition During the experimental period, all mice showed no mortality, lethargy, or abnormal eating habits, and exhibited normal weight gain and no toxic reactions. In the UVB model group, the skin on the backs of mice gradually developed photoaging symptoms such as dryness, roughness, peeling, sagging, increased wrinkles, and pigmentation. The skin condition of mice in the SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 postbiotic microcapsule group was significantly better than that of the UVB model group; their skin was smooth and firm, with significant improvement in peeling, wrinkles, and dullness, approaching the condition of the blank control group.

[0142] Eight weeks after administration, the mice were sacrificed, and skin tissue from the lesion area on the back was collected. The skin moisture content was measured using a kit and instruments.

[0143] Skin hydration testing procedure: After euthanizing the mice, the skin on their backs was quickly dissected, excess hair and attached impurities were removed, and tissue fluid and blood were blotted dry with sterile filter paper. Using a high-precision skin hydration meter, under standard conditions of constant room temperature, no strong light, and no ventilation, three different testing points were selected in the central area of ​​each skin sample. Each point was tested three times consecutively, and the hydration content was recorded. The average value was taken as the skin hydration content of a single sample. The mean and standard deviation of each group were statistically analyzed to compare and analyze the skin moisturizing and repair effects.

[0144] The results are shown in Table 16. The skin moisture content of the UVB model group was as low as 45.31%, with poor moisturizing effect, which was significantly different from the blank control group (P < 0.05). The skin moisture content of the SuperAKK 001 live bacteria microcapsule preparation group and the SuperAKK 001 post-biotic microcapsule preparation group was significantly increased, higher than that of the UVB model group, and there was a significant difference compared with the UVB model group (P < 0.05). There was no significant difference compared with the blank control group (P > 0.05), indicating that the SuperAKK 001 microcapsule preparation and the post-biotic microcapsule preparation have a certain effect on improving skin dryness caused by UVB.

[0145] Table 16 Results of Skin Moisture Content Detection

[0146] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0147] Experimental Example 7 The effects of SuperAKK 001 live bacteria microcapsule formulation (prepared in Example 6) and postbiotic microcapsule formulation (prepared in Example 9) on improving UVB-induced collagen loss in mouse skin tissue. Hydroxyproline is a characteristic amino acid unique to collagen. It exists in trace amounts in elastin and a small amount of complement protein, and more than 90% of it is concentrated in collagen in skin tissue. The human body cannot obtain large amounts of it directly from food. It is produced by the hydroxylation of proline through prolyl hydroxylase and is the most classic and commonly used indicator for measuring the total collagen content in the skin.

[0148] The hydroxyproline content in the skin tissue from the dorsal lesion area of ​​mice sacrificed in Experiment Example 6 was detected.

[0149] Procedure for detecting hydroxyproline content in skin: Accurately weigh 30 mg of fresh skin tissue samples from each group of mice, cut them into small pieces, and add 9 times the volume of sterile physiological saline to prepare a 10% tissue homogenate. Place the homogenate in a centrifuge tube and centrifuge at 3000 rpm for 10 min at 4°C. Collect the supernatant for later use. Strictly follow the instructions of the hydroxyproline detection kit, adding the sample supernatant, reagent buffer, and chromogenic solution sequentially. Mix well and react in a boiling water bath for 20 min. After cooling to room temperature, centrifuge at 3500 rpm for 10 min. Measure the absorbance of each well at 550 nm using the supernatant. Calculate the hydroxyproline content in the sample according to the standard curve, in μg / mg, to characterize the total collagen content of the skin tissue.

[0150] The results are shown in Table 17. The UVB model group had low hydroxyproline content in the skin, indicating low total collagen content, which was significantly different from the blank control group (P < 0.05). The SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 post-biotic microcapsule group showed significantly increased hydroxyproline content in the skin, with significant differences compared to the UVB model group (P < 0.05), but no significant difference compared to the blank control group (P > 0.05). This indicates that the SuperAKK 001 microcapsule and post-biotic microcapsule preparations have a certain effect on improving collagen loss in skin tissue.

[0151] Table 17 Results of Skin Hydroxyproline Content Detection

[0152] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0153] Experimental Example 8 The effects of SuperAKK 001 live bacteria microcapsule formulation (prepared in Example 6) and postbiotic microcapsule formulation (prepared in Example 9) on improving UVB-induced oxidative aging of mouse skin. Superoxide dismutase (SOD) is a core antioxidant enzyme in the body that specifically removes excess superoxide anion free radicals in the skin, inhibits oxidative stress, and reduces the oxidative damage to collagen and elastin. It is one of the most classic indicators for evaluating skin's antioxidant and anti-photoaging properties.

[0154] MDA is the end metabolite of lipid peroxidation. Skin cell membranes and organelle membranes are rich in unsaturated fatty acids, which undergo lipid oxidation and degradation under ultraviolet radiation, oxidative stress, and free radical attack, ultimately producing malondialdehyde (MDA). MDA can not only directly damage cell membranes, but also cross-link with proteins and nucleic acids, causing dermal fibroblast aging, collagen degeneration, and dull skin due to aging. It is the most classic marker reflecting the degree of oxidative damage to the skin.

[0155] Skin SOD activity and MDA content were detected in the skin tissue of the dorsal lesion area of ​​mice sacrificed in Experiment Example 6.

[0156] Skin SOD activity assay procedure: Take the supernatant of the 10% skin tissue homogenate prepared above and use a superoxide dismutase (SOD) assay kit to detect SOD activity. Add the sample, substrate solution, and enzyme working solution sequentially according to the kit ratio, mix thoroughly, and incubate at 37℃ for 40 min. Add the stop solution to terminate the reaction, mix well, and let stand for 5 min. Measure the absorbance at 450 nm using a microplate reader. Calculate the skin tissue SOD activity according to the standard formula of the kit, with the unit being U / mg. The level of SOD activity directly reflects the endogenous antioxidant capacity of skin tissue.

[0157] Steps for detecting skin MDA content: Take the supernatant of skin tissue homogenate and use a malondialdehyde (MDA) detection kit to determine lipid peroxidation levels. Add the sample and MDA detection working solution sequentially, vortex to mix, seal, heat in a boiling water bath for 40 min, cool to room temperature under running water, centrifuge at 4000 rpm for 10 min, and measure the absorbance of the supernatant at 532 nm. Calculate the MDA content of the sample using a standard curve, expressed in nmol / mg. Higher MDA content indicates more severe oxidative damage and lipid peroxidation in the skin tissue.

[0158] The results are shown in Table 18. The UVB model group showed significantly decreased SOD activity and significantly increased MDA content, with significant differences compared to the blank control group (P < 0.05). The SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 postbiotic microcapsule group showed significantly increased SOD activity and decreased MDA content, with significant differences compared to the UVB model group (P < 0.05), but no significant differences compared to the blank control group (P > 0.05). This indicates that the SuperAKK 001 microcapsule and postbiotic microcapsule preparations have a certain effect on improving skin tissue oxidative aging.

[0159] Table 18 Results of Skin SOD Activity and MDA Content Detection

[0160] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0161] Experimental Example 9 Effects of SuperAKK 001 live bacteria microcapsule formulation (prepared in Example 6) and postbiotic microcapsule formulation (prepared in Example 9) on UVB-induced mouse skin tissue morphology. The epidermis consists of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. Normal, healthy skin has a thin and uniform epidermis. The dermis is the core of skin function, mainly composed of collagen fibers, elastic fibers, fibroblasts, and extracellular matrix, determining skin firmness. Dermal collagen fibers are large, cord-like, densely and neatly arranged, exhibiting an orderly wavy pattern. The gaps between collagen fibers are small, and they appear as a bright, deep blue when stained by Masson's stain. Fibroblasts are abundant in the dermis.

[0162] Skin tissue from the same area on the back of mice euthanized in Experiment 6 was fixed in 4% paraformaldehyde for 24 hours, then routinely dehydrated, cleared, and embedded in paraffin to prepare 4μm thick continuous sections.

[0163] HE staining: Sections were routinely dewaxed to water, stained with hematoxylin for 5 min, rinsed with tap water, differentiated with 1% hydrochloric acid alcohol for 3 s, rinsed with tap water to regain blue color, stained with eosin for 3 min, dehydrated with graded alcohols, cleared with xylene, and mounted with neutral resin. The skin tissue structure was observed under an optical microscope, and the thickness of the epidermis, dermis, and collagen fiber arrangement were measured.

[0164] The results are shown in Table 19 and Figure 1 .Depend on Figure 1 It can be seen that the epidermis of the blank control group (A) is thin and uniform, composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale, with regular cell arrangement; the dermis is thicker, with dense and orderly collagen fibers arranged in an orderly wavy pattern, small gaps between collagen fibers, and abundant fibroblasts. The UVB model group (B) shows significant epidermal thickening (stratum spinosum hypertrophy), a significant increase in the number of cell layers, exhibiting typical characteristics of acanthosis; the dermis is thinner, with disordered, sparse, and broken collagen fibers, and increased gaps, showing typical pathological changes of photoaging. The SuperAKK 001 live bacteria microcapsule preparation group (C) and the post-biotic microcapsule preparation group (D) show epidermal thickness restored to near-normal levels, with cell arrangement becoming more regular; the dermis is thickened, collagen fiber arrangement is significantly improved, density is restored, gaps are reduced, and morphology is close to the blank control group. Combined with... Figure 1 As shown in Table 19, the UVB model group showed significant differences in epidermal thickening (a typical characteristic of photoaging), dermal thinning, and disordered and sparse collagen fiber arrangement compared to the blank control group (P < 0.05). The SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 postbiotic microcapsule group showed significant differences in epidermal and dermal thickness and collagen fiber arrangement compared to the UVB model group (P < 0.05), but no significant differences compared to the blank control group (P > 0.05). This indicates that after intervention with SuperAKK 001 microcapsule preparations and postbiotic microcapsule preparations, epidermal thickness was significantly reduced, dermal thickness increased, and collagen fiber arrangement became more dense and orderly. Therefore, SuperAKK 001 microcapsule preparations and postbiotic microcapsule preparations have a certain effect on improving the morphological changes of mouse skin tissue caused by UVB.

[0165] Table 19 Statistical analysis of HE staining results

[0166] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0167] Masson trichrome staining: Sections were dewaxed to water, stained with Weigert iron hematoxylin for 5 min, differentiated with acidic ethanol, rinsed with running water to regain blue color, stained with Ponceau S and acidic fuchsin for 8 min, rinsed with 0.2% glacial acetic acid solution, differentiated with phosphomolybdic acid solution for 5 min, counterstained with aniline blue for 5 min, rinsed with 0.2% glacial acetic acid solution, dehydrated, cleared, and mounted. Under a microscope, the distribution and arrangement of collagen fibers were observed; collagen fibers appeared blue, muscle fibers appeared red, and cell nuclei appeared bluish-black. Image-Pro Plus software was used to quantitatively analyze the area ratio of collagen fibers in the dermis.

[0168] COL1A1 immunohistochemical staining: Sections were dewaxed to water, subjected to high-temperature antigen retrieval with citrate buffer, incubated with 3% H2O2 for 10 min to block endogenous peroxidase, blocked with 5% BSA for 30 min, and COL1A1 primary antibody (1:200 dilution) was added and incubated overnight at 4°C. The next day, horseradish peroxidase-labeled secondary antibody was added, and the sections were incubated at 37°C for 30 min, followed by DAB staining, hematoxylin counterstaining of the nuclei, dehydration, clearing, and mounting. The distribution of positive staining was observed under a microscope, and the average optical density (AOD) was analyzed using Image-Pro Plus software.

[0169] The results are shown in Table 20 and... Figure 2 and Figure 3 .

[0170] In Masson's trichrome staining, collagen fibers appear blue, muscle fibers appear red, and cell nuclei appear bluish-black. Figure 2 As can be seen, the dermis of the blank control group (A) showed a large number of dark blue, coarse, cord-like collagen fibers, which were densely and orderly arranged and accounted for a high proportion of collagen fiber area. In the UVB model group (B), the blue collagen fibers in the dermis were significantly reduced, the fibers were thinner and broken, and the arrangement was disordered and sparse, while the red matrix component was significantly increased, indicating severe collagen degradation. In the SuperAKK 001 live bacteria microcapsule preparation group (C) and the post-biotic microcapsule preparation group (D), the blue collagen fibers in the dermis were significantly restored, the fibers were coarse and the arrangement was relatively dense and orderly, which was significantly different from the UVB model group and close to the blank control group, suggesting that microcapsule preparation intervention can effectively promote the recovery and remodeling of collagen fibers.

[0171] COL1A1 positive staining appears brownish-yellow (DAB staining), and the cell nucleus appears blue (hematoxylin counterstaining). From Figure 3As can be seen, the dermis of the blank control group (A) showed a large amount of brownish-yellow positive staining and abundant COL1A1 expression, indicating vigorous type I collagen synthesis. In the UVB model group (B), the brownish-yellow positive staining in the dermis was significantly reduced, with only a small amount scattered, indicating significant inhibition of type I collagen synthesis. In the SuperAKK 001 live bacteria microcapsule preparation group (C) and the post-biotic microcapsule preparation group (D), the brownish-yellow positive staining in the dermis was significantly restored, with a distribution density similar to the blank control group, suggesting that type I collagen synthesis was significantly restored after microcapsule intervention, demonstrating a significant protective and repairing effect against UVB-induced skin photoaging.

[0172] As shown in Table 20, the UVB model group showed a significant reduction in blue collagen fibers in the dermis and a significant downregulation of type I collagen expression compared to the blank control group (P < 0.05). The SuperAKK 001 live bacteria microcapsule group and the SuperAKK 001 post-biotic microcapsule group showed a recovery in collagen fiber area ratio to over 62%, with larger, denser collagen fibers and significantly restored type I collagen expression, showing a significant difference compared to the UVB model group (P < 0.05). However, there was no significant difference compared to the blank control group (P > 0.05). This indicates that intervention with SuperAKK 001 microcapsule and post-biotic microcapsule formulations demonstrated protective and collagen-promoting effects on skin collagen, meaning that SuperAKK 001 microcapsule and post-biotic microcapsule formulations have a certain effect on improving UVB-induced skin morphology in mice.

[0173] Table 20. Statistics of Masson trichrome staining and COL1A1 positive staining results

[0174] Note: # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the model group (P < 0.05).

[0175] comprehensive Figures 1-3 As shown in Tables 19 and 20, the three staining methods consistently confirmed from different perspectives (tissue structure / collagen distribution / target protein expression) that UVB irradiation can cause typical pathological changes in mouse skin photoaging (epidermal thickening, dermal thinning, collagen fiber degradation, and downregulation of COL1A1 expression). The intervention of SuperAKK 001 live bacteria microcapsule preparation and post-biotic microcapsule preparation can significantly improve the above-mentioned photoaging pathological changes, restore skin tissue morphology, collagen fiber distribution and type I collagen expression level to near normal, and further verify the protective and repairing effect of SuperAKK 001 microcapsule preparation on UVB-induced skin photodamage from the tissue morphology level.

[0176] Experimental Example 10 Parallel comparison experiment between the myxotrophic Akkermansia superAKK 001 obtained in Example 1 and the standard strain ATCC BAA-835 To demonstrate that the *Ackermania* SuperAKK 001 provided by this invention possesses significant physiological and efficacy advantages compared to the standard type strain ATCC BAA-835 of the same genus, the following three sets of parallel comparative experiments were conducted. In all comparative experiments, the two strains were operated in parallel under identical culture conditions, coating processes, gavage dosages, and detection methods; the only variable was the strain itself.

[0177] Standard control strain: Akkermansia myxophilus ATCC BAA-835, purchased from the American Type Culture Collection (ATCC), is an internationally recognized model strain of Akkermansia myxophilus.

[0178] 1. Comparative experiment on acid resistance SuperAKK 001 and ATCC BAA-835 were activated and cultured to the end-log phase. After centrifugation to collect the bacterial cells, they were washed twice with sterile PBS and resuspended at a concentration of 1.0 × 10⁻⁶. 9 CFU / mL available for use.

[0179] Preparation of simulated gastric juice (SGF): NaCl 0.2g, pepsin 0.32g, distilled water to a final volume of 100mL, adjust pH to 2.0 and 1.5 respectively, and sterilize by filtration through a 0.22μm membrane. Preparation of simulated intestinal juice (SIF): KH₂PO₄ 0.68g, trypsin 7.72g, ox bile salts 0.3g, adjust pH to 6.8, distilled water to a final volume of 100mL, and sterilize by filtration through a 0.22μm membrane.

[0180] Add 1 mL of bacterial culture to 9 mL of simulated gastric or intestinal fluid and incubate anaerobically at 37°C. Take 1 mL samples at 0 h, 1 h, 2 h, 3 h, and 4 h, and immediately dilute each sample 10-fold. Spread 100 μL of each sample onto BHI plates containing 0.2% mucin and incubate anaerobically at 37°C for 72 h. Count the number of viable colonies. Survival rate (%) = (N...) t / N0)×100%, where N t The values ​​represent the number of viable bacteria measured after incubation time t (t corresponds to CFU / mL at time points 1h, 2h, 3h, and 4h), and N0 represents the number of viable bacteria at 0h (i.e., the initial colony count when simulated gastric / intestinal fluid is added, which is CFU / mL at t=0). Each group has 3 biological replicates. The results are shown in Table 21.

[0181] Table 21 Results of simulated gastric and intestinal fluids

[0182] Note: ND indicates not detected (below the detection limit); This indicates a comparison with the ATCC BAA-835 group (P < 0.05).

[0183] As shown in Table 21, after 4 hours of treatment in simulated gastric fluid at pH 2.0, the survival rate of SuperAKK 001 was 56.18%, significantly higher than that of ATCC BAA-835 (12.35%) (P < 0.05), representing an increase of approximately 4.5 times. Under extremely acidic conditions (pH 1.5), the survival rate of ATCC BAA-835 was only 1.25% after 3 hours and completely undetectable after 4 hours, while SuperAKK 001 maintained a survival rate of 15.28% after 4 hours, demonstrating that SuperAKK 001 possesses extremely acid-resistant physiological characteristics. After 4 hours of treatment in simulated intestinal fluid containing 0.3% bile salts, the survival rate of SuperAKK 001 (72.56%) was also significantly higher than that of ATCC BAA-835 (36.25%) (P < 0.05), representing an increase of approximately 2.0 times. This characteristic allows SuperAKK 001 to survive a higher proportion of the gastrointestinal tract after oral administration, providing a physiological basis for subsequent intestinal colonization and efficacy.

[0184] 2. Comparative experiment on intestinal colonization Fifty healthy male SPF-grade ICR mice, weighing (20±2) g and 6 weeks old, were purchased and randomly divided into 5 groups of 10 mice each after 1 week of acclimatization feeding: blank control group, ATCC BAA-835 uncoated live bacteria group, ATCC BAA-835 live bacteria microcapsule preparation group, SuperAKK 001 uncoated live bacteria group, and SuperAKK 001 live bacteria microcapsule preparation group.

[0185] The gavage dose for each group was uniformly 5×10 6 CFU / mouse / day (based on viable count), administered by gavage for 14 consecutive days, followed by 14 days of observation after drug withdrawal. Fresh feces were collected from mice in each group on days 1, 3, 7, and 14 after gavage, and on days 17, 21, and 28 after drug withdrawal. The feces were weighed and mixed with 9 volumes of sterile PBS, vortexed to prepare a 10% fecal homogenate. After a 10-fold serial dilution, 100 μL was spread onto BHI selective plates containing 0.2% mucin and anaerobically incubated at 37°C for 72 hours. The results are expressed as CFU / g feces. Results are shown in Table 22.

[0186] Table 22 Fecal Test Results

[0187] Note: ND indicates not detected; # indicates a comparison with the control group (P < 0.05), and # indicates a comparison with the ATCC BAA-835 treatment group (P < 0.05). 17d, 21d, and 28d are the monitoring points after drug withdrawal.

[0188] Fourteen days after gavage, five mice from each group were randomly selected and sacrificed. Under aseptic conditions, the contents of the duodenum, jejunum, ileum, and colon were collected. The abundance of AKK bacteria in each intestinal segment was detected using qPCR (primers: forward 5'-CAGCACGTGAAGGTGGGGAC-3' (SEQ ID NO.4), reverse 5'-CCTTGCGGTTGGCTTCAGAT-3' (SEQ ID NO.5)). Results were expressed as 16S rRNA gene copy number / g of contents. The results are shown in Table 23.

[0189] Table 23. Abundance of AKK bacteria in each intestinal segment.

[0190] Note: ND indicates not detected (below the detection limit); # indicates a comparison with the blank group (P < 0.05), and # indicates a comparison with the same treatment group of ATCCBAA-835 (P < 0.05).

[0191] As shown in Tables 22 and 23, under the condition of equal-dose gavage, the fecal viable bacteria count in the uncoated SuperAKK 001 group reached 8.85 × 10⁻⁶ on day 7. 4 The CFU / g was significantly higher than that of the uncoated ATCC BAA-835 group (1.21 × 10⁻⁶). 4 The CFU / g (P < 0.05) was approximately 7.3 times that of the standard strain, demonstrating that SuperAKK 001 itself has a stronger intestinal colonization capacity. After microencapsulation, the colonic colonization of the SuperAKK 001 microencapsulation group reached 3.25 × 10⁻⁶. 5 The copy number / g is 4.25 × 10⁻⁶ for the ATCC BAA-835 microcapsule group. 4 The number of copies / g was approximately 7.6 times higher than that of the control group (P < 0.05). 14 days after drug withdrawal (Day 28), 1.52 × 10⁻⁶ copies / g were still detectable in the SuperAKK 001 microcapsule group. 4 The CFU / g level was significantly lower in the uncoated ATCC BAA-835 group than in the microencapsulated ATCC BAA-835 group, with only 8.25 × 10⁻⁶ CFU / g detected. 2 The CFU / g result demonstrates that SuperAKK 001 possesses a stronger ability to persistently colonize the gut. This advantage is directly related to its extremely acid-resistant physiological characteristics; higher gastrointestinal survival rates allow more live bacteria to reach the intestinal colonization site.

[0192] 3. Comparative Experiment on Anti-Photoaging Effect Fifty healthy SPF-grade ICR mice, female, weighing (20±2) g, 6 weeks old, were purchased and randomly divided into 5 groups of 10 mice each after 1 week of acclimatization feeding: blank control group, UVB model group, ATCC BAA-835 live bacteria microcapsule preparation group (prepared in the same way as in Example 6), SuperAKK 001 live bacteria microcapsule preparation group (prepared in Example 6), and SuperAKK 001 postbiotic microcapsule preparation group (prepared in Example 9).

[0193] Except for the blank control group, a 2cm × 3cm area was marked on the back of mice in the other groups and the hair was shaved off. The mice were then given 40mJ / cm² of medication daily. 2 UVB irradiation was administered once daily for 8 weeks. The gavage dose for all groups was uniformly 10 mg / kg body weight / day (microcapsule preparations were administered by mass, with a live bacteria content of 10 billion / g in both groups, and the same volume was administered by gavage). The control group and the UVB model group were given the same volume of physiological saline, and the gavage was continued for 8 weeks.

[0194] Eight weeks after drug administration, mice were sacrificed, and skin tissue from the dorsal lesion area was collected. The following indicators were measured: skin moisture content (high-precision skin moisture analyzer), hydroxyproline content (alkaline hydrolysis kit), SOD activity (WST-1 method kit), MDA content (TBA method kit), epidermal and dermal thickness (HE staining microscopy), collagen fiber area ratio, and COL1A1 expression (Masson trichrome staining + immunohistochemistry). The detection methods were the same as those in the corresponding sections above and will not be repeated here. The results are shown in Table 24.

[0195] Table 24 Comparison of Anti-photoaging Effects Experiment Results

[0196] Note: # indicates comparison with the blank group (P < 0.05), # indicates comparison with the UVB model group (P < 0.05), and △ indicates comparison with the ATCC BAA-835 microcapsule group (P < 0.05).

[0197] As shown in Table 24, under identical coating process and gavage dosage conditions, the SuperAKK 001 microcapsule formulation group was significantly superior to the ATCC BAA-835 microcapsule formulation group in all detection dimensions (P < 0.05). Specifically: skin moisture content increased by 19.9%, hydroxyproline content increased by 27.8%, SOD activity increased by 25.7%, MDA content decreased by 28.9%, dermal thickness recovered to 84.2% of the control group, collagen fiber area ratio increased by 31.5%, and COL1A1 expression increased by 37.8%. These results indicate that SuperAKK 001 has significantly better anti-UVB photoaging efficacy than the standard strain ATCC BAA-835 of the same genus. This advantage is not due to the microcapsule coating process or dosage, but rather to the inherent biological characteristics of the strain itself. Based on the results of the acid resistance comparison and intestinal colonization comparison above, the excellent anti-photoaging effect of SuperAKK 001 is directly related to its stronger gastrointestinal survival rate and intestinal colonization ability. After more live bacteria colonize the intestine, it exerts a more significant systemic anti-photoaging effect through the gut-epidermal axis.

[0198] Through the above three sets of parallel comparative experiments, it was demonstrated that SuperAKK 001 has the following significant advantages over the standard strain ATCC BAA-835: (1) Extremely acid-resistant physiological characteristics, maintaining a survival rate of 15.28% after 4 hours under extremely acidic conditions of pH 1.5, while the standard strain could not be detected; (2) Stronger intestinal colonization ability, with a colonization amount 7.3 times that of the standard strain after 7 days under the same dose of gavage, and 1.52×10⁻⁶ can still be detected 14 days after drug withdrawal. 4 CFU / g, while the uncoated group of the standard strain has dropped below the detection limit; (3) Better anti-UVB photoaging effect. Under the same coating process and administration conditions, the improvement effect of 7 skin photoaging indicators is significantly better than that of the standard strain (P<0.05), with each indicator increasing by 19.9%~37.8%.

[0199] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A type of Akkermansia muciniphilis with cosmetic effects, SuperAKK 001, characterized in that, The strain of *Ackermania superakk 001* is classified as *Ackermania viride*. Akkermansia muciniphila It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC NO.47119.

2. A bacterial powder with cosmetic effects, characterized in that, The bacterial powder contains the *Ackermania superakok* 001 as described in claim 1.

3. The bacterial powder according to claim 2, characterized in that, When the bacterial powder is freeze-dried, it also contains a cryoprotectant composed of trehalose and glycerol; the concentration of the bacterial powder suspension is 1.0 × 10⁻⁶. 9 The volume-to-weight ratio of the bacterial powder suspension, trehalose, and glycerol, based on CFU / mL, is 100 mL: 10 g: 1 g. The bacterial powder suspension is obtained by mixing bacterial cells with skim milk.

4. An after-growth powder with cosmetic effects, characterized in that, The post-biotic powder is prepared from the *Ackermania superakans* SuperAKK 001 described in claim 1 through anaerobic culture, inactivation, concentration, and drying.

5. The epigenic powder according to claim 4, characterized in that, The anaerobic culture medium is BHI liquid medium, which contains, per 1L, 38g of brain heart extract powder, 5g of yeast extract, 5g of sodium chloride, 2g of porcine gastric mucin, 0.5g of L-cysteine ​​hydrochloride, and the remainder water; the pH of the BHI liquid medium is 6.5; the concentration is to concentrate the bacterial solution obtained from the anaerobic culture to 1 / 5 of the original volume; the drying includes freeze-drying, which includes the following steps: pre-freezing at -80℃ for 4h, then drying at -40℃ under vacuum <10Pa for 18h, and then drying at 26℃ for 6h.

6. A microcapsule formulation with cosmetic effects, characterized in that, The coating is obtained by coating the bacterial powder according to any one of claims 2 to 3 or the post-biotic powder according to any one of claims 4 to 5 as the core material; the coating solution used for coating is composed of 8% to 12% ethyl cellulose, 0.5% to 1.0% magnesium stearate, 75% to 85% anhydrous ethanol and the balance water in a mass ratio.

7. The microencapsulation formulation according to claim 6, characterized in that, The viscosity of the ethyl cellulose is 10-20 mPa·s; the weight gain of the coating is 15%-25% based on the core material mass; during coating, the inlet air temperature is 40-50℃, the coating solution temperature is 30-35℃, the atomization pressure is 1.5-2.5 bar, the coating solution spray rate is 2-5 mL / min, and the ventilation volume is 80-120 m³ / min. 3 / h.

8. The use of the Akkermansia melanophila SuperAKK 001 of claim 1, the bacterial powder of any one of claims 2-3, the post-biotic powder of any one of claims 4-5, or the microcapsule preparation of any one of claims 6-7 in the preparation of oral cosmetic preparations.

9. The application according to claim 8, characterized in that, The cosmetic treatments include inhibiting photoaging of the skin, repairing photoaging of the skin, promoting the repair of skin wounds and barriers, firming the skin, improving wrinkles, whitening and / or moisturizing.

10. The application according to claim 8, characterized in that, The cosmetic benefits include improving skin matrix loss and dermal thinning caused by photoaging, alleviating UV-induced skin oxidative stress damage, inhibiting collagen loss in skin tissue, improving skin tissue oxidative aging, promoting collagen fiber recovery and remodeling, improving dry skin, promoting skin cell regeneration, promoting the migration and repair of photodamaged skin cells, and / or blocking fibroblast apoptosis.

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