HK40090601A - Lacticaseibacillus paracasei and its application in anti-aging and improving skin anti-oxidation - Google Patents

Description

Lactobacillus paracasei and its application in anti-aging and improving skin antioxidant function

Technical Field

This invention belongs to the field of microbiology, specifically relating to Lactobacillus paracasei and its application in anti-aging and improving skin antioxidant activity.

Background Technology

As human lifespan increases, aging is inevitable, a consequence of stress and fatigue, injury and infection, impaired immune response, malnutrition, metabolic disorders, and negligence and drug abuse. Aging is closely linked to many diseases, including hypertension, type 2 diabetes, atherosclerosis, and Alzheimer's disease, the onset of which can be slowed by delaying aging.

Antioxidants are a key aspect of anti-aging, but synthetic antioxidants can have adverse effects on the liver, lungs, and spleen. Some people experience methemoglobinemia after taking oral antioxidants. Excessive antioxidant dosage can lead to symptoms such as diarrhea, increased intestinal motility, abdominal pain, and gastrointestinal bleeding, and can also cause side effects like hyperuricemia. Therefore, developing safe and effective anti-oxidative and anti-aging products is essential, and probiotics are currently a promising area of research.

Lactic acid bacteria (LAB) are among the most commonly used probiotics. LAB plays a crucial role in yogurt because the organic acids produced by these bacteria, including lactic acid, act as natural preservatives and flavor enhancers. LAB is gaining increasing acceptance as a probiotic, as it helps stimulate the host's immune system, reduces cancer risk, and provides antihypertensive, anti-inflammatory, anti-diabetic, antioxidant, immunomodulatory, cholesterol-lowering, or microbiome-regulating effects, while also preventing infection by pathogenic microorganisms. Lactic acid bacteria, including Lactobacillus and Bifidobacterium, have been shown to prevent D-galactose-induced oxidative stress.

Chinese patent application number 202211367692.4 discloses a strain of *Lactobacillus paracasei* and its applications, relating to the field of microbial technology. The disclosed * Lactobacillus paracasei * strain is named GforU-31, with accession number CCTCC No: M20221354. Experiments show that GforU-31 has functions such as promoting cell proliferation, anti-aging, and enhancing skin's antibacterial ability, and can be used in the preparation of food, pharmaceuticals, cosmetics, etc.

Chinese Patent Application No. 202011038787.2 discloses * Lactobacillus paracasei * ZJUIDS05 with antioxidant functions and its applications. The patent has accession number CGMCC NO. 20515 and exhibits strong in vivo and in vitro antioxidant capabilities. The uses of this *Lactobacillus paracasei* ZJUIDS05 are: to prepare live bacterial preparations with antioxidant functions, and to prepare various products with antioxidant functions.

However, the existing technologies mentioned above are all aimed at hydrogen peroxide-induced oxidative damage, which is far removed from the mechanism of natural aging, and their actual antioxidant and anti-aging effects cannot meet expectations.

Summary of the Invention

To address the aforementioned issues, this invention provides a novel strain, specifically *Lactobacillus paracasei*. Analysis of mouse serum and tissues revealed that *Lactobacillus paracasei* 3Q225 inhibits oxidative aging, validating its antioxidant and anti-aging effects. This aims to expand probiotic germplasm resources in the areas of anti-aging and antioxidant properties, laying a theoretical foundation for further in-depth human research and industrial development.

On the one hand, the present invention provides a strain of Lacticaseibacillus paracasei .

The *Lactobacillus paracasei* with accession number CGMCC NO.26439 was deposited at the China General Microbiological Culture Collection Center on January 9, 2023.

The physiological characteristics of *Lactobacillus paracasei* are: Gram-positive, rod-shaped, non-spore-forming, round colonies, catalase-negative, facultatively anaerobic, and with high proteolytic activity.

On the other hand, the present invention provides a method for culturing the aforementioned Lactobacillus paracasei.

The culture method includes inoculating the aforementioned *Lactobacillus paracasei* into a culture medium for culture.

The type of culture medium can be solid culture medium, semi-solid culture medium or liquid culture medium.

The culture media include, but are not limited to: MRS medium, BHI medium, and M17 medium.

The culture medium may also be a culture medium optimized for the aforementioned Lactobacillus paracasei based on existing culture media.

The culture conditions can be: 37℃, anaerobic or facultative anaerobic culture for 24 hours.

The culture conditions described can also be optimized based on existing technologies for the above-mentioned Lactobacillus paracasei, including but not limited to temperature, time, feeding, inoculum size, agitation, oxygen content, and carbon dioxide content.

The present invention also provides products prepared by the aforementioned culture method.

The product includes Lactobacillus paracasei.

The products include, but are not limited to, any one or more of the following: fermentation broth, fermentation broth supernatant, fermentation broth precipitate, freeze-dried powder, live bacteria, and dead bacteria.

In another aspect, the present invention provides the application of the aforementioned Lactobacillus paracasei or its products in anti-aging or anti-skin oxidation.

Specifically, it is used to prepare anti-aging or anti-skin oxidation products.

The signs of aging include, but are not limited to: loose skin, decreased digestive function, memory loss, graying hair, and degeneration of bones and joints.

The oxidation mentioned can be skin oxidation, and the manifestations of skin oxidation include, but are not limited to: sallow skin, dark corners of the mouth, enlarged pores, pigmentation, and dry and rough skin.

The present invention also provides oral products containing the aforementioned Lactobacillus paracasei or its products for anti-aging or anti-oxidation purposes.

Specifically, the oral product may be a medicine, food, or health product.

The aforementioned medicines, food products, and health products meet national quality standards.

Preferably, the oral product may contain more than ^10⁷ CFU/g or ^10⁷ CFU/mL of Lactobacillus paracasei.

The oral product may contain ^10⁸ - ^10¹¹ CFU/g or ^10⁸ - ^10¹¹ CFU/mL of *Lactobacillus paracasei*. Preferably, the oral product may contain ^10⁹ - ^10¹⁰ CFU/g or ^10⁹ - ^10¹⁰ CFU/mL of *Lactobacillus paracasei*.

In fact, the content of Lactobacillus paracasei in the oral product can be adjusted, and the specific intake of Lactobacillus paracasei can be adjusted according to the dosage of the oral product. When the content of Lactobacillus paracasei in the oral product is low, the intake of sufficient Lactobacillus paracasei can be ensured by increasing the dosage of the oral product.

The oral products may also include other antioxidant or anti-aging ingredients that work together.

The combined effect can be additive or synergistic.

Specifically, it may also include one or more of the following: soy isoflavones, lycopene, astaxanthin, vitamins, coenzyme Q10, and nicotinamide.

The antioxidant or anti-aging ingredients also include those selected from plant extracts, animal extracts, or traditional Chinese medicine extracts.

The oral products may also include excipients.

The excipients may be pharmaceutically acceptable carriers or excipients, or they may be conventional excipients or additives in the food or health product industry.

The excipients may be selected from one or more of the following: starch, oligosaccharides, proteins, dietary fiber, spices, seasonings, flavorings, and colorings.

In some embodiments, the oral product may be an oral liquid or an oral capsule.

The beneficial effects of this invention are:

The *Lactobacillus paracasei* 3Q225 provided by this invention can increase the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and glutathione (GSH) in a D-galactose-induced oxidative aging mouse model, while reducing the levels of malondialdehyde (MDA) in the serum and liver of the oxidative aging mouse model. Pathological observation showed that *Lactobacillus paracasei* 3Q225 alleviated liver, spleen, and skin damage in oxidatively aged mice. *Lactobacillus paracasei* 3Q225 of this invention can protect the D-galactose-induced aging model from oxidative stress better than *Lactobacillus bulgaricus* (LB) and vitamin C.

Preservation Instructions

Strain name: Lactobacillus paracasei 3Q225 (TMPC 3Q225);

Classification and nomenclature: Lacticaseibacillus paracasei ;

Accession number: CGMCC NO.26439;

Preservation date: January 9, 2023;

Preservation Center: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee;

Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

Attached Figure Description

Figure 1 shows the levels of SOD, GSH, GSH-Px, CAT, and MDA in mouse serum.

Figure 2 shows the levels of SOD, GSH, GSH-Px, CAT, and MDA in mouse liver.

Figure 3 shows the H&E pathological observation of mouse liver (historical micrograph).

Figure 4 shows the H&E pathological observation of mouse spleen (historical micrograph).

Figure 5 shows the H&E pathological observation of mouse skin (historical micrograph).

Figure 6 shows the mRNA expression levels of SOD1, SOD2, CAT, eNOS, nNOS, and iNOS in mouse liver tissue.

Figure 7 shows the mRNA expression levels of SOD1, SOD2, CAT, eNOS, nNOS, and iNOS in mouse spleen tissue.

Detailed Implementation

The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

In the following examples, the animal validation model used is a D-galactose-induced aging model. D-galactose is a drug that can control aging. The D-galactose-induced aging model is similar to natural aging and has been widely used for animal modeling. The mechanism of this model is that intracellular galactose is reduced to galactitol and cannot be further degraded. The accumulation of galactitol disrupts cellular metabolism and induces the production of reactive oxygen species (ROS), leading to cell damage and aging.

Example 1

(1) Laboratory strains

Lactobacillus paracasei 3Q225 was isolated from pickled bamboo shoots in Mangshi, Dehong Dai and Jingpo Autonomous Prefecture, Yunnan Province, China. After identification, it was deposited at the China Culture Collection Center (CGMCC) and classified as * Lacticaseibacillus paracasei *, with accession number CGMCC NO.26439. *Lactobacillus delbrueckii* subsp. bulgaricus (LB, CGMCC 1.16075) was obtained from the CGMCC and used as a control strain to compare with *Lactobacillus paracasei* 3Q225.

(2) Animal models of oxidation-induced aging

Sixty Kunming (KM) mice (6 weeks old, female, 20-25g) were purchased from the Laboratory Animal Center of Chongqing Medical University (Chongqing, China). They were given free access to food and water and allowed to acclimatize to the laboratory environment. After one week, all mice were randomly divided into 6 groups (n=10/group): normal control group, control group (model group), vitamin C (Vc) group, *Lactobacillus delbrueckii* subsp. bulgaricus (LB) group, low-dose *Lactobacillus paracasei* 3Q225 treatment group (3Q225-L), and high-dose *Lactobacillus paracasei* 3Q225 treatment group (3Q225-H). For the first three weeks, the normal and control groups received no treatment. Mice in the LB group were administered LB via gavage daily at a concentration of 2.0 × ^10⁹ cfu/kg body weight; mice in the 3Q225-L and 3Q225-H groups were administered Lactobacillus paracasei 3Q225 via gavage daily at a concentration of 2.0 × ^10⁸ cfu/kg body weight and 2.0 × ^10⁹ cfu/kg body weight, respectively; mice in the Vc group were administered vitamin C via gavage daily at a concentration of 100 mg/kg body weight (the volume administered was comparable to that of the other groups). After three weeks, except for the normal group, all mice in all groups received intraperitoneal injections of D-galactose at a concentration of 120 mg/kg body weight daily for six weeks. Finally, all mice were fasted for 24 hours and sacrificed by cervical dislocation. Blood, spleen, skin, and liver were obtained for subsequent experiments.

(3) Determination of SOD, GSH, GSH-Px, CAT and MDA levels in serum and liver

Mouse blood was collected and centrifuged at 4500 r/min for 15 min at 4℃ to prepare serum. Serum samples were stored at -80℃ for later use. The levels of superoxide dismutase (SOD, catalog number: A001-1-1), glutathione (GSH, catalog number: A005-1-1), glutathione peroxidase (GSH-Px, catalog number: A005-1-1), catalase (CAT, catalog number: A007-2-1), and malondialdehyde (MDA, catalog number: A003-1-1) in serum and liver were determined using a kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu).

(4) Pathological observation of skin, liver and spleen tissues

Skin, liver, and spleen samples (0.5 cm² ⁾ were taken from each mouse and immediately fixed in 10% formalin solution for 48 hours. After dehydration, clearing, paraffin embedding, embedding, and sectioning, H&E staining was performed, and morphological changes in these tissues were observed under an optical microscope.

(5) Quantitative PCR (qPCR) detection

Total RNA was extracted from liver and spleen tissues using TRIzol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer's instructions. The purity and concentration of total RNA were determined using a nano-100 spectrophotometer (All For Life Science, Hangzhou, China). Total RNA samples were used as templates for cDNA generation via reverse transcription; the cDNA concentration in all samples was adjusted to the same level of 1 pg/μL according to the transcription kit (Thermo Fisher Scientific). The total volume of the PCR reaction was 20 μL, including 1 μL of cDNA, 10 μL of fluorescent nucleic acid dye premix (SYBR Green PCRMaster Mix, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 7.8 μL of _ddH₂O , and 0.6 μL of each primer. PCR parameters were as follows: 95℃ for 60 seconds; 95℃ for 15 seconds, 55℃ for 30 seconds, 72℃ for 35 seconds, 40 cycles; 95℃ for 30 seconds, 55℃ for 35 seconds. GAPDH was used as an internal control, and the relative expression level of the target gene was calculated using the 2 ^-ΔΔCt method (Zhang et al., 2018).

(6) Statistical analysis

The experiment was repeated three times, and the results are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS software (IBM, North Castle, NY, USA), employing one-way ANOVA and the LSD test. A p-value < 0.05 was considered significant. All measurements were repeated at least three times, and data are expressed as mean ± standard deviation. All figures were plotted using Origin 8.0 software.

(7) Results

Levels of SOD, GSH, GSH-Px, CAT, and MDA:

As shown in Figures 1-2, compared with the normal group of mice, the model group mice had the highest MDA content in serum and liver, and the lowest levels of SOD, GSH, GSH-Px, and CAT. Treatment with LB, *Lactobacillus paracasei* 3Q225, or vitamin C decreased MDA content and increased SOD, GSH, GSH-Px, and CAT levels in oxidatively induced aging mice. In particular, the MDA, SOD, GSH, GSH-Px, and CAT levels in the high-dose *Lactobacillus paracasei* 3Q225 group were very close to those in the normal group, indicating that *Lactobacillus paracasei* 3Q225 had a better effect on delaying aging indicators in mice than LB and vitamin C.

Pathological observation of mouse liver, spleen, and skin:

Histological micrographs of the liver are shown in Figure 3. In the normal group of mice, hepatocytes were regular in morphology, size, and staining, showing no signs of inflammation. The hepatocyte lines were arranged in an orderly manner with clear boundaries, and the central veins were radially distributed. However, compared with the normal group, the control group mice contained disordered hepatocytes with irregular morphology, indistinct cell boundaries, irregular central vein shape, swollen cells, and extensive inflammatory infiltration. Mice treated with vitamin C, LB, or Lactobacillus paracasei 3Q225 showed a reduction in this chemical damage compared with the control group. In the Lactobacillus paracasei 3Q225 treatment group, the degree of liver damage was milder than that in the control group (treated with D-galactose only), therefore Lactobacillus paracasei 3Q225 was more effective in preventing liver atrophy. Furthermore, the hepatoprotective effect was dose-dependent.

Histological micrographs of the spleen are shown in Figure 4. In the normal group, the spleen sac was flat, the trabecular structure was normal, the boundary between the red and white pulp was clear, and no obvious ocular disease was observed. Compared with the normal group, the spleen in the control group lost its normal histological morphology and cellular structure, accompanied by dilation of the red pulp sinuses, a large number of red blood cells, a decrease in white pulp lymphocytes, narrowing of the red pulp cords, and decreased cell density. Compared with the control group, the spleen histological morphology was significantly improved after treatment with vitamin C, LB, or Lactobacillus paracasei 3Q225, showing a clear boundary between the red and white pulp, a flattened spleen sac, obvious germinal centers, and good cellular structure. Each treatment group showed micrographs similar to those of the normal group mice, with Lactobacillus paracasei 3Q22 showing the best effect.

Histological micrographs of the skin are shown in Figure 5. The epidermal structure of normal skin tissue is clear and intact. The dermis is rich in collagen fibers. The boundary between the dermis and epidermis is clear and intact. The dermis is rich in collagen fibers, and the number of fat vacuoles is normal. Compared with the normal group, the control group mice had reduced collagen fiber content, increased fat vacuoles, intradermal infiltration, and blurred boundaries between the dermis and epidermis. Compared with the control group mice, treatment with vitamin C, LB, or *Lactobacillus paracasei* 3Q225 significantly improved lipid vacuoles, intradermal infiltration, and the boundary between the epidermis and dermis in skin tissue, alleviating D-galactose-induced skin lesions in aging mice. Furthermore, the micrographs of mice in the *Lactobacillus paracasei* 3Q225-H group were closer to those of normal mice than those in the LB and Vc groups, indicating that *Lactobacillus paracasei* 3Q225 is more effective in preventing skin lesions.

Expression of oxidation-related genes in mouse liver:

The mRNA expression levels of SOD1, SOD2, CAT, eNOS, nNOS and iNOS in mouse liver tissue are shown in Figure 6.

The results showed that the mRNA levels of SOD1, SOD2, CAT, eNOS, and nNOS were highest in the liver of normal mice, while the mRNA level of iNOS was lowest. The mRNA expression levels of these genes in the control group showed the opposite trend to those in the normal group. Treatment with vitamin C, LB, or 3Q225 significantly improved the expression of SOD1, SOD2, CAT, eNOS, nNOS, and iNOS in aging mice (P < 0.05), with high-dose 3Q225 showing the best effect.

Expression of oxidation-related genes in mouse spleen

The mRNA expression levels of SOD1, SOD2, CAT, eNOS, nNOS and iNOS in mouse spleen tissue are shown in Figure 7.

qPCR results for spleen tissue were similar to those for liver. In the control group, the mRNA expression of SOD1, SOD2, CAT, eNOS, and nNOS was weakest in the spleen tissue, but strongest in the normal control group. Treatment with vitamin C, LB, or 3Q225 increased expression compared to the control group. 3Q225-treated mice showed stronger expression than vitamin C and LB-treated mice, approaching that of normal mice; vitamin C-treated mice also showed stronger expression than LB-treated mice.

(8) Discussion

Lactic acid bacteria (LAB) are widely used in the food industry due to their numerous probiotic properties, becoming a relatively indispensable health food in daily life. LAB strains isolated from fermented foods may possess antioxidant activity. Therefore, this invention focuses on the antioxidant activity of *Lactobacillus paracasei* strain 3Q225 isolated from traditional fermented foods. Continuous subcutaneous injection of D-galactose has been shown to induce an aging model in mice, leading to an increase in reactive oxygen species (ROS) and a decrease in the activity of antioxidant enzymes. The increase in free radicals may lead to the degeneration of aging-related mechanisms, ultimately resulting in increased oxidative stress and accelerating the aging process.

Pathological observation involves examining morphological changes in organs, tissues, or cells to identify pathological changes in the body. The skin, spleen, and liver can reflect characteristic changes that occur due to aging. According to this study, compared to normal mice, the aging group showed atrophy of the liver and spleen tissues and damage to the skin. Treatment with vitamin C, LB, or *Lactobacillus paracasei* 3Q225 significantly improved the abnormal morphology of the tissues, with *Lactobacillus paracasei* 3Q225 showing the most significant improvement. Mice treated with *Lactobacillus paracasei* 3Q225 had morphological tissues most closely resembling those of the normal group.

During the biological oxidation process in the human body, a large number of free radicals are generated. The balance of free radicals in the body is maintained by several antioxidant defense systems, including GSH-Px, SOD, CAT, reduced GSH, and vitamin C. Their synergistic effect converts excess free radicals into oxygen and water molecules, thus playing an antioxidant role. Lactic acid bacteria possess antioxidant activity and the ability to scavenge free radicals, assisting antioxidant enzymes in their antioxidant functions. Furthermore, bacteria release antioxidant enzymes, such as superoxide dismutase and glutathione peroxidase, during their metabolism to inhibit oxidation. Clinical studies have shown that the levels of SOD, GSH-Px, GSH, CAT, and MDA change in patients receiving antioxidant therapy. SOD protects cells from oxidative damage, indirectly reflecting its antioxidant activity. GSH-Px can scavenge lipid peroxidation induced by reactive oxygen species and hydroxyl free radicals, protecting the integrity of cell membrane structure and function. In this invention, *Lactobacillus paracasei* 3Q225 significantly increased SOD levels and GSH-Px activity in mouse serum and liver tissue. As a non-enzymatic antioxidant defense system, GSH is responsible for reducing hydrogen peroxide under the catalysis of glutathione peroxidase (GSH-PX). Compared with the normal group, the liver GSH content was significantly reduced, indicating that D-galactose caused severe cellular oxidative stress. In addition, CAT levels were also significantly reduced, indicating a decrease in the total antioxidant capacity of aging model mice. In this invention, *Lactobacillus paracasei* 3Q225, *Lactobacillus bulgaricus* LB, and vitamin C were shown to inhibit the decrease in liver and serum GSH, GSH-Px, and CAT levels. The antioxidant activity of *Lactobacillus paracasei* 3Q225 was higher than that of other experimental groups. Studies have shown that probiotics can significantly improve the antioxidant capacity of hyperlipidemic mice and increase the activity of antioxidant enzymes such as SOD, GSH-PX, GPT, and GOT. Therefore, this further demonstrates that lactic acid bacteria have the ability to enhance the synthesis of antioxidant enzymes. MDA is one of the final products of lipid peroxidation, which can cause cross-linking polymerization of biomolecules such as proteins and nucleic acids, and has cytotoxic and genotoxic effects. Therefore, the level of MDA is an important indicator of oxidative damage and aging. In this invention, compared with the D-galactose model group, the MDA content in the liver tissue of mice in the low-dose group of *Lactobacillus paracasei* 3Q225 was significantly reduced, while the MDA content in the liver tissue of mice in the high-dose group was close to normal levels. This invention demonstrates that *Lactobacillus paracasei* 3Q225 can reduce MDA content in serum and liver, further proving that 3Q225 can enhance the body's antioxidant defense system. This study confirms that *Lactobacillus paracasei* 3Q225 can alleviate the effects of aging on mice, restoring the levels of SOD, GSH-Px, GSH, CAT, and MDA in aging mouse models to levels similar to those in normal mice.

Claims (9)

1. A strain of Lacticaseibacillus paracasei , characterized by its preservation number CGMCC NO.26439. 2. The method for culturing Lactobacillus paracasei according to claim 1, characterized in that it includes inoculating Lactobacillus paracasei into a culture medium for cultivation. 3. The product prepared by the cultivation method according to claim 2 is characterized in that it includes live Lactobacillus paracasei, selected from any one or more of fermentation broth, fermentation broth precipitate, and lyophilized powder. 4. An oral product comprising the Lactobacillus paracasei of claim 1 or the product of claim 3, for anti-aging or anti-skin oxidation purposes. 5. The oral product according to claim 4, wherein the oral product is a medicine or health product. 6. The oral product according to claim 5, characterized in that it comprises Lactobacillus paracasei at a concentration of ^10⁷ CFU/g or ^10⁷ CFU/mL or higher. 7. The oral product according to claim 5, characterized in that it further comprises one or more of soy isoflavones, lycopene, astaxanthin, vitamins, coenzyme Q10, and nicotinamide. 8. The oral product according to claim 5, characterized in that it further comprises plant extracts, animal extracts or traditional Chinese medicine extracts; and further comprises excipients, wherein the excipients are selected from one or more of starch, oligosaccharides, proteins, dietary fiber, spices, flavoring agents, fragrance agents and coloring agents. 9. The oral product according to claim 5, characterized in that it is an oral liquid or an oral capsule.