Probiotics capable of regulating and controlling host intestinal cysteine metabolism and application

Abstract

The invention relates to the technical field of probiotics, in particular to probiotics capable of regulating and controlling host intestinal cysteine metabolism and application. The probiotics are Lactobacillus gasseri BKR-017, the classification name is Lactobacillus gasseri, the Lactobacillus gasseri is preserved in the China General Microbiological Culture Collection Center on September 26, 2025, the address of the preservation unit is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No: 36098. The invention provides a brand-new precise intervention strategy taking intestinal Cys metabolism as a target spot. The lactobacillus gasseri BKR-017 strain disclosed by the invention has the advantages of clear mechanism, high safety, remarkable effect and the like, and a novel safe, effective and feasible adjuvant therapy and prevention scheme is provided for obesity and related metabolic diseases thereof.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of probiotics, in particular to a probiotic capable of regulating host intestinal cysteine metabolism and application thereof. BACKGROUND

[0002] Traditional obesity intervention methods mainly include diet control, exercise therapy and drug treatment, which can alleviate symptoms to a certain extent, but have significant limitations: the efficacy is often limited, the durability is poor, and may be accompanied by adverse reactions, such as gastrointestinal discomfort, cardiovascular risk or drug dependence [ADCES in Practice, 2022, 10(1): 34-38., Cardiovascular Research, 2022, 118(3): 686-715.]. Therefore, it is urgent to develop a safe, effective and mechanism-specific innovative intervention strategy.

[0003] In recent years, dietary cysteine restriction has attracted attention as a new metabolic intervention strategy. Several studies have shown that among various essential amino acid restrictions, cysteine (Cys) restriction has the most significant effect on weight loss [Nutrition & Metabolism, 2020, 17(1): 20., Nature communications, 2014, 5(1): 3592.]. For example, in a mouse model, conditional cysteine restriction can cause a rapid weight loss of up to 30% in just one week, and this effect is reversible [Nature, 2025: 1-9.]. The core of this mechanism is that cysteine deficiency activates both the integrated stress response (ISR) and the oxidative stress response (OSR), which amplify each other, leading to the induction of key metabolic regulators such as fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15), thereby promoting energy consumption and fat metabolism [Journal of Biological Chemistry, 2012, 287(10): 7603-7614., Trends in Endocrinology & Metabolism, 2021, 32(11): 904-915.]. In addition, cysteine deficiency also leads to a significant decrease in tissue coenzyme A (CoA) levels, which in turn triggers mitochondrial dysfunction and metabolic reprogramming, reducing energy utilization efficiency [Nature, 2025: 1-9.]. Cysteine depletion can also trigger the "browning" of adipose tissue, i.e., the transformation of white adipose tissue (WAT) into heat-producing brown adipocytes, increasing energy consumption [Nature Metabolism, 2025: 1-19., Nature Reviews Endocrinology, 2025: 1-1.]. This cysteine depletion-induced fat browning and weight loss requires the mediation of the β3-adrenergic receptor (β3-AR) signaling pathway [Nature Metabolism, 2025: 1-19.]. In a high-fat diet-induced obese mouse model, cysteine deprivation can also induce rapid fat browning, significantly increase energy consumption, reverse metabolic inflammation, and ultimately achieve a weight loss of about 30% [Nature Reviews Endocrinology, 2025: 1-1., Drug Metabolism Letters, 2021, 14(3): 177-192.].

[0004] However, the above studies mainly rely on extreme cysteine-free diets or gene knockout models (such as cystathionine gamma-lyase Cth knockout mice), which are difficult to popularize in actual clinical applications and may cause potential health risks such as nutritional imbalance or metabolic disorders [Nature, 2025: 1-9., Nature Metabolism, 2025: 1-19.]. At the same time, intestinal microbiota plays an important role in regulating host amino acid metabolism, for example, some strains can metabolize sulfur-containing amino acids through cysteine-dependent biosynthetic pathways [Gut microbes, 2024, 16(1): 2419565.]. Studies have shown that the regulatory mechanism of cysteine-dependent methionine biosynthesis in Bifidobacterium longum (BL-04®) Bifidobacterium longum ) reveals that this strain can metabolize cysteine or its degradation product hydrogen sulfide (H2S), and the metabolic pathway is highly dependent on the concentration of environmental cysteine: at low concentrations, a dual pathway is activated, while at high concentrations, the H2S pathway is preferred [Nutrients, 2023, 15(8): 1966., BMC microbiology, 2017, 17(1): 61.]. This suggests that by regulating intestinal microbiota, it may be possible to simulate cysteine metabolism regulation without extreme dietary intervention, thereby obtaining benefits similar to cysteine deficiency, such as promoting fat burning and weight loss [Cell Host & Microbe, 2025, 33(6): 836-853.].

[0005] Existing research on weight loss probiotics has focused on the production of short-chain fatty acids (SCFAs) or the regulation of host inflammation, while research on promoting fat burning and weight loss through the regulation of cysteine metabolism is still in its infancy [DARU Journal of pharmaceutical sciences, 2019, 27(2): 827-837., Food Science & Nutrition, 2025, 13(8): e70434.]. Some literature reports that Paracasei Lactobacillus gasseri (LGG® Lactobacillus paragasseri ), as Lactobacillus gasseri (LGG® Lactobacillus gasseriA sister taxon of Lactobacillus gasseri SBT0271, which can significantly reduce body weight, fasting blood glucose and insulin resistance index, and improve glucose tolerance in high-fat diet-induced obese mice after administration [Beneficial Microbes, 2025, 1(aop): 1-14.]. The strain improves metabolic disorders by inhibiting oxidative stress and inflammation. These studies provide strong evidence for the regulation of host metabolism by probiotics, but the specific mechanisms still need to be further explored [Nature Aging, 2024, 4(4): 584-594.].

[0006] In summary, the prior art still has significant deficiencies in safely and effectively intervening obesity by utilizing the new mechanism of cysteine metabolism. There is an urgent need for a new intervention strategy with clear mechanism, high safety and easy standardization to accurately alleviate the problem of obesity. SUMMARY

[0007] The purpose of the present application is to address the deficiencies of the prior art in safely and effectively intervening obesity and related metabolic disorders, and to provide a probiotic that can regulate the metabolism of cysteine in the host's intestinal tract and its application.

[0008] To achieve the above-mentioned purpose, the technical solutions of the present application are as follows: A probiotic that can regulate the metabolism of cysteine (Cys) in the host's intestinal tract, the probiotic being Lactobacillus gasseri BKR-017, classified and named as Lactobacillus gasseri which was deposited with the China General Microbiological Culture Collection Center on September 26, 2025, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number CGMCC No: 36098.

[0009] The application of the probiotic that can regulate the metabolism of cysteine (Cys) in the host's intestinal tract, the application of the probiotic in glutathione (GSH) and / or coenzyme A (CoA) in the host.

[0010] The application of the probiotic in regulating the metabolism of cysteine by reducing the level of glutathione (GSH) and / or coenzyme A (CoA) in the host.

[0011] The application of the probiotic in preparing a composition for reducing body weight, preventing and / or treating obesity-related metabolic disorders.

[0012] The obesity-related metabolic disorders are selected from one or more of insulin resistance, type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD).

[0013] The above-mentioned strain regulates the metabolism of cysteine (Cys) in the host's intestinal tract, thereby gently simulating the Cys deficiency state, avoiding the risks brought by extreme diet or genetic intervention.

[0014] The Cys deficiency state works by activating the host's integrated stress response (ISR) and / or oxidative stress response (OSR), thereby causing adipose tissue browning, increasing energy expenditure and promoting fat burning.

[0015] The adipose tissue browning and / or fat burning is achieved by activating the beta3-adrenergic receptor (beta3-AR) signaling pathway.

[0016] A probiotic preparation containing the Lactobacillus gasseri BKR-017.

[0017] The probiotic preparation contains a culture, a bacterial suspension or a fermentation broth of the strain.

[0018] The preparation is in the form of a capsule, a tablet, a powder, a granule or a liquid.

[0019] The preparation can be a medicament.

[0020] Compared with the prior art, the present application has the following advantages: The present application discloses for the first time a novel mechanism by which a specific probiotic promotes fat burning and weight loss by regulating intestinal cysteine metabolism, activating the host's stress response and adipose tissue browning. This pathway is completely different from the mechanism of existing probiotics through short-chain fatty acids or inflammation regulation, providing a novel target for obesity intervention.

[0021] The strain of the present application achieves a similar weight loss effect as extreme dietary intervention by mildly simulating a cysteine deficiency state, but avoids the nutritional imbalance or potential health risks caused by long-term extreme diet, thereby providing a new strategy with high safety and mild effect.

[0022] The Lactobacillus gasseri BKR-017 strain of the present application can be used as a safe and effective live bacterial preparation, and can be widely used in medicines, providing a novel solution for the prevention and adjuvant therapy of obesity and related metabolic diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The VITEK® 221347 anaerobic bacteria and coryneform bacteria identification card (ANC, 21347) before and after incubation in the physiological and biochemical detection of the Lactobacillus gasseri BKR-017 provided in Example 1 of the present application; wherein A is the effect picture before incubation, and B is the effect picture after incubation.

[0024] Figure 2Effect diagram of inhibitory effect of Lactobacillus gasseri BKR-017 on body weight, body fat and lean body mass of mice in a high-fat diet-induced obesity model provided for Example 2 of the present application; wherein A is the effect on inhibition of body weight of mice, B is the effect on inhibition of body fat of mice, and C is the effect on inhibition of lean body mass of mice.

[0025] Figure 3 Effect diagram of regulating effect of Lactobacillus gasseri BKR-017 on GSH levels in serum and liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application; wherein A is the regulating effect on GSH levels in serum, and B is the regulating effect on GSH levels in liver.

[0026] Figure 4 Effect diagram of regulating effect of Lactobacillus gasseri BKR-017 on CoA levels in liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0027] Figure 5 Effect diagram of regulating effect of Lactobacillus gasseri BKR-017 on Cys levels in intestine of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0028] Figure 6 Effect diagram of regulating effect of Lactobacillus gasseri BKR-017 on SCFAs levels in intestine of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0029] Figure 7 Effect diagram of up-regulating effect of Lactobacillus gasseri BKR-017 on mRNA expression levels of Atf4 in liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0030] Figure 8 Effect diagram of up-regulating effect of Lactobacillus gasseri BKR-017 on mRNA expression levels of Chop gene in liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0031] Figure 9 Effect diagram of up-regulating effect of Lactobacillus gasseri BKR-017 on mRNA expression levels of Fgf21 gene in liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0032] Figure 10 Effect diagram of up-regulating effect of Lactobacillus gasseri BKR-017 on mRNA expression levels of Gdf15 gene in liver of mice in a high-fat diet-induced obesity model provided for Example 3 of the present application.

[0033] Figure 11Figure for up-regulation effect of Lactobacillus gasseri BKR-017 on mRNA expression level of Nrf2 gene in eWAT of mice in high-fat diet-induced obesity model provided in Example 3 of the present application.

[0034] Figure 12 Figure for up-regulation effect of Lactobacillus gasseri BKR-017 on mRNA expression level of Nqo1 gene in eWAT of mice in high-fat diet-induced obesity model provided in Example 3 of the present application.

[0035] Figure 13 Figure for up-regulation effect of Lactobacillus gasseri BKR-017 on mRNA expression level of Gstm1 gene in eWAT of mice in high-fat diet-induced obesity model provided in Example 3 of the present application.

[0036] Figure 14 Figure for up-regulation effect of Lactobacillus gasseri BKR-017 on mRNA expression level of Ho-1 gene in eWAT of mice in high-fat diet-induced obesity model provided in Example 3 of the present application.

[0037] Figure 15 Figures for up-regulation effect of Lactobacillus gasseri BKR-017 on UCP1 positive area and mRNA expression level of UCP1 gene in sWAT of mice in high-fat diet-induced obesity model provided in Example 4 of the present application; wherein, A is the effect figure of UCP1 positive area, and B is the effect figure of mRNA expression level of UCP1 gene.

[0038] Figure 16 Figure for up-regulation effect of Lactobacillus gasseri BKR-017 on mRNA expression level of Prdm16 gene in sWAT of mice in high-fat diet-induced obesity model provided in Example 4 of the present application. DETAILED DESCRIPTION

[0039] The specific embodiments of the present application are further described in conjunction with examples, it should be noted that the specific embodiments described herein are only for the purpose of illustration and explanation of the present application, and are not limited to the present application.

[0040] The present application utilizes the precise and multi-effect synergistic mechanism of Lactobacillus gasseri BKR-017 to address obesity and its related metabolic disorders, which makes up for the shortcomings of traditional intervention methods and existing probiotic therapies. The probiotic of the present application can activate the integrated stress response (ISR) and oxidative stress response (OSR) from the source by regulating intestinal cysteine (Cys) metabolism and mildly simulating Cys deficiency, thereby precisely intervening in the core link of obesity-related metabolic disorders; and the Lactobacillus gasseri BKR-017 proposed in the present application not only targets specific types of obesity models, but also shows significant improvement effects on various obesity-related complications induced by high-fat diet, including weight gain, body fat accumulation, insulin resistance and non-alcoholic fatty liver disease (NAFLD), etc. In addition, the probiotic can effectively induce fat tissue browning, increase energy consumption and promote fat burning; significantly reduce fasting blood glucose levels, thereby reducing the risk of insulin resistance and secondary diabetes. In addition, by down-regulating the levels of pro-inflammatory factors (such as IL-1β), the potential low-grade inflammation state that may accompany obesity is effectively alleviated, the immune and inflammatory state is regulated, and the overall metabolic health is further improved.

[0041] The present application activates stress response and fat browning from the source by regulating intestinal cysteine metabolism, thereby significantly improving obesity and its related metabolic disorders.

[0042] The strain BKR-017 involved in the following examples was deposited with the China General Microbiological Culture Collection Center on September 26, 2025, at the address of No. 1, Beichen West Road, Chaoyang District, Beijing, with the preservation number of CGMCC No: 36098.

[0043] The culture medium involved in the following examples is as follows: MRS culture medium (g / L): 10 g / L of proteose peptone, 10 g / L of beef extract, 20 g / L of glucose, 5 g / L of yeast powder, 2 g / L of diammonium hydrogen citrate, 2 g / L of K2HPO4, 0.1 g / L of MgSO4·7H2O, 0.05 g / L of MnSO4·H2O, 1 mL / L of Tween 80, and 0.5 g / L of L-cysteine hydrochloride.

[0044] The preparation method of the probiotic suspension involved in the following examples is as follows: The strain BKR-017 stored in a glycerol tube was inoculated in MRS culture medium and cultured anaerobically at 37℃ until OD 600 >1.2. The fermentation broth after culture was loaded into a centrifuge tube and centrifuged at 5000×g for 15 min, and the supernatant was discarded to recover the bacterial cells. The bacterial cells were resuspended to 1×10 10 CFU / mL for mixing.

[0045] The following examples relate to test animals: Healthy male 8-week-old SPF C57BL / 6J mice, weighing 20-22 g. The mice were placed in standard transparent plastic cages at 22-24°C, and were free to eat and drink. The animal experiments involved in the present application were strictly in accordance with the relevant laws and regulations and national standards of our country on the management and use of experimental animals. The experimental animals were provided by qualified suppliers holding the "Experimental Animal Production License", and obtained the quality qualified certificate of experimental animals. All experimental operations comply with GB / T 35823-2018 "General Requirements for Animal Experiments", and the experimental animal feeding and environmental facilities meet the requirements of GB 14925 "Experimental Animal Environment and Facilities". The principles of animal welfare and ethics were fully followed throughout the experiment.

[0046] The experimental results data were statistically analyzed using GraphPad Prism 9.0, ***p<0.001 represents a very significant difference compared with the ND control group, **p<0.01 represents a very significant difference compared with the ND control group, *p<0.05 represents a significant difference compared with the ND control group. ##p<0.001 represents a very significant difference compared with the HFD control group, ##p<0.01 represents a very significant difference compared with the HFD control group, #p<0.05 represents a significant difference compared with the HFD control group.

[0047] Example 1: Isolation, identification and biological characteristics analysis of Lactobacillus gasseri BKR-017 1. Isolation and identification of the strain Samples were taken from the milk of a healthy lactating woman in Shenyang, and after gradient dilution, they were spread on MRS agar plates and incubated anaerobically at 37°C for 48 hours. Single colonies with different morphologies were picked and purified to obtain a pure culture strain, designated as BKR-017.

[0048] The strain obtained above was identified by 16S rDNA, The universal primers used for 16S rDNA identification were: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (Sequence 1) 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (Sequence 2).

[0049] After PCR amplification of the strain DNA, it was sent to Shanghai Sangon Biological Engineering (Shanghai) Co., Ltd. for sequencing, and the sequences were compared for similarity using EzBioCloud (https: / / www.ezbiocloud.net / ), and the species were determined.

[0050] The strain was identified as Lactobacillus gasseri by 16S rDNA and named BKR-017. The strain has been preserved in the China General Microbiological Culture Collection Center (CGMCC, located at No. 1, Yihuangyuan, Beichenxi Road, Chaoyang District, Beijing) with a preservation number of CGMCC No. 36098 on September 26, 2025. Lactobacillus gasseri

[0051] 2. Genetic stability analysis and physiological and biochemical characteristics The strain BKR-017 obtained by the above separation was continuously subcultured for 10 times on an MRS plate in a conventional manner, and the F10 strain was detected by using a VITEK® 2 21347 anaerobic bacteria and corynebacterium identification card (ANC, 21347). The identification card contains 64 different biochemical test reaction holes. By using a VITEK® 2 full-automatic microbial analysis system and a matching software, a biochemical phenotype atlas such as enzyme activity, sugar fermentation and assimilation shown on the identification card was read, and the identification results of the strain were compared and analyzed. The results are shown in Table 1. Figure 1

[0052] The comprehensive biochemical profile of the strain on the biochemical indicators did not change, and the identification confidence remained above 99%. This proves that the strain has excellent genetic and physiological stability, and does not have trait degradation or variation in the continuous subculture process.

[0053] The main physiological and biochemical characteristics of the strain are as follows: (1) Morphological and staining characteristics: Gram-positive (G+), short rod-shaped or rod-shaped under a microscope, both ends are blunt, mostly arranged in pairs or short chains, no spores, no flagella (non-motile), and facultative anaerobic.

[0054] (2) VITEK® 2 biochemical metabolism characteristics (ANC card reaction results): Enzyme activity: leucine arylamidase (LeuA), alpha-glucosidase (dGLU), beta-galactosidase (dGAL), and arginine dihydrolase (ADH) are positive; urease (URE), hydrogen peroxide (Catalase), and alkaline phosphatase (PHOS) are negative.

[0055] Carbon source utilization: Strong positive (+): can efficiently utilize glucose, sucrose, fructose, maltose, galactose, cellobiose, trehalose and salicin to produce acid.

[0056] ​​Negative (-): Cannot utilize arabinose, xylose, rhamnose (this is an important characteristic that distinguishes Lactobacillus gasseri from Lactobacillus casei), sorbitol, and mannitol.

[0057] Other biochemical reactions: Indole test negative, H2S production test negative, aescin hydrolysis positive.

[0058] (3) Growth characteristics and environmental tolerance: Temperature tolerance: It has a wide growth temperature range, with the optimal growth temperature being 37℃; this strain exhibits heat resistance and can still grow at 45℃, but its growth is inhibited at 15℃.

[0059] Salt tolerance: It grows well in 2%~4% NaCl, and its growth is limited but not completely inhibited at 5% NaCl concentration, indicating that it has a certain degree of salt tolerance.

[0060] Bile salt tolerance: It grows well in an environment containing 0.3% (w / v) ox gall and has the potential to cross the intestinal barrier.

[0061] Acid tolerance: It has strong acid resistance, with an 80% survival rate after treatment at pH 3.0 for 4 hours.

[0062] Example 2: The intervention effect of Lactobacillus gasseri BKR-017 on body weight, body fat and energy metabolism in high-fat diet-induced obese mice. Experimental objective: This embodiment aims to evaluate whether Lactobacillus gasseri BKR-017 bacterial suspension has a significant ameliorative effect on obesity (increased body weight and body fat) induced by a high-fat diet (HFD) in C57BL / 6J mice, and to explore whether it exerts its intervention effect through mechanisms such as increasing energy expenditure and promoting lipid oxidation, and to evaluate the specificity of this effect.

[0063] Test method: 1. Animals and Grouping: Forty healthy 8-week-old male C57BL / 6J mice, weighing 20-22g, were randomly divided into 4 groups of 10 mice each after one week of acclimatization feeding, as follows: Group 1: Normal diet control group (ND control group): fed standard chow diet (D12450B, ResearchDiets, USA; 10% fat calories), and administered 100 μL PBS daily via oral gavage as a carrier control.

[0064] Group 2: Normal diet + probiotic group (ND + BKR-017 group): fed standard chow diet, and orally administered 100 μL of BKR-017 bacterial suspension (containing 1×10⁻⁶ bacteria) daily. 9 CFU (live bacteria).

[0065] Group 3: High-fat diet model group (HFD control group): 60% fat energy high-fat feed (D12492, Research Diets, USA) was given, and 100 μL PBS was orally gavaged daily.

[0066] Group 4: High-fat diet model group + probiotic group (HFD + BKR-017 group) as a probiotic model intervention group: high-fat feed was given, and 100 μL BKR-017 bacterial suspension (containing 1×10 9 CFU viable bacteria) was orally gavaged daily.

[0067] 2. Intervention scheme: The probiotic intervention group (groups 2 and 4) was given Lactobacillus gasseri BKR-017 bacterial suspension 100 μL (strain viable bacteria: 1×10 9 CFU per mouse per day) by gavage daily. The rest of the groups were given the same volume of PBS by gavage daily. The high-fat diet model group and the probiotic model intervention group (i.e., groups 3 and 4) first received 8 weeks of high-fat feed to induce an obesity model (more than 25% higher in body weight than the normal diet group). From the 9th week, each group began the corresponding intervention for 8 weeks. The body weight and feed intake of each group of mice were recorded every week during the period. The probiotic intervention scheme referred to the study of the effect of probiotics on obesity.

[0068] 3. Sample collection and index detection: At the end of the experiment, the mice were fasted for 12 hours, and the final body weight was measured. The whole body composition of the mice, including body fat mass and lean body mass (lean body mass (LBM), also commonly known as fat-free mass (FFM), was measured using a dual-energy X-ray absorptiometry (DEXA, Lunar PIXImus, GE Healthcare). Subsequently, the oxygen consumption (VO2), carbon dioxide production (VCO2), heat production, and voluntary activity of the mice were monitored by the Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments) within 48 hours, the respiratory exchange ratio (RER = VCO2 / VO2) was calculated, and the energy expenditure was calculated based on the Weir formula.

[0069] Body weight, body fat mass, and lean body mass were measured at the beginning of the experiment (week 0), after the model was established (week 8), and at the end of the intervention (week 16), respectively.

[0070] Test results: 1. Improvement of Lactobacillus gasseri BKR-017 on body weight and body fat increase induced by high-fat diet AsFigure 2 As shown, at the beginning of the experiment (week 0), there was no significant difference in body weight, body fat mass and lean body mass among the mice in each group. After 8 weeks of high-fat diet feeding (week 8), the body weight and body fat mass of the mice in the high-fat diet model group and the high-fat diet + probiotic group were significantly increased (***p < 0.001 vs. normal diet control group), indicating that the obesity model was successfully established, while the lean body mass did not change significantly. After 8 weeks of probiotic intervention (week 16), compared with the normal diet control group, the body weight and body fat mass of the mice in the high-fat diet model group were further significantly increased (***p < 0.001), and the lean body mass increased slightly but not statistically significantly. Compared with the high-fat diet model group, the body weight of the mice in the group intervened with Lactobacillus gasseri BKR-017 was significantly suppressed, with a decrease of 20.2% in the final body weight (###p < 0.001), a significant decrease in body fat mass (###p < 0.001), and no significant difference in lean body mass. Under normal diet conditions, there was no significant difference in the indicators between the probiotic group and the control group, indicating that BKR-017 is specific to high-fat diet-induced obesity and has good safety.

[0071] 2. Effect of Lactobacillus gasseri BKR-017 on energy metabolism The results showed that compared with the model group, the energy consumption of the mice in the intervention group during the dark period increased significantly by 15% (###p < 0.001), the RER decreased significantly (0.75 vs. 0.85), and the heat production increased by 10% (#p < 0.05), indicating that the energy source shifted to fat oxidation. The specific data are shown in Table 1: Table 1 Energy metabolism indicators of mice in each group (week 16)

[0072] Note: The data are expressed as mean ± standard deviation (n = 10). ***p < 0.001, **p < 0.01 vs. ND control group; ###p < 0.001, #p < 0.05 vs. HFD control group.

[0073] Conclusion: The results of this example demonstrate that Lactobacillus gasseri BKR-017 has a significant intervention effect on high-fat diet-induced obesity. Its core mechanism of action is closely related to reducing fat accumulation, increasing energy consumption and promoting fat oxidation.

[0074] Example 3: Lactobacillus gasseri BKR-017 activates host stress response by regulating intestinal cysteine metabolism Objective of the experiment: This example aims to evaluate whether Lactobacillus gasseri BKR-017 activates the integrated stress response (ISR) and oxidative stress response (OSR) by affecting host cysteine (Cys) metabolism at the molecular level, and to explore the independence of this mechanism. In addition, the functional specificity of BKR-007 strain group compared with BKR-017 strain group is discussed.

[0075] Test method: In addition to the mice from each group in Example 2, a fifth group of mice (n=10) was added: high-fat diet model group + probiotic group (HFD + BKR-007 group) as another Lactobacillus gasseri control intervention group: given high-fat feed, 100 μL of BKR-007 bacterial suspension (containing 1×10 9 CFU viable bacteria) was administered orally by gavage daily.

[0076] The BKR-007 is described in detail in Chinese Patent ZL202210440892.1, with publication number CN 114908006B, and patent name A Lactobacillus gasseri and its use in reducing blood uric acid, which is described in detail. Lactobacillus gasseri ( Lactobacillus gasseri ) SYP-B4432 (BKR-007) has been deposited with the China General Microbiological Culture Collection Center (CGMCC for short, address: No. 3, Beichen West Road, Chaoyang District, Beijing), with the accession number CGMCC No. 22950, and the deposit date is July 26, 2021.

[0077] 1. Sample collection: from the mice in each group in Example 2 (n=10 / group), after the intervention, the blood was taken by heart puncture after isoflurane anesthesia, and then the liver, epididymal white adipose tissue (eWAT), inguinal subcutaneous white adipose tissue (sWAT), and cecal contents were quickly separated. The sample was quickly frozen in liquid nitrogen and stored at -80°C.

[0078] 2. Targeted metabolomics analysis: accurately weigh 50 mg of liver or cecal content tissue, add extraction solution containing stable isotope internal standard (methanol / water / chloroform, 2:1:1 v / v / v) for homogenization, ultrasonic disruption for 30 minutes, then centrifuge (4°C, 12000xg, 15 minutes) to take the supernatant. Absolute quantitative analysis of Cys, glutathione (GSH), coenzyme A (CoA) and related sulfur metabolites in serum, liver and intestinal samples was performed using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS, Waters ACQUITY UPLC I-Class / Xevo TQ-S micro). Short-chain fatty acids (SCFAs) in cecal contents were additionally analyzed as controls.

[0079] 3. Gene expression analysis: Total RNA was extracted from liver and eWAT using TRIzol reagent (Invitrogen), and the concentration and purity were determined using a NanoDrop One (Thermo Fisher). 2 μg of total RNA was used to synthesize cDNA using a PrimeScript RT kit (Takara). qPCR was performed using TB Green Premix Ex Taq II (Takara) on a QuantStudio 6 Flex real-time PCR instrument with cDNA as a template. The expression of ISR key genes (Atf4, Chop, Fgf21, Gdf15) and OSR key genes (Nrf2, Nqo1, Gstm1, Ho-1) was detected. Gapdh was used as an internal reference, and the relative expression was calculated using the 2 -ΔΔCt method.

[0080] Primer sequences: Atf4 forward primer: 5'-AGCAAAACAAGACAGCAGCC-3' (SEQ ID NO: 3) Atf4 reverse primer: 5'-ACTCTCTTCTTCCCCCTTGC-3' (SEQ ID NO: 4) Chop forward primer: 5'-GAGTCCCTGCCTTTCACCTT-3' (SEQ ID NO: 5) Chop reverse primer: 5'-TTCCTCTTCGTTTCCTGGGG-3' (SEQ ID NO: 6) Fgf21 forward primer: 5'-TGACGACCAAGACACTGAAGC-3' (SEQ ID NO: 7) Fgf21 reverse primer: 5'-TTTGAGCTCCAGGAGACTTTCTG-3' (SEQ ID NO: 8) Gdf15 forward primer: 5'-GAGAGGACTCGAACTCAGAAC-3' (SEQ ID NO: 9) Gdf15 reverse primer: 5'-GACCCCAATCTCACCTCTG-3' (SEQ ID NO: 10) Nrf2 forward primer: 5'-CACATTCCCAAACAAGATGCCT-3' (SEQ ID NO: 11) Nrf2 reverse primer: 5'-TATCCAGGGCAAGCGACTCA-3' (SEQ ID NO: 12) Nqo1 forward primer: 5'-AGGATGGGAGGTACTCGAATC-3' (SEQ ID NO: 3) Nqol reverse primer: 5'-TGCTAGAGATGACTCGGAAGG-3' (SEQ ID NO: 14) Gstm1 forward primer: 5'-GATTGGTGCAGGGTTGGGAG-3' (SEQ ID NO: 15) Gstm1 reverse primer: 5'-GCTGGTGCTGTGGTCTTCTC-3' (SEQ ID NO: 16) Ho-1 forward primer: 5'-TTAAGCTGGTGATGGCTTCCT-3' (SEQ ID NO: 17) Ho-1 reverse primer: 5'-AGTGGGGCATAGACTGGGTT-3' (SEQ ID NO: 18) Gapdh (internal reference gene) forward primer: 5'-AACGACCCCTTCATTGAC-3' (SEQ ID NO: 19) Gapdh (internal reference gene) reverse primer: 5'-TCCACGACATACTCAGCAC-3' (SEQ ID NO: 20).

[0081] Specific operation steps of qPCR: After RNA extraction, the RNA concentration and purity (A260 / A280 > 1.8) were determined using NanoDrop One. 2 μg of total RNA was used to synthesize cDNA by reverse transcription using the PrimeScript RT kit (Takara), and the reaction conditions were 37°C for 15 min and 85°C for 5 s. Using cDNA as a template (diluted to 50 ng / μL), each reaction system was 20 μL, containing: 10 μL TB Green Premix Ex Taq II, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 2 μL cDNA template, 6.4 μL sterile water. PCR conditions: pre-denaturation at 95°C for 30 s; followed by 40 cycles of amplification (95°C for 5 s, 60°C for 30 s); finally, melting curve analysis (95°C for 15 s, 60°C for 60 s, 95°C for 15 s) was performed to confirm the specificity of the product. Each sample was triplicated, and the experiment was repeated three times.

[0082] Test results: 1. Changes in the levels of cysteine downstream metabolites As Figures 3 to 6As shown, compared with the high-fat diet model group, the HFD+BKR-017 probiotic model intervention group showed a 32% and 38% decrease in serum and liver GSH levels, respectively (###p<0.001), a 25% decrease in CoA levels (###p<0.001), and an 18% decrease in intestinal Cys concentration (###p<0.001). SCFAs levels showed no significant change, indicating that the mechanism is independent of the traditional probiotic pathway. In the BKR-007 intervention group, there were no significant differences in the above indicators except for SCFAs levels, but SCFAs levels were significantly increased, indicating that the BKR-017 mechanism is independent of other Lactobacillus gasseri strains.

[0083] 2. Stress response pathway gene activation like Figures 7 to 14 As shown, compared with the high-fat diet model group, the expression of Atf4, Chop, Fgf21, and Gdf15 in the liver of the HFD+BKR-017 probiotic model intervention group was upregulated by an average of 3.1-fold, 2.8-fold, 5.2-fold, and 3.5-fold (###p<0.001); and the expression of Nrf2, Nqo1, Gstm1, and Ho-1 in the OSR pathway of eWAT was upregulated by 2.2-fold, 3.5-fold, 2.7-fold, and 4.0-fold (###p<0.001). No significant differences were found in the above indicators in the HFD+BKR-007 intervention group.

[0084] in conclusion: The results of this embodiment confirm that *Lactobacillus gasseri* BKR-017 activates ISR and OSR through the regulation of Cys metabolism, revealing a novel upstream molecular mechanism for its weight loss efficacy. This mechanism is independent and specific, representing a novel mechanism distinct from the traditional probiotic SCFAs pathway, and also different from other *Lactobacillus gasseri* strains, exhibiting significant specificity and providing a new target for obesity intervention.

[0085] Example 4: Lactobacillus gasseri BKR-017 induces browning of adipose tissue via the β3-adrenergic receptor signaling pathway Experimental objective: This embodiment aims to verify whether Lactobacillus gasseri BKR-017 induces browning of white adipose tissue by activating the β3-adrenergic receptor (β3-AR) signaling pathway, and to assess the dependence of this effect.

[0086] Test method: 1. Animal grouping and intervention: Based on Example 2, an additional mechanism verification group (Group 5, n=10) was established: animals were given a high-fat diet and simultaneously administered BKR-017 via gavage (1×10⁻⁶). 9 The intervention lasted for 8 weeks and involved the β3-AR specific antagonist SR59230A (CAS No.: 174689-39-5, 1 mg / kg body weight / day, intraperitoneal injection).

[0087] 2. Histology and immunohistochemistry analysis: sWAT tissues were fixed with 4% paraformaldehyde for 24 hours, embedded in paraffin and made into 4 pm sections. H&E staining was used to observe the morphology of adipocytes. The primary antibody was rabbit anti-UCP1 (Abeam, ab10983, 1:200), and the secondary antibody was HRP-labeled goat anti-rabbit IgG (Abeam, 1:500), and DAB was used for color development. ImageJ 1.53 software was used to quantify the UCP1 positive area.

[0088] 3. Gene expression analysis: TRIzol reagent (Invitrogen) was used to extract sWAT total RNA, and NanoDrop One (Thermo Fisher) was used to determine the concentration and purity. 2 pg of total RNA was used to synthesize cDNA using the PrimeScript RT kit (Takara). Using cDNA as a template, TB Green Premix Ex Taq II (Takara) was used to perform qPCR on a QuantStudio 6 Flex real-time PCR instrument. The expression of fat browning key genes (Ucp1, Prdm16) was detected. Using Gapdh as an internal reference, the relative expression was calculated using the 2 -ΔΔCt method.

[0089] Primer sequences: Ucp1 forward primer: 5'-GTGAAGGTCAGAATGCAAGC-3' (SEQ ID NO: 21) Ucp1 reverse primer: 5'-AGGGCCCCCTTCATGAGGTC-3' (SEQ ID NO: 22) Prdm16 forward primer: 5'-CAGCACGGTGAAGCCATTC-3' (SEQ ID NO: 23) Prdm16 reverse primer: 5'-GCGTGCATCCGCTTGTG-3' (SEQ ID NO: 24) Gapdh (internal reference gene) forward primer: 5'-AACGACCCCTTCATTGAC-3' (SEQ ID NO: 25) Gapdh (internal reference gene) reverse primer: 5'-TCCACGACATACTCAGCAC-3' (SEQ ID NO: 26).

[0090] Specific steps for qPCR: After RNA extraction, the RNA concentration and purity were determined using NanoDrop One (A260 / A280 > 1.8). 2 μg of total RNA was used for reverse transcription to synthesize cDNA using the PrimeScript RT kit (Takara). The reaction conditions were 37℃ for 15 min and 85℃ for 5 s. Using cDNA as a template (diluted to 50 ng / μL), each 20 μL reaction volume contained: 10 μL TB Green Premix Ex Taq II, 0.8 μL forward primer (10 μM), 0.8 μL reverse primer (10 μM), 2 μL cDNA template, and 6.4 μL sterile water. PCR conditions: pre-denaturation at 95℃ for 30 s; followed by 40 cycles of amplification (95℃ for 5 s, 60℃ for 30 s); finally, melting curve analysis (95℃ for 15 s, 60℃ for 60 s, 95℃ for 15 s) was performed to confirm product specificity. Each sample was tested in triplicate, and the experiment was repeated three times.

[0091] Experimental results: 1. Lactobacillus gasseri BKR-017 induces fatty browning The model group sWAT cells were large unilocular lipid droplet cells, while the probiotic model intervention group showed multilocular beige cells. Figure 15 , Figure 16 As shown, the positive area of ​​UCP1 was significantly increased (###p<0.001). The average expression of Ucp1 and Prdm16 mRNA was upregulated by 4.8-fold and 3.2-fold, respectively (###p<0.001).

[0092] 2. The effect of adipose browning depends on the β3-AR pathway. In the mechanism validation group, weight loss was blocked (p>0.05 vs. model group), sWAT showed no obvious browning, and the positive area of ​​UCP1 and the expression of Ucp1 and Prdm16 returned to the level of the model group. Specific data are shown in Table 2. Table 2. Mechanism validation group adipose browning index

[0093] Note: Data are expressed as mean ± standard deviation (n=10). ###p<0.001 vs. HFD control group (no significant difference between the mechanism validation group and the HFD control group).

[0094] in conclusion: The results of this example suggest that L. gasseri BKR-017 is ineffective against the specific antagonist SR59230A of β3-AR induced by high-fat diet in mice, and it is believed that L. gasseri BKR-017 plays a key downstream mechanism in the reduction of obesity by inducing fat browning dependent on the β3-AR pathway. This effect is dependent and specific, providing a new strategy for obesity intervention.

[0095] Example 5: Improvement of L. gasseri BKR-017 on obesity-related metabolic syndrome Purpose of the test: This example aims to evaluate the comprehensive improvement effect of L. gasseri BKR-017 on obesity-related metabolic syndrome, such as insulin resistance and non-alcoholic fatty liver disease.

[0096] Test method: 1. Sample collection: Use the samples (n=8 / group) of the high-fat diet model group and the probiotic model intervention group in Example 2.

[0097] 2. Index detection: Use glucose oxidase method (Glucometer, Roche) to measure fasting blood glucose; ELISA kit (Millipore EZRMI-13K) to determine the content of serum insulin (INS), and calculate the insulin resistance index HOMA-IR = (fasting blood glucose mmol / L x fasting insulin mU / L) / 22.5; Use biochemical reagent kit to determine the level of serum low density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT) level, aspartate aminotransferase (AST) level; Take liver tissue, use tissue total cholesterol (TC) detection kit, triglyceride (TG) detection kit, superoxide dismutase (SOD) activity kit to determine the liver TC, TG and SOD level; Use ELISA kit (eBioscience) to determine the concentration of interleukin-1β (IL-1β) in liver, and correct with liver protein concentration.

[0098] Test results: 1. Improvement of insulin resistance and blood glucose The results show that the fasting blood glucose of the probiotic model intervention group is reduced to 7.1±0.9 mmol / L (model group 11.5±1.3, ###p<0.001), the INS content is reduced, and the HOMA-IR is decreased by 48% (###p<0.001). The specific data is shown in Table 3, which shows an effective inhibitory effect on liver steatosis.

[0099] Table 3: Insulin resistance-related indicators of mice in each group

[0100] Note: Data are expressed as mean ± SD (n = 8). ***p < 0.001 vs. ND control group; ###p < 0.001 vs. HFD control group.

[0101] 2. Improvement of liver lipid metabolism, antioxidant and inflammation The results show that the serum LDL-C, ALT and AST levels of the probiotic model intervention group are significantly reduced (###p < 0.001); the liver TC, TG and IL-1β levels are reduced, and the SOD level is increased (###p < 0.001). The specific data are shown in Table 4: Table 4 Liver-related indicators of mice in each group

[0102] Note: Data are expressed as mean ± SD (n = 8). ***p < 0.001 vs. ND control group; ###p < 0.001 vs. HFD control group.

[0103] Conclusion: The results of this example demonstrate that Lactobacillus gasseri BKR-017 can effectively improve insulin resistance, non-alcoholic fatty liver and chronic low-grade inflammation caused by obesity. This strain has industrialization potential and provides a safe and effective new solution for the prevention and adjuvant treatment of obesity and its related metabolic diseases.

[0104] In summary, the strain of the present application can modulate the metabolism of intestinal cysteine (Cys) to mildly simulate the Cys deficiency state. This mechanism of action activates the integrated stress response (ISR) and oxidative stress response (OSR) of the host, thereby inducing adipose tissue browning, increasing energy consumption and promoting fat burning. Unlike existing probiotics, which use short-chain fatty acids (SCFAs) or inflammation regulation to reduce weight, the present application provides a new and precise intervention strategy targeting intestinal Cys metabolism. The Lactobacillus gasseri BKR-017 strain described in the present application has the advantages of clear mechanism, high safety and significant effect, and provides a safe, effective and feasible new solution for the adjuvant treatment and prevention of obesity and its related metabolic diseases.

Claims

1. A probiotic that can regulate cysteine ​​metabolism in the host gut, characterized in that: The probiotic is Lactobacillus gasseri (Lactobacillus gasseri) Lactobacillus gasseri The strain BKR-017 was deposited on September 26, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No: 36098.

2. The application of the probiotic described in claim 1 that can regulate host intestinal cysteine ​​metabolism, characterized in that: The application of the probiotics in the host glutathione (GSH) and / or coenzyme A (CoA).

3. The application of the probiotics that can regulate host intestinal cysteine ​​metabolism according to claim 2, characterized in that: The probiotics play a role in regulating cysteine ​​metabolism by reducing host glutathione (GSH) and / or coenzyme A (CoA) levels.

4. The application of the probiotics that can regulate host intestinal cysteine ​​metabolism according to claim 2 or 3, characterized in that: The use of the probiotics in the preparation of compositions for weight loss, prevention and / or treatment of obesity-related metabolic disorders.

5. The application of the probiotics that can regulate host intestinal cysteine ​​metabolism according to claim 4, characterized in that: The obesity-related metabolic disorder is selected from one or more of insulin resistance, type 2 diabetes, cardiovascular disease, and non-alcoholic fatty liver disease (NAFLD).

6. A probiotic preparation, characterized in that, It contains the probiotics as described in claim 1.

7. The probiotic preparation according to claim 6, characterized in that, The probiotic preparation contains a culture of the strain, a culture suspension, or a fermentation broth.

8. The probiotic preparation according to claim 6, characterized in that, The dosage form of the preparation is capsule, tablet, powder, granule or liquid.

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