A probiotic capable of regulating host intestinal cysteine metabolism and application thereof

Abstract

The present application relates to the technical field of probiotics, and particularly relates to a probiotic capable of regulating host intestinal cysteine metabolism and application. Lactobacillus gasseri The probiotic is Lactobacillus gasseri BKR-017, which is classified and named as Lactobacillus gasseri and has been preserved in the China General Microbiological Culture Collection Center on September 26, 2025, the address of the preservation unit is No. 1, Xili, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No: 36098. The present application provides a new precise intervention strategy targeting intestinal Cys metabolism. The Lactobacillus gasseri BKR-017 strain has the advantages of clear mechanism, high safety and significant effect, and provides a safe, effective and feasible new scheme for the auxiliary treatment and prevention of obesity and related metabolic diseases.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a probiotic that can regulate the metabolism of cysteine ​​in the host gut and its applications. Background Technology

[0002] Traditional obesity interventions, mainly including diet control, exercise therapy, and drug treatment, can alleviate symptoms to some extent, but they have significant limitations: their efficacy is often limited and poorly sustained, and they may be accompanied by adverse reactions, such as drug treatment leading to gastrointestinal discomfort, cardiovascular risk, or drug dependence [ADCES in Practice, 2022, 10(1):34-38., Cardiovascular Research, 2022, 118(3): 686-715.]. Therefore, there is an urgent need to develop a safe, effective, and mechanistic innovative intervention strategy.

[0003] In recent years, dietary cysteine ​​restriction has attracted much attention as an emerging metabolic intervention strategy. Multiple 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 mouse models, conditional cysteine ​​restriction can lead to a rapid weight loss of up to 30% within just one week, and this effect is reversible [Nature, 2025: 1-9.]. The core of this mechanism lies in the fact that cysteine ​​deficiency simultaneously activates the integrated stress response (ISR) and the oxidative stress response (OSR). These two stress responses 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 lipid 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 causes mitochondrial dysfunction and metabolic reprogramming, reducing energy utilization efficiency [Nature, 2025: 1-9.]. Cysteine ​​depletion can also trigger "browning" in adipose tissue, the transformation of white adipose tissue (WAT) into thermogenic beige adipocytes, increasing energy expenditure [Nature Metabolism, 2025: 1-19., Nature Reviews Endocrinology, 2025: 1-1.]. This cysteine ​​depletion-induced adipose browning and weight loss requires the β3-adrenergic receptor (β3-AR) signaling pathway to be mediated [Nature Metabolism, 2025: 1-19.]. In a high-fat diet-induced obese mouse model, cysteine ​​deprivation also induced rapid adipose browning, significantly increased energy expenditure, reversed metabolic inflammation, and ultimately achieved approximately 30% weight loss [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 γ-lyase Cth knockout mice). These methods are difficult to promote 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.]. Meanwhile, the gut 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 revealed that *Bifidobacterium longum* (… Bifidobacterium longum The regulation mechanism of cysteine-dependent methionine biosynthesis in this strain indicates that it can utilize cysteine ​​or its degradation product hydrogen sulfide (H2S) for metabolism, and the metabolic pathway is highly dependent on the environmental cysteine ​​concentration: at low concentrations, a dual pathway is activated, while at high concentrations, the H2S pathway is preferentially utilized [Nutrients, 2023, 15(8): 1966., BMC microbiology, 2017, 17(1): 61.]. This suggests that by regulating the gut microbiota, it may be possible to mildly mimic cysteine ​​metabolic regulation without extreme dietary interventions, thereby obtaining benefits similar to cysteine ​​deficiency, such as promoting fat burning and weight loss [Cell Host&Microbe, 2025, 33(6): 836-853.].

[0005] Current research on probiotics for weight loss mainly focuses on the production of short-chain fatty acids (SCFAs) or their regulation of host inflammation. However, research on promoting fat burning and weight loss through the novel mechanism of regulating cysteine ​​metabolism is still lacking [DARU Journal of pharmaceutical sciences, 2019, 27(2): 827-837., Food Science & Nutrition, 2025, 13(8): e70434.]. Some literature reports that *Lactobacillus paracasei* (… Lactobacillus paragasseri ), as Lactobacillus gasseri ( Lactobacillus gasseriA sister taxa of [a specific species], when administered to mice with high-fat diet-induced obesity, significantly reduced body weight, fasting blood glucose, and insulin resistance index, and improved glucose tolerance [Beneficial Microbes, 2025, 1(aop): 1-14.]. This strain improves metabolic disorders by inhibiting oxidative stress and inflammation. These studies provide strong evidence for probiotics regulating host metabolism, but the specific mechanisms still need further investigation [Nature Aging, 2024, 4(4): 584-594.].

[0006] In summary, current technologies still have significant limitations in safely and effectively intervening in obesity using the novel mechanism of cysteine ​​metabolism. There is an urgent need for a new intervention strategy with a clearly defined mechanism, high safety profile, and ease of standardization to precisely alleviate obesity. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies in safely and effectively intervening in obesity and related metabolic disorders by providing a probiotic that can regulate host intestinal cysteine ​​metabolism and its applications.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A probiotic that can regulate cysteine ​​(Cys) metabolism in the host gut, the probiotic is Lactobacillus gasseri BKR-017, classified as... Lactobacillus gasseri It 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.

[0010] The application of a probiotic that can regulate host intestinal cysteine ​​(Cys) metabolism, wherein the probiotic is used in host glutathione (GSH) and / or coenzyme A (CoA).

[0011] The probiotics play a role in regulating cysteine ​​metabolism by reducing host glutathione (GSH) and / or coenzyme A (CoA) levels.

[0012] The use of the probiotics in the preparation of compositions for weight loss, prevention and / or treatment of obesity-related metabolic disorders.

[0013] 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).

[0014] The strains described above gently mimic a Cys deficiency state by regulating the metabolism of cysteine ​​(Cys) in the host gut, thus avoiding the risks associated with extreme diets or genetic interventions.

[0015] The Cys deficiency state works by activating the host’s integrated stress response (ISR) and / or oxidative stress response (OSR), thereby leading to adipose tissue browning, increased energy expenditure and promotion of fat burning.

[0016] The browning and / or fat burning of adipose tissue is achieved by activating the β3-adrenergic receptor (β3-AR) signaling pathway.

[0017] A probiotic preparation containing the aforementioned Lactobacillus gasseri BKR-017.

[0018] The probiotic preparation contains a culture of the strain, a culture suspension, or a fermentation broth.

[0019] The dosage form of the preparation is capsule, tablet, powder, granule or liquid.

[0020] The above-mentioned preparations can be drugs.

[0021] Compared with the prior art, the advantages of the present invention are as follows:

[0022] This invention reveals for the first time a novel mechanism by which specific probiotics promote fat burning and weight loss by regulating intestinal cysteine ​​metabolism, activating host stress response and fat browning. This pathway is entirely different from existing probiotic mechanisms that regulate fat through short-chain fatty acids or inflammation, providing a novel target for obesity intervention.

[0023] The strain described in this invention achieves weight loss effects similar to extreme dietary interventions by mildly simulating a state of cysteine ​​deficiency, but avoids the nutritional imbalances or potential health risks caused by long-term extreme diets, thus providing a new strategy that is both safe and mild.

[0024] The Lactobacillus gasseri BKR-017 strain of the present invention can be used as a safe and effective live bacterial preparation, and can be widely used in pharmaceuticals, providing a novel solution for the prevention and adjuvant treatment of obesity and related metabolic diseases. Attached Figure Description

[0025] Figure 1 The images shown are before and after incubation of the VITEK® 221347 anaerobic bacteria and corynebacterium identification card (ANC, 21347) in the physiological and biochemical detection of Lactobacillus gasseri BKR-017 provided in Example 1 of this invention; where A is the image before incubation and B is the image after incubation.

[0026] Figure 2The graph shows the inhibitory effects of Lactobacillus gasseri BKR-017 on mouse weight, body fat mass, and lean body mass in the high-fat diet-induced obesity model provided in Example 2 of this invention; where A represents the effect on mouse weight inhibition, B represents the effect on mouse body fat mass inhibition, and C represents the effect on mouse lean body mass inhibition.

[0027] Figure 3 The diagram shows the regulatory effect of Lactobacillus gasseri BKR-017 on GSH levels in mouse serum and liver in a high-fat diet-induced obesity model provided in Example 3 of this invention; where A represents the regulatory effect on serum GSH levels and B represents the regulatory effect on liver GSH levels.

[0028] Figure 4 The image shows the effect of Lactobacillus gasseri BKR-017 on the regulation of CoA levels in mouse liver in a high-fat diet-induced obesity model provided in Example 3 of this invention.

[0029] Figure 5 The image shows the effect of Lactobacillus gasseri BKR-017 on the regulation of Cys levels in the mouse intestine in a high-fat diet-induced obesity model provided in Example 3 of this invention.

[0030] Figure 6 The image shows the effect of Lactobacillus gasseri BKR-017 on the level of SCFAs in the mouse intestine in a high-fat diet-induced obesity model provided in Example 3 of the present invention.

[0031] Figure 7 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of Atf4 mRNA expression level in mouse liver in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0032] Figure 8 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of the mRNA expression level of the Chop gene in the liver of mice in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0033] Figure 9 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of the mRNA expression level of the Fgf21 gene in the mouse liver in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0034] Figure 10 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of the mRNA expression level of the Gdf15 gene in the mouse liver in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0035] Figure 11The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of Nrf2 gene mRNA expression in mouse eWAT in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0036] Figure 12 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of Nqo1 gene mRNA expression in mouse eWAT in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0037] Figure 13 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of Gstm1 gene mRNA expression in mouse eWAT in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0038] Figure 14 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of the Ho-1 gene mRNA expression level in mouse eWAT in the high-fat diet-induced obesity model provided in Example 3 of this invention.

[0039] Figure 15 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of UCP1 positive area and UCP1 gene mRNA expression level in mouse sWAT in the high-fat diet-induced obesity model provided in Example 4 of this invention; where A is the effect of UCP1 positive area and B is the effect of UCP1 gene mRNA expression level.

[0040] Figure 16 The image shows the effect of Lactobacillus gasseri BKR-017 on the upregulation of the mRNA expression level of the Prdm16 gene in mouse sWAT in the high-fat diet-induced obesity model provided in Example 4 of this invention. Detailed Implementation

[0041] The following examples further illustrate specific embodiments of the present invention. It should be noted that the specific embodiments described herein are merely for illustration and explanation and are not intended to limit the scope of the present invention.

[0042] This invention utilizes the precise and synergistic mechanism of *Lactobacillus gasseri* BKR-017 to address obesity and related metabolic disorders, overcoming the shortcomings of traditional interventions and existing probiotic therapies. The probiotics of this invention can regulate intestinal cysteine ​​(Cys) metabolism, gently mimicking a Cys-deficient state, and fundamentally activate the host integrated stress response (ISR) and oxidative stress response (OSR), precisely intervening in the core aspects of obesity-related metabolic disorders. Furthermore, the *Lactobacillus gasseri* BKR-017 proposed in this invention not only targets specific types of obesity models but also shows significant improvement effects on various obesity-related complications induced by high-fat diets, including weight gain, body fat accumulation, insulin resistance, and non-alcoholic fatty liver disease (NAFLD). In addition, the probiotics can effectively induce browning of adipose tissue, increase energy expenditure, promote fat burning, and significantly reduce fasting blood glucose levels, thereby reducing the risk of insulin resistance and secondary diabetes. In addition, by downregulating the levels of pro-inflammatory factors (such as IL-1β), it can effectively alleviate the potential low-grade inflammatory state that may accompany obesity, regulate the immune and inflammatory state, and further improve overall metabolic health.

[0043] This invention significantly improves obesity and its related metabolic disorders by regulating intestinal cysteine ​​metabolism, thereby activating stress response and adipose browning at the source.

[0044] The strain BKR-017 involved in the following examples 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.

[0045] The culture media involved in the following examples are as follows:

[0046] MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 20g / L, yeast extract 5g / L, diammonium hydrogen citrate 2g / L, K2HPO4 2g / L, MgSO4·7H2O 0.1g / L, MnSO4·H2O 0.05g / L, Tween 80 1mL / L, L-cysteine ​​hydrochloride 0.5g / L.

[0047] The following examples illustrate the method for preparing probiotic suspensions:

[0048] The strain BKR-017, preserved in glycerol tubes, was inoculated into MRS medium and anaerobically cultured at 37°C until OD500. 600 If the concentration is >1.2, the fermentation broth after culture is transferred to centrifuge tubes and centrifuged at 5000×g for 15 min. The supernatant is discarded to recover the bacterial cells. The bacterial cells are resuspended in sterile phosphate-buffered saline (PBS, pH 7.4) to a concentration of 1×10⁻⁶. 10 Mix CFU / mL and set aside.

[0049] The experimental animals involved in the following examples are:

[0050] Healthy male 8-week-old SPF-grade C57BL / 6J mice, weighing 20-22g, were used. The mice were housed in standard transparent plastic cages at 22-24℃ with free access to food and drink. All animal experiments involved in this invention were strictly conducted in accordance with relevant Chinese laws, regulations, and national standards regarding the management and use of laboratory animals. Laboratory animals were provided by qualified suppliers holding a "Laboratory Animal Production License" and obtained certificates of quality. All experimental procedures followed GB / T 35823-2018 "General Requirements for Laboratory Animal Experiments," and the animal husbandry and environmental facilities met the requirements of GB 14925 "Laboratory Animal Environment and Facilities." Animal welfare and ethical principles were fully adhered to throughout the entire experimental process.

[0051] The experimental results were statistically analyzed using GraphPad Prism 9.0. ***p<0.001 indicates an extremely significant difference compared to the ND control group, **p<0.01 indicates a highly significant difference compared to the ND control group, and *p<0.05 indicates a significant difference compared to the ND control group. ##p<0.001 indicates an extremely significant difference compared to the HFD control group, ##p<0.01 indicates a highly significant difference compared to the HFD control group, and #p<0.05 indicates a significant difference compared to the HFD control group.

[0052] Example 1: Isolation, identification and biological characteristics analysis of Lactobacillus gasseri BKR-017

[0053] 1. Isolation and Identification of Strains

[0054] Samples were taken from the breast milk of a healthy lactating woman in Shenyang, serially diluted, and plated onto MRS agar plates. The samples were then anaerobically incubated at 37°C for 48 hours. Single colonies with different morphologies were picked and purified to obtain a pure culture strain, named BKR-017.

[0055] The strains obtained above were identified using 16S rDNA.

[0056] The universal primers used for 16S rDNA identification are:

[0057] 27F:5'-AGAGTTTGATCCTGGCTCAG-3' (Sequence 1)

[0058] 1492R:5'-TACGGCTACCTTGTTACGACTT-3' (Sequence 2).

[0059] After PCR amplification, the strain DNA was sent to Shanghai Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The similarity of the sequenced data was compared using EzBioCloud (https: / / www.ezbiocloud.net / ) to determine its species.

[0060] The strain was identified as *Lactobacillus gasseri* by 16S rDNA analysis. Lactobacillus gasseri The strain was named BKR-017. It has been deposited at the China General Microbiological Culture Collection Center (CGMCC, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 36098 and deposit date of September 26, 2025.

[0061] 2. Genetic stability analysis and physiological and biochemical characteristics

[0062] The isolated strain BKR-017 was passaged 10 times consecutively on MRS plates using standard methods. The F10 strain was then analyzed using the bioMérieux VITEK® 2 21347 Anaerobic and Corynebacterium Identification Card (ANC, 21347). This card contains 64 wells for different biochemical assays. The biochemical phenotypic profiles, including enzyme activity, sugar fermentation, and assimilation, displayed on the identification card were read using the VITEK® 2 fully automated microbial analysis system and its accompanying software. The identification results were then compared and analyzed. The results are as follows: Figure 1 As shown.

[0063] The overall biochemical profile of the strain remained unchanged, with identification confidence levels consistently above 99%. This demonstrates that the strain of this invention possesses excellent genetic and physiological stability, and no phenotypic degeneration or variation occurred during continuous subculturing.

[0064] The main physiological and biochemical characteristics of the strain of this invention are as follows:

[0065] (1) Morphological and staining characteristics:

[0066] Gram-positive (G+), appearing as short rods or clubs under microscopic examination, with blunt and rounded ends, mostly arranged in pairs or short chains, without spores, without flagella (non-motile), and facultatively anaerobic.

[0067] (2) Biochemical metabolic characteristics of VITEK® 2 (ANC card reaction results):

[0068] Enzyme activity: Leucine arylamidinase (LeuA), α-glucosidase (dGLU), β-galactosidase (dGAL), and arginine dihydrolase (ADH) were positive; urease (URE), catalase, and alkaline phosphatase (PHOS) were negative.

[0069] Carbon source utilization:

[0070] Strong positive (+): Can efficiently utilize glucose, sucrose, fructose, maltose, galactose, cellobiose, trehalose and salicin to produce acid.

[0071] Negative (-): Cannot utilize arabinose, xylose, rhamnose (this is an important characteristic that distinguishes Lactobacillus gasseri from Lactobacillus casei), sorbitol, and mannitol.

[0072] Other biochemical reactions: Indole test negative, H2S production test negative, aescin hydrolysis positive.

[0073] (3) Growth characteristics and environmental tolerance:

[0074] 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℃.

[0075] 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.

[0076] 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.

[0077] Acid tolerance: It has strong acid resistance, with an 80% survival rate after treatment at pH 3.0 for 4 hours.

[0078] 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.

[0079] Experimental objective:

[0080] 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.

[0081] Test method:

[0082] 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:

[0083] 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.

[0084] 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).

[0085] Group 3: High-fat diet model group (HFD control group): fed a high-fat diet (D12492, Research Diets, USA) with 60% fat as the energy source, and orally administered 100 μL PBS daily.

[0086] Group 4: High-fat diet model group + probiotic group (HFD+BKR-017 group) as the probiotic model intervention group: given a high-fat diet, and orally administered 100 μL of BKR-017 bacterial suspension (containing 1×10⁻⁶ bacteria) daily. 9 CFU (live bacteria).

[0087] 2. Intervention plan:

[0088] The probiotic intervention groups (groups 2 and 4) were given 100 μL of Lactobacillus gasseri BKR-017 bacterial suspension (live count: 1×10⁻⁶) by gavage daily. 9 CFU / mouse / day). The remaining groups were administered an equal volume of PBS via gavage daily. The high-fat diet model group and the probiotic model intervention group (i.e., groups 3 and 4) were initially fed a high-fat diet for 8 weeks to induce an obesity model (body weight more than 25% higher than the normal diet group). From week 9 onwards, each group began the corresponding 8-week intervention. Mouse body weight and feed intake were recorded weekly during this period. The probiotic intervention protocol referenced research on the effects of probiotics on obesity.

[0089] 3. Sample collection and index testing:

[0090] At the end of the experiment, the mice were fasted for 12 hours and their final body weight was measured. The total body composition of the mice, including body fat mass and lean body mass (LBM, also often called fat-free mass, FFM, calculated as: lean body mass = body weight - fat weight), was determined using a dual-energy X-ray absorptiometry (DEXA, Lunar PIXImus, GE Healthcare). Subsequently, the mice's oxygen consumption (VO2), carbon dioxide production (VCO2), thermogenesis, and spontaneous activity were monitored over 48 hours using a comprehensive laboratory animal monitoring system (CLAMS, Columbus Instruments). The respiratory exchange rate (RER = VCO2 / VO2) was calculated, and energy expenditure was calculated based on the Weir formula.

[0091] Body weight, body fat mass, and lean body mass were measured at the beginning of the trial (week 0), after the model was established (week 8), and at the end of the intervention (week 16).

[0092] Experimental results:

[0093] 1. The effect of Lactobacillus gasseri BKR-017 on improving weight and body fat gain induced by a high-fat diet.

[0094] like Figure 2 As shown, at the beginning of the experiment (week 0), there were no significant differences in body weight, body fat mass, and lean body mass among the groups of mice. After 8 weeks of high-fat diet feeding (week 8), the body weight and body fat mass of mice in both the high-fat diet model group and the high-fat diet + probiotic group increased significantly (***p<0.001 vs. normal diet control group), indicating that the obesity model was successfully established, while 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 mice in the high-fat diet model group increased significantly further (***p<0.001), while lean body mass increased slightly but without statistical difference. Compared with the high-fat diet model group, the weight gain trend of mice in the group treated with Lactobacillus gasseri BKR-017 was significantly curbed, with a 20.2% reduction in final body weight (###p<0.001), a significant reduction in body fat mass (###p<0.001), and no significant difference in lean body mass. Under normal dietary conditions, there were no significant differences in the indicators between the probiotic group and the control group, indicating that BKR-017 specifically targets obesity induced by a high-fat diet and has good safety.

[0095] 2. Effects of Lactobacillus gasseri BKR-017 on energy metabolism

[0096] The results showed that, compared with the model group, the intervention group mice had a significantly increased energy expenditure of 15% during the dark period (###p<0.001), a significantly decreased RER (0.75 vs. 0.85), and a 10% increased thermogenesis (#p<0.05), indicating that the energy source shifted to lipid oxidation. Specific data are shown in Table 1.

[0097] Table 1. Energy metabolism indicators of mice in each group (week 16)

[0098]

[0099] Note: 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.

[0100] in conclusion:

[0101] The results of this embodiment confirm that Lactobacillus gasseri BKR-017 exhibits a significant intervention effect on obesity induced by a high-fat diet. Its core mechanism of action is closely related to reducing fat accumulation, increasing energy expenditure, and promoting fat oxidation.

[0102] Example 3: Lactobacillus gasseri BKR-017 activates host stress response by regulating intestinal cysteine ​​metabolism.

[0103] Experimental objective:

[0104] This embodiment aims to evaluate at the molecular level whether Lactobacillus gasseri BKR-017 activates integrated stress response (ISR) and oxidative stress response (OSR) by affecting host cysteine ​​(Cys) metabolism, and to explore the independence of this mechanism; in addition, the functional specificity of the BKR-017 strain was compared with that of the BKR-007 strain group.

[0105] Test method:

[0106] In addition to the mice from each group in Example 2, a fifth group of mice (n=10) was added: a high-fat diet model group + probiotic group (HFD+BKR-007 group) as other Lactobacillus gasseri control intervention groups: given a high-fat diet, and orally administered 100 μL BKR-007 bacterial suspension (containing 1×10⁻⁶) daily. 9 CFU (live bacteria).

[0107] The details of BKR-007 can be found in Chinese Patent ZL202210440892.1, Publication No. CN 114908006B, entitled "A strain of Lactobacillus gasseri and its effect in lowering blood uric acid," which describes it in detail. Lactobacillus gasseriSYP-B4432 (BKR-007) has been deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing), with accession number CGMCC No. 22950 and deposit date of July 26, 2021.

[0108] 1. Sample Collection: Samples were collected from mice in each group (n=10 / group) in Example 2. After the intervention, blood was collected by cardiac puncture under isoflurane anesthesia. Subsequently, the liver, periepididymal white adipose tissue (eWAT), groin subcutaneous white adipose tissue (sWAT), and cecal contents were rapidly separated. Samples were flash-frozen in liquid nitrogen and stored at -80°C.

[0109] 2. Targeted Metabolomics Analysis: 50 mg of liver or cecal contents were accurately weighed and homogenized in an extraction buffer (methanol / water / chloroform, 2:1:1 v / v / v) containing a stable isotope internal standard. The homogenate was ultrasonically disrupted for 30 minutes and then centrifuged (4℃, 12000×g, 15 minutes), and the supernatant was collected. Absolute quantification 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, WatersACQUITY UPLC I-Class / Xevo TQ-S micro). Short-chain fatty acids (SCFAs) in cecal contents were additionally analyzed as a control.

[0110] 3. Gene Expression Analysis: Total RNA was extracted from liver and eWAT cells using TRIzol reagent (Invitrogen), and its concentration and purity were determined using NanoDrop One (Thermo Fisher). 2 μg of total RNA was used to synthesize cDNA using the PrimeScript RT kit (Takara). Using the cDNA as a template, qPCR was performed on a QuantStudio 6 Flex real-time PCR instrument using TB Green Premix Ex Taq II (Takara). The expression of key ISR genes (Atf4, Chop, Fgf21, Gdf15) and key OSR genes (Nrf2, Nqo1, Gstm1, Ho-1) was detected. Gapdh was used as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0111] Primer sequences:

[0112] Atf4 forward primer: 5'-AGCAAAACAAGACAGCAGCC-3' (Sequence 3)

[0113] Atf4 reverse primer: 5'-ACTCTCTTCTTCCCCCTTGC-3' (Sequence 4)

[0114] Chop forward primer: 5'-GAGTCCCTGCCTTTCACCTT-3' (Sequence 5)

[0115] Chop reverse primer: 5'-TTCCTCTTCGTTTCCTGGGG-3' (sequence 6)

[0116] Fgf21 forward primer: 5'-TGACGACCAAGACACTGAAGC-3' (Sequence 7)

[0117] Fgf21 reverse primer: 5'-TTTGAGCTCCAGGAGACTTTCTG-3' (Sequence 8)

[0118] Gdf15 forward primer: 5'-GAGAGGACTCGAACTCAGAAC-3' (Sequence 9)

[0119] Gdf15 reverse primer: 5'-GACCCCAATCTCACCTCTG-3' (sequence 10)

[0120] Nrf2 forward primer: 5'-CACATTCCCAAACAAGATGCCT-3' (Sequence 11)

[0121] Nrf2 reverse primer: 5'-TATCCAGGGCAAGCGACTCA-3' (Sequence 12)

[0122] Nqo1 forward primer: 5'-AGGATGGGAGGTACTCGAATC-3' (Sequence 3)

[0123] Nqo1 reverse primer: 5'-TGCTAGAGATGACTCGGAAGG-3' (Sequence 14)

[0124] Gstm1 forward primer: 5'-GATTGGTGCAGGGTTGGGAG-3' (sequence 15)

[0125] Gstm1 reverse primer: 5'-GCTGGTGCTGTGGTCTTCTC-3' (Sequence 16)

[0126] Ho-1 forward primer: 5'-TTAAGCTGGTGATGGCTTCCT-3' (sequence 17)

[0127] Ho-1 reverse primer: 5'-AGTGGGGCATAGACTGGGTT-3' (Sequence 18)

[0128] Gapdh (internal reference gene) forward primer: 5'-AACGACCCCTTCATTGAC-3' (sequence 19)

[0129] Gapdh (internal reference gene) reverse primer: 5'-TCCACGACATACTCAGCAC-3' (sequence 20).

[0130] 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.

[0131] Experimental results:

[0132] 1. Changes in the levels of downstream metabolites of cysteine

[0133] like Figures 3 to 6 As 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.

[0134] 2. Stress response pathway gene activation

[0135] like Figures 7 to 14As 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.

[0136] in conclusion:

[0137] 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.

[0138] Example 4: Lactobacillus gasseri BKR-017 induces adipose tissue browning via the β3-adrenergic receptor signaling pathway

[0139] Experimental objective:

[0140] 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.

[0141] Test method:

[0142] 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).

[0143] 2. Histological and Immunohistochemical Analysis: sWAT tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, and sectioned into 4μm sections. Adipocyte morphology was observed by H&E staining. The primary antibody was rabbit anti-UCP1 (Abcam, ab10983, 1:200), and the secondary antibody was HRP-labeled goat anti-rabbit IgG (Abcam, 1:500). DAB staining was performed. The area of ​​UCP1-positive tissue was quantified using ImageJ 1.53 software.

[0144] 3. Gene Expression Analysis: Total RNA from sWAT was extracted using TRIzol reagent (Invitrogen), and its concentration and purity were determined using NanoDropOne (Thermo Fisher). 2 μg of total RNA was used to synthesize cDNA using the PrimeScript RT kit (Takara). Using the cDNA as a template, qPCR was performed on a QuantStudio 6Flex real-time PCR instrument with TB Green Premix Ex Taq II (Takara). The expression of key genes for adipose browning (Ucp1, Prdm16) was detected. Gapdh was used as an internal control. -ΔΔCt The relative expression level is calculated using this method.

[0145] Primer sequences:

[0146] Ucp1 forward primer: 5'-GTGAAGGTCAGAATGCAAGC-3' (Sequence 21)

[0147] Ucp1 reverse primer: 5'-AGGGCCCCCTTCATGAGGTC-3' (Sequence 22)

[0148] Prdm16 forward primer: 5'-CAGCACGGTGAAGCCATTC-3' (Sequence 23)

[0149] Prdm16 reverse primer: 5'-GCGTGCATCCGCTTGTG-3' (sequence 24)

[0150] Gapdh (internal reference gene) forward primer: 5'-AACGACCCCTTCATTGAC-3' (sequence 25)

[0151] Gapdh (internal reference gene) reverse primer: 5'-TCCACGACATACTCAGCAC-3' (sequence 26).

[0152] 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.

[0153] Experimental results:

[0154] 1. Lactobacillus gasseri BKR-017 induces fatty browning

[0155] 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).

[0156] 2. The effect of adipose browning depends on the β3-AR pathway.

[0157] 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.

[0158] Table 2. Mechanism validation group adipose browning index

[0159]

[0160] 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).

[0161] in conclusion:

[0162] The results of this embodiment suggest that *Lactobacillus gasseri* BKR-017 is ineffective against high-fat diet mice induced by the β3-AR specific antagonist SR59230A. Therefore, it is believed that *Lactobacillus gasseri* BKR-017 exerts its weight-loss effect through the β3-AR pathway, which is induced by adipose browning. This effect is dependent and specific, providing a new strategy for obesity intervention.

[0163] Example 5: The ameliorative effect of Lactobacillus gasseri BKR-017 on obesity-related metabolic syndrome

[0164] Experimental objective:

[0165] This embodiment aims to evaluate the comprehensive improvement effect of Lactobacillus gasseri BKR-017 on obesity-related metabolic syndromes, such as insulin resistance and non-alcoholic fatty liver disease.

[0166] Test method:

[0167] 1. Sample collection: Samples from the high-fat diet model group and the probiotic model intervention group in Example 2 were used (n=8 / group).

[0168] 2. Indicator Detection: Fasting blood glucose was measured using the glucose oxidase method (Glucometer, Roche); serum insulin (INS) levels were measured using an ELISA kit (Millipore EZRMI-13K), and the insulin resistance index HOMA-IR was calculated as (fasting blood glucose mmol / L × fasting insulin mU / L) / 22.5; serum low-density lipoprotein cholesterol (LDL-C), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels were measured using biochemical kits; liver tissue was collected, and liver TC, TG, and SOD levels were measured using a tissue total cholesterol (TC) assay kit, a triglyceride (TG) assay kit, and a superoxide dismutase (SOD) activity assay kit; the concentration of interleukin-1β (IL-1β) in the liver was measured using an ELISA kit (eBioscience), corrected for liver protein concentration.

[0169] Experimental results:

[0170] 1. Insulin resistance and improved blood sugar

[0171] The results showed that the fasting blood glucose level in the probiotic model intervention group decreased to 7.1±0.9 mmol / L (model group 11.5±1.3, p<0.001), INS content decreased, and HOMA-IR decreased by 48% (p<0.001). Specific data are shown in Table 3, demonstrating an effective inhibitory effect on hepatic steatosis.

[0172] Table 3. Insulin resistance-related indicators in each group of mice

[0173]

[0174] Note: Data are expressed as mean ± standard deviation (n=8). ***p<0.001 vs. ND control group; ###p<0.001 vs. HFD control group.

[0175] 2. Improvement in liver lipid metabolism, antioxidant activity, and inflammation.

[0176] The results showed that serum LDL-C, ALT, and AST levels were significantly decreased in the probiotic model intervention group (###p<0.001); liver TC, TG, and IL-1β levels were decreased, while SOD level was increased (###p<0.001). Specific data are shown in Table 4.

[0177] Table 4 Liver-related indicators in each group of mice

[0178]

[0179] Note: Data are expressed as mean ± standard deviation (n=8). ***p<0.001 vs. ND control group; ###p<0.001 vs. HFD control group.

[0180] in conclusion:

[0181] The results of this embodiment confirm that *Lactobacillus gasseri* BKR-017 can effectively improve obesity-induced insulin resistance, non-alcoholic fatty liver disease, and chronic low-grade inflammation. This strain has industrialization potential, providing a safe and effective new solution for the prevention and adjunctive treatment of obesity and related metabolic diseases.

[0182] In summary, the strain of this invention can mildly mimic a Cys-deficient state by regulating intestinal cysteine ​​(Cys) metabolism. This mechanism of action induces adipose tissue browning, increases energy expenditure, and promotes fat burning by activating the host's integrated stress response (ISR) and oxidative stress response (OSR). Unlike existing probiotic weight loss mechanisms mediated by short-chain fatty acids (SCFAs) or inflammation, this invention provides a novel, precise intervention strategy targeting intestinal Cys metabolism. The *Lactobacillus gasseri* BKR-017 strain described in this invention has advantages such as a clear mechanism, high safety, and significant efficacy, providing a safe, effective, and feasible new adjunctive treatment and prevention approach for obesity and 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 probiotics are used in the preparation of drugs for weight loss, treatment of type 2 diabetes and / or non-alcoholic fatty liver disease.

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

4. The probiotic preparation according to claim 3, characterized in that, The probiotic preparation contains a culture suspension of the strain.

5. The probiotic preparation according to claim 4, characterized in that, The dosage form of the preparation is capsule, tablet, powder, granule or liquid.

Images