Screening method of double-target targeting lipid-lowering and weight-reducing probiotics, probiotics and application thereof

Abstract

The application discloses a screening method of double-target-point lipid-lowering and weight-reducing probiotics, the probiotics and application thereof, and belongs to the technical field of biotechnology. The application constructs a UCP1 and GLP1R double-luciferase reporter gene cell screening model, and combines with alpha-glucosidase inhibition activity detection to first establish a precise screening system of double-target-point synergistic lipid-lowering and weight-reducing probiotics. Through the above screening system, lactococcus lactis LTJ47 and plantarum SLJ451 are obtained, and the animal experiment verifies that the two probiotics can simultaneously activate the UCP1-mediated brown fat thermogenesis pathway and the GLP1R-mediated intestinal sugar metabolism pathway, significantly inhibit the body weight growth induced by a high-fat diet, improve sugar and lipid metabolism disorders and chronic inflammation, and the postbiotic preparations also have excellent effects. The application provides new high-quality strain resources for preparing food, health products or drugs with the functions of lipid-lowering, weight-reducing and blood sugar maintaining.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for screening probiotics that target lipid reduction and weight loss with dual targets, the probiotics themselves, and their applications. Background Technology

[0002] Obesity has become a serious public health problem worldwide, closely related to gut microbiota dysbiosis and imbalances in energy metabolism. It easily induces various chronic metabolic diseases such as type II diabetes, hyperlipidemia, and non-alcoholic fatty liver disease, seriously threatening human physical and mental health and placing a heavy burden on public health systems. Probiotics and post-biotics, as novel functional factors that can regulate host metabolism, have shown significant application potential in improving host metabolic homeostasis and intervening in obesity and related complications. Constructing an efficient, targeted, and precise functional strain screening system is the core prerequisite and key foundation for the industrialization of lipid-lowering and weight-loss probiotics and the promotion of their clinical application.

[0003] Currently, traditional screening systems for functional probiotics mainly rely on in vitro physicochemical tolerance tests (such as acid and bile salt tolerance tests), in vitro enzyme activity inhibition experiments, and non-specific cell function assays, ultimately verifying in vivo efficacy through animal experiments. However, these traditional screening methods have significant core technical defects, making it difficult to meet the needs of the industrialization of lipid-lowering and weight-loss probiotics. Specifically: First, the screening cycle is lengthy, the detection throughput is low, and the cost is high, making it impossible to achieve rapid and targeted screening of large numbers of strains, severely restricting the efficiency of functional strain screening; Second, the functional targeting is weak, failing to directly correlate the strain's intervention on the core regulatory pathways of obesity at the cellular level, resulting in a low match between in vitro screening results and actual in vivo efficacy, easily leading to the phenomenon of "effective in vitro, ineffective in vivo"; Third, existing screening methods cannot simultaneously achieve synergistic evaluation of both lipid-lowering and glucose-lowering functions, resulting in strains with relatively singular efficacy, making it difficult to meet the precise intervention needs of obese individuals with combined glucose and lipid metabolism disorders, thus limiting the application of probiotics in complex metabolic disorder scenarios.

[0004] In recent years, molecular screening technologies, represented by reporter gene systems, have been widely applied in gene regulation mechanism research and functional factor screening due to their outstanding advantages such as high sensitivity, precise quantification, and high-throughput detection. They can directly reflect the activation effect of target genes at the transcriptional level, providing a new technical approach for constructing precise targeted probiotic screening systems. With the deepening research into the molecular mechanisms of obesity and related metabolic disorders, the industry has placed higher demands on the precision of targeted screening and efficacy evaluation of probiotics. Currently, synergistic screening systems adapted to obesity metabolic regulation targets still need improvement, and high-quality probiotic resources with both clear target regulatory activity and stable lipid-lowering and weight-loss effects are still lacking systematic exploration. Therefore, establishing a targeted, precise, efficient, and industrially viable lipid-lowering and weight-loss probiotic screening system is of significant theoretical and practical value for promoting the innovative application of functional probiotics in obesity intervention and filling technological gaps in the industry. Summary of the Invention

[0005] The purpose of this invention is to provide a screening method for probiotics targeting lipid-lowering and weight loss, the probiotics themselves, and their applications, to address the problems existing in the prior art. This invention constructs a UCP1 and GLP1R dual-luciferase reporter gene screening model, combined with α-glucosidase inhibitory activity detection, to achieve precise and efficient screening of probiotics targeting lipid-lowering and weight loss. The obtained *Pediococcus lactis* LTJ47 and *Lactobacillus plantarum* SLJ451 and their metabiotics can simultaneously activate dual targets of thermogenesis and glucose metabolism, significantly improving obesity-related glucose and lipid metabolism disorders.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for screening probiotics that target lipid reduction and weight loss on dual targets, comprising the following steps: Construct a dual-luciferase reporter gene cell screening model driven by the UCP1 promoter and the GLP1R promoter; The fermentation supernatant of the probiotic strain to be tested was co-cultured with the dual-luciferase reporter gene cell screening model, and the relative activities of firefly luciferase and kidney luciferase were detected to screen candidate strains that can simultaneously activate the UCP1 promoter and the GLP1R promoter. The α-glucosidase inhibitory activity of the fermentation supernatant of the candidate strain was determined; Using a blank control without the fermentation supernatant of the probiotics to be tested as the control group, strains that simultaneously meet the requirements of having relative luciferase activities of both the UCP1 promoter and the GLP1R promoter ≥ 2.0 times that of the control group and an α-glucosidase inhibition rate ≥ 60% are selected as the dual-target lipid-lowering and weight-loss probiotics.

[0007] Optionally, the amplification primer sequences for the UCP1 promoter are shown in SEQ ID NO.1-2; and the amplification primer sequences for the GLP1R promoter are shown in SEQ ID NO.3-4.

[0008] This invention also provides a probiotic strain that helps control body fat and maintain healthy blood sugar levels, said probiotic being *Pediococcus lactis* (…). Pediococcus acidilactici LTJ47; The lactic acid cocci LTJ47 was deposited at the China General Microbiological Culture Collection Center on April 3, 2026, with accession number CGMCC No. 38041.

[0009] This invention also provides a probiotic that helps control body fat and maintain healthy blood sugar levels, said probiotic being *Lactobacillus plantarum* (…). Lactiplantibacillus plantarum SLJ451; The Lactobacillus plantarum SLJ451 was deposited at the China General Microbiological Culture Collection Center on April 3, 2026, with accession number CGMCC No. 38029.

[0010] The present invention also provides a probiotic preparation, wherein the probiotic preparation is a live bacteria preparation or a postbiotic preparation; the postbiotic preparation is prepared by inactivation treatment of the *Pediococcus lactis* LTJ47 and / or the *Lactobacillus plantarum* SLJ451.

[0011] Optionally, the inactivation treatment is boiling at 100°C for 30 minutes.

[0012] The present invention also provides the application of the probiotic or the probiotic preparation described above in the preparation of products that help control body fat and help maintain healthy blood sugar levels. The products can simultaneously activate dual targets of thermogenesis and glucose metabolism, and significantly improve obesity-related glucose and lipid metabolism disorders.

[0013] Optionally, the products include health supplements and pharmaceuticals.

[0014] The present invention also provides a product that helps control body fat and helps maintain healthy blood sugar levels, wherein the active ingredients include the probiotics or the probiotic preparations described herein.

[0015] Optional, other pharmaceutically or health-adaptive excipients may also be included.

[0016] The present invention discloses the following technical effects: This invention constructs a UCP1 and GLP1R dual-luciferase reporter gene cell screening model and, combined with α-glucosidase inhibitory activity detection, establishes for the first time a precise screening system for lipid-lowering and weight-loss probiotics with dual-target synergy. This system overcomes the shortcomings of traditional screening methods, such as weak functional targeting, low matching degree between in vitro and in vivo efficacy, and inability to simultaneously evaluate lipid-lowering and glucose-lowering functions. It can directly quantify the ability of strains to intervene in the core regulatory pathways of obesity at the cellular level, significantly improving screening efficiency and accuracy, and providing technical support for industrial development.

[0017] The *Pediococcus lactis* LTJ47 and *Lactobacillus plantarum* SLJ451 strains obtained through the above screening system were verified in animal experiments to simultaneously activate the UCP1-mediated brown adipose tissue thermogenesis pathway and the GLP1R-mediated intestinal glucose metabolism pathway, significantly inhibiting high-fat diet-induced weight gain, improving glucose and lipid metabolism disorders and chronic inflammation. Furthermore, their postbiotic formulations also exhibit excellent efficacy. Both probiotic strains demonstrate good gastrointestinal tolerance and industrial-scale fermentation characteristics, providing new high-quality strain resources for the preparation of foods, health products, or pharmaceuticals with lipid-lowering, weight-loss, and blood sugar-maintaining functions. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 The results show the in vitro screening results of probiotics; where A represents the relative luciferase activity of the UCP1 promoter; B represents the relative luciferase activity of the GLP1R promoter; C represents the in vitro inhibitory activity of α-glucosidase; and D represents the Venn diagram analysis of the multidimensional functional screening results. Figure 2 Phylogenetic tree of target strains based on 16S rRNA gene sequence; Figure 3 The growth characteristics and gastrointestinal tolerance of strains LTJ47 and SLJ451 were detected. Among them, A is the growth curve and pH change curve of strains LTJ47 and SLJ451; B is the survival rate of strains under different pH conditions; C is the survival rate of strains under different bile salt concentrations; D is the change in viable bacteria count after treatment with artificial gastrointestinal fluid. Figure 4 This is a curve showing the change in body weight of mice during the 10-week intervention period; Figure 5 The blood glucose change curves in the intraperitoneal glucose tolerance test (A) and insulin tolerance test (B) of mice in different treatment groups are shown. Figure 6 The rectal temperature of mice in different treatment groups under different ambient temperatures; Figure 7 The images show organ indices for mice in different treatment groups; where A represents the liver, B the spleen, C the kidney, D the epididymal fat, and E the brown adipose tissue. Figure 8 The levels of four lipid parameters in the serum of mice in different treatment groups are shown; where A is TC level; B is TG level; C is LDL-C level; and D is HDL-C level. Figure 9 Serum inflammatory factor levels in mice under different treatment groups; where A represents TNF-α level; B represents IL-1β level; C represents IL-6 level; and D represents IL-10 level. Figure 10 The results of H&E staining of mouse liver (A) and epididymal adipose tissue (B); Figure 11 The relative mRNA expression levels of thermogenesis-related genes UCP1, PGC-1α, PRDM16, and PPARγ in brown adipose tissue of mice in different treatment groups; Figure 12 The relative mRNA expression levels of glucose metabolism-related genes GLP1R, Gcg, GK, and PEPCK in the colon tissue of mice in different treatment groups. Detailed Implementation

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0022] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0023] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0024] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0025] Example 1: A screening system for dual-target lipid-lowering and weight-loss probiotics and the acquisition of target strains. 1. Construction of a UCP1 and GLP1R dual-luciferase reporter gene cell screening model 1.1 Primer design and amplification for the target gene promoter The upstream promoter regions of the transcription start sites of the human UCP1 and GLP1R genes were retrieved from the NCBI gene database. The 349 bp UCP1 promoter region and the 1020 bp GLP1R promoter region were selected as amplification target sequences. Specific PCR primers were designed using SnapGene software, and the primer sequences are shown in Table 1.

[0026] Table 1 Primer sequences for PCR amplification of the target gene promoter PCR amplification was performed using HEK293T cell genomic DNA as a template. The reaction system and procedure are shown in Tables 2 and 3, respectively. The amplification products were detected by 1% agarose gel electrophoresis, excised from the gel, and purified to obtain the target promoter fragment.

[0027] Table 2 PCR reaction system for the target gene Table 3 PCR reaction procedure for the target gene 1.2 Construction of recombinant expression plasmids The purified UCP1 promoter fragment and GLP1R promoter fragment were ligated into the linear pGL4.23 reporter gene vector, which had been digested with restriction endonucleases KpnI and HindIII, respectively, for homologous recombination. The homologous recombination reaction system is shown in Table 4. After reacting the reaction system in a 50℃ water bath for 15 min, the cells were transformed into DH5α competent cells, plated on LB solid medium containing ampicillin, and incubated overnight at 37℃. Single colonies were picked for colony PCR identification. The colony PCR reaction system and reaction procedure are shown in Tables 5 and 6. Positive clones were sent to Suzhou Genewise Biotechnology Co., Ltd. for Sanger sequencing. After sequence alignment confirmed that the promoter fragment was correctly inserted, without mutations or deletions, plasmids were extracted using an endotoxin-free plasmid extraction kit and stored at -20℃ for later use.

[0028] Table 4 Homologous recombination reaction system Table 5 Colony PCR Reaction System Table 6 Colony PCR Reaction Procedure 1.3 Cell transfection and model construction After resuscitation, HEK293T cells were cultured in DMEM complete medium (containing a mixture of 10% fetal bovine serum and 1% penicillin-streptomycin) at 37°C in a 5% CO2 incubator. Cells in the logarithmic growth phase from passages 3 to 8 were harvested, and the cell density was adjusted to 1 × 10⁻⁶ cells / year. 5 Cells were seeded at 2 mL / well in 6-well cell culture plates and cultured until cell confluence reached 70%–80% before plasmid transfection. Using liposome transfection reagent, 1 μg of recombinant UCP1 or GLP1R reporter plasmid and 0.1 μg of internal control plasmid pRL-TK (Renex luciferase reporter plasmid, Promega) were co-transfected into each well. Ten h after transfection, the culture medium was discarded, and the supernatant of the probiotics to be tested or the control sample was added. The cells were cultured for another 24 h to obtain the UCP1 and GLP1R dual-luciferase reporter gene cell screening model.

[0029] 2. Precise in vitro screening of beneficial probiotics with lipid-lowering and weight-loss effects. 2.1 Screening of probiotic strains All 15 probiotic strains tested were strains previously screened by the inventors and preserved in the laboratory. Each strain was streaked on MRS agar plates, incubated at 37°C for 24 h for activation, and single colonies were picked and inoculated into MRS liquid medium, incubated at 37°C for 12 h, and then transferred at a 2% inoculum for further culture for 12 h. The bacterial culture was centrifuged at 8000 r / min for 10 min, the supernatant was collected, filtered through a 0.22 μm filter for sterilization, and stored at 4°C for later use.

[0030] Ten h after cell transfection, the culture medium in the 6-well plates was discarded, and 200 μL of bacterial supernatant was added to each well. An equal volume of MRS liquid culture medium was used as a blank control, and the cells were cultured for another 24 h. The culture medium was then discarded, and 200 μL of cell lysis buffer was added to each well for complete lysis. The cells were centrifuged at 12000 r / min for 5 min, and the supernatant was collected. The activities of firefly luciferase and Renilla luciferase were measured using a dual-luciferase assay kit, and the relative luciferase activities were calculated.

[0031] The results are as follows Figure 1 As shown in Figures A and B: Compared with the blank control group, strain SLJ451 exhibited the strongest activation activity in UCP1 promoter transcriptional activation, with a relative luciferase activity 4.3 times that of the control group; in GLP1R promoter transcriptional activation, strains LTJ47 and SLJ456 showed the best activation activity, with relative luciferase activities approximately 2.5 times that of the control group. Among them, strains PR88, PR93, SLJ453, and SLJ456 showed strong activation ability for single-target promoters, while strains LTJ47, SLJ451, and SLJ455 showed significant activation effects on both UCP1 and GLP1R dual promoters.

[0032] 2.2 Detection of α-glucosidase inhibitory activity The α-glucosidase inhibitory activity of 15 probiotic strains was detected. 25 μL of bacterial supernatant, 50 μL of 0.5 U / mL α-glucosidase solution, and 25 μL of 0.1 mol / L PBS buffer were added sequentially to 96-well plates and incubated at 37°C for 10 min. Then, 50 μL of PNPG substrate was added, and the reaction was continued at 37°C for 15 min. The reaction was terminated by adding 100 μL of 0.1 mol / L Na₂CO₃, and the absorbance at 405 nm was measured using a microplate reader. Acarbose (1 mg / mL) was used as a positive control. PBS was used instead of enzyme solution in the blank group, and PBS was used instead of the sample in the control group. The enzyme activity inhibition rate was calculated.

[0033] The results are as follows Figure 1 As shown in Figure C, strains LTJ47, PR98, PR99, ZC198, and SLJ451 all exhibited inhibition rates of over 60% against α-glucosidase, demonstrating good potential for in vitro hypoglycemic activity.

[0034] 2.3 Comprehensive screening and target strain identification The three indicators are: UCP1 promoter activation activity, GLP1R promoter activation activity, and α-glucosidase inhibitory activity. Figure 1Based on the D Venn diagram analysis, the strains that simultaneously met the criteria of having significant activation effects on both UCP1 and GLP1R dual promoters (relative luciferase activity ≥ 2.0 times that of the control group) and α-glucosidase inhibition rate ≥ 60% were LTJ47 and SLJ451, and these two strains were finally identified as the target functional strains.

[0035] 3. Molecular biological identification of the target strain Strains LTJ47 and SLJ451 were inoculated into MRS liquid medium and incubated overnight at 37°C. Genomic DNA was extracted using a bacterial genomic DNA extraction kit and amplified by PCR using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO.5) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO.6). After electrophoresis verification, the products were sent to Suzhou Genewiz Biotechnology Co., Ltd. for Sanger sequencing. The sequencing results were compared with the NCBI database for BLAST homology, and a phylogenetic tree was constructed using MEGA 12 to complete species-level identification.

[0036] The results are as follows Figure 2 As shown, strain LTJ47 and Pediococcus acidilactici 24NL38 clustered into the same branch, with sequence homology ≥99%, and were identified as Pediococcus lactis ( Pediococcus acidilactici The strain SLJ451 was deposited on April 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38041, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Lactiplantibacillus plantarum GA5 and HBUAS52357 clustered into the same branch, with sequence homology ≥99%, and were identified as *Lactobacillus plantarum*. Lactiplantibacillus plantarum It was deposited on April 3, 2026, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38029, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0037] Example 2: Growth characteristics and stress resistance evaluation of the target strain 1. Growth curve and acid production capacity determination Activated strains LTJ47 and SLJ451 were inoculated into MRS liquid medium and incubated at 37°C. Samples were taken every 4 hours to measure OD. 600 The pH value of the culture medium was measured using a pH meter, and the measurement was carried out continuously for 36 h. Growth curves and pH change curves were plotted.

[0038] The results are as follows Figure 3 As shown in Figure A: the growth trends of strains LTJ47 and SLJ451 are consistent. The growth lag phase is 0–8 h, the rapid logarithmic growth phase begins 8–16 h, and the stationary phase is reached 20–24 h, with the endpoint OD... 600 The pH values ​​stabilized at approximately 0.8 and 1.2, respectively. As the strains proliferated, the pH of the culture medium continuously decreased, reaching approximately 3.6 at the end of 36 hours of cultivation. This indicates that both strains possess good growth and reproductive capabilities as well as acid-producing properties.

[0039] 2. Determination of acid and bile salt resistance Two bacterial strains were inoculated at a 2% inoculum into MRS liquid medium at pH 2.0, pH 3.0, and pH 4.0, and into MRS medium containing 0.1%, 0.3%, and 0.5% (w / v) ox bile salts, respectively. The cultures were incubated statically at 37°C for 12 h, and the OD was measured. 600 Values ​​and calculate survival rate.

[0040] The results are as follows Figure 3 As shown in Figures B and C: at pH 4.0, the survival rate of strain LTJ47 was 47.56%, and that of strain SLJ451 was 46.22%; the survival rate decreased significantly at pH 3.0; at bile salt concentrations of 0.1% and 0.3%, the survival rates of both strains remained above 80%; in a high-concentration bile salt environment of 0.5%, the survival rate of strain LTJ47 was 75.95%, and that of strain SLJ451 was 78.86%. This indicates that both strains possess good acid and bile salt tolerance.

[0041] 3. Artificial gastrointestinal fluid tolerance test The activated bacterial strain was inoculated into artificial gastric fluid at a 2% inoculum and incubated at 37°C for 3 h. Then, it was transferred to artificial intestinal fluid at a 10% inoculum and incubated at 37°C for another 4 h. Samples were taken at 0 h, 3 h, 5 h, and 7 h, and the viable bacterial count was determined using the plate count method to calculate the survival rate. For the artificial gastric fluid preparation: 9.0 g of NaCl and 3.0 g of pepsin were dissolved in an appropriate amount of water, the pH was adjusted to 3.0 with 0.1 mol / L HCl, and the volume was brought to 1 L with water. The solution was then filtered through a 0.22 μm filter for sterilization. For the artificial intestinal fluid preparation: 9.0 g of NaCl, 9.0 g of ox bile salts, and 1.0 g of trypsin were dissolved in an appropriate amount of water, the pH was adjusted to 8.0 with 0.1 mol / L NaOH, and the volume was brought to 1 L with water. The solution was then filtered through a 0.22 μm filter for sterilization.

[0042] The results are as follows Figure 3As shown in Figure D: after 3 hours of treatment with artificial gastric fluid, the survival rate of both strains remained above 90%; after further culture in artificial intestinal fluid for 7 hours, the survival rate of both strains remained above 69%. This confirms that both strains can tolerate continuous stress from gastrointestinal digestive fluids.

[0043] Example 3: Verification of the lipid-lowering and weight-loss effects of the target strain and its postbiotic on high-fat diet-induced obese mice. 1. Preparation of bacterial strains and postbiotic preparations Activated strains LTJ47 and SLJ451 were inoculated into MRS liquid medium and incubated at 37°C for 12 h. The bacterial cells were collected by centrifugation at 4000 r / min for 10 min, washed three times with sterile physiological saline, resuspended in sterile physiological saline, and the bacterial concentration was adjusted to prepare low-dose (2×10⁻⁶) solutions. 7 CFU / mL) and high doses (2×10) 9 CFU / mL live bacterial preparation. A separate high-dose live bacterial preparation was boiled at 100℃ for 30 min to inactivate the bacteria. After verification of no live bacterial growth by plate spreading, this was used as a post-biotic preparation. All preparations were prepared and used immediately, and thoroughly mixed before gavage.

[0044] 2. Grouping and intervention protocols for experimental animals Fifty-four 5-week-old SPF-grade male C57BL / 6J mice were randomly divided into 9 groups of 6 mice each after one week of acclimatization. The grouping details and treatments for each group are shown in Table 7. All mice had free access to food and water and were administered 0.2 mL of fluid by gavage daily for 10 consecutive weeks. Their body weight was monitored weekly.

[0045] Table 7. Grouping and Intervention Protocols for Obese Mouse Models Weight monitoring results as follows Figure 4 As shown: After 10 weeks of high-fat diet feeding, the HFD group mice gained 73.4% of their body weight, and the obesity model was successfully established; from the 3rd week of intervention, the weight gain of mice in each strain intervention group was significantly lower than that in the HFD group, and the weight inhibition effect continued to increase with the extension of intervention time; the high-dose strain group had a better weight control effect than the low-dose group, and the post-biotic group had the best effect. The weight gain inhibition rate of the P-LTJ47 group was 40.2%, and the weight gain inhibition rate of the P-SLJ451 group was 36.9%, which was similar to that of the OLYM group.

[0046] The above results confirm that the strains LTJ47 and SLJ451 of the present invention and their postbiotics can significantly inhibit the increase in body weight induced by a high-fat diet in mice.

[0047] 3. Detection of physiological and biochemical indicators 3.1 Glucose tolerance and insulin tolerance test In week 8 of the intervention, an intraperitoneal glucose tolerance test (IPGTT) was performed: mice were fasted for 12 h and then injected intraperitoneally with glucose solution (2 g / kg). Blood samples were collected from the tail tip before injection (0 min) and at 15, 30, 60, 90, and 120 min after injection. Blood glucose levels were measured using a glucometer, and a blood glucose curve was plotted and the area under the curve (AUC) was calculated. In week 9 of the intervention, an insulin tolerance test (ITT) was performed: mice were fasted for 4 h and then injected intraperitoneally with human insulin (0.75 U / kg). Blood glucose levels were measured from the tail tip before injection (0 min) and at 15, 30, 60, 90, and 120 min after injection.

[0048] The results are as follows Figure 5 As shown in Figures A and B: the peak blood glucose level in the IPGTT of mice in the HFD group was significantly higher than that in the NFD group, and the rate of blood glucose decline within 120 min was significantly slower. In the ITT test, the rate of blood glucose decline in mice in the HFD group was significantly slower than that in the NFD group, and the blood glucose level had not recovered to the baseline level after 150 min, indicating that a high-fat diet induces significant glucose tolerance and insulin resistance in mice. All strain intervention groups can significantly reduce the peak blood glucose level in the IPGTT, accelerate the rate of blood glucose clearance, and improve glucose tolerance and insulin sensitivity in mice. The high-dose group showed significantly better improvement than the low-dose group. The post-biotic group of the two strains showed the best effect, which was close to that of the OLYM positive drug group.

[0049] 3.2 Body temperature and heat production capacity detection Before the intervention ended, the mice were placed in a 25°C room temperature environment for 30 min to stabilize and their rectal temperature was measured. Then the mice were transferred to a 4°C cold stimulation environment and their rectal temperature was measured again after 60 min.

[0050] The results are as follows Figure 6 As shown: Under normal temperature conditions of 25℃, there was no significant difference in rectal temperature among the groups of mice (stable at 36~38℃); after cold stimulation at 4℃, the rectal temperature of mice in the HFD group dropped significantly to about 33℃, while the rectal temperature of mice in the NFD group remained basically stable, indicating that the high-fat diet led to impaired thermogenesis function of brown adipose tissue and energy metabolism disorders in mice; the intervention groups of each strain could significantly reverse the decrease in body temperature under cold stimulation and maintain the stability of the core body temperature of mice, among which the post-genetic group had the most prominent thermoprotective effect, confirming that the strain can improve the body's energy metabolism by activating thermogenesis of brown adipose tissue.

[0051] 3.3 Collection of serum and tissue samples and detection of indicators After 10 weeks of intervention, mice were fasted for 12 hours, and blood was collected from their eyeballs. After standing at room temperature for 30 minutes, serum was separated by centrifugation at 3000 r / min for 15 minutes at 4℃. Serum levels of total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were detected using a fully automated biochemical analyzer. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) were detected using an ELISA kit. Mice were euthanized by cervical dislocation after blood collection, and the liver, spleen, kidneys, brown adipose tissue (interscapular region), and epididymal adipose tissue were separated, weighed, and organ indices were calculated. Liver and epididymal adipose tissue were fixed with 10% neutral formaldehyde for H&E staining; brown adipose tissue and colon tissue were flash-frozen in liquid nitrogen and stored at -80℃.

[0052] Organ index results as follows Figure 7 As shown: Compared with the NFD group, the HFD group mice showed significantly increased adipose tissue indices in the liver, spleen, kidneys, and epididymis, and significantly decreased brown adipose tissue indices, exhibiting a typical phenotype of obesity-related organ damage and abnormal lipid accumulation. The intervention groups of each strain could significantly reverse the abnormal organ indices of obese mice, effectively alleviate liver lipid deposition, inflammation-related organ hyperplasia, and abnormal fat accumulation, with the high-dose group and the post-biotic group showing more prominent improvement effects.

[0053] Blood lipid test results as follows Figure 8 As shown, the HFD group mice exhibited significantly elevated serum TC, TG, and LDL-C levels, and significantly decreased HDL-C levels, displaying a typical hyperlipidemia phenotype. All strain intervention groups significantly downregulated TC, TG, and LDL-C levels and upregulated HDL-C levels, effectively improving high-fat diet-induced dyslipidemia. Furthermore, the lipid-regulating effect of the post-biotic group was significantly better than that of the low- and high-dose groups of the same strain.

[0054] Serum inflammatory factor test results as follows Figure 9 As shown, the serum levels of pro-inflammatory factors TNF-α, IL-1β, and IL-6 were significantly increased, while the level of anti-inflammatory factor IL-10 was significantly decreased in the HFD group mice, confirming that a high-fat diet induced a state of systemic chronic inflammation in the mice. All strain intervention groups significantly downregulated the levels of pro-inflammatory factors and upregulated the levels of anti-inflammatory factors, effectively alleviating obesity-related chronic inflammation. Among them, SLJ451 showed a more significant anti-inflammatory effect than LTJ47.

[0055] 3.4 Histopathological staining H&E staining results are as follows Figure 10As shown: In the NFD group, mouse hepatocytes exhibited regular morphology and no obvious lipid droplet vacuoles; epididymal adipocytes were uniform in size and had clear boundaries, maintaining a normal physiological volume. In the HFD group, mice showed typical obesity-related pathological damage, with severe steatosis in the liver tissue, numerous macrovesicular lipid droplet vacuoles visible within hepatocytes, and significantly enlarged and highly heterogeneous epididymal adipocytes, exhibiting characteristic changes of excessive lipid accumulation. LTJ47, SLJ451, and post-biotic intervention could alleviate the above pathological changes. The low-dose group showed limited improvement, while the high-dose group and the post-biotic group significantly reduced lipid droplet deposition in hepatocytes and decreased the volume of epididymal adipocytes, with the post-biotic group showing better improvement. The morphology of liver tissue and epididymal adipose tissue in the P-SLJ451 group was basically restored to normal levels, confirming that it can effectively alleviate liver damage and abnormal lipid accumulation induced by a high-fat diet.

[0056] 4. Detection of mRNA expression levels of genes related to brown adipose tissue and colonic tissue metabolism Total RNA was extracted from mouse brown adipose tissue and colon tissue using the Trizol method, and cDNA was synthesized by reverse transcription. The mRNA expression levels of thermogenesis-related genes UCP1, PGC-1α, PRDM16, and PPARγ in brown adipose tissue, and glucose metabolism-related genes GLP1R, Gcg, GK, and PEPCK in colon tissue were detected using RT-qPCR. -ΔΔCt The relative expression levels of each target gene can be calculated.

[0057] The results are as follows Figure 11 and Figure 12 As shown, compared with the NFD group, the mRNA expression levels of thermogenesis-related genes UCP1, PGC-1α, PRDM16, and PPARγ in brown adipose tissue of the HFD group were significantly reduced, indicating that a high-fat diet inhibited the thermogenesis function of brown adipose tissue. Simultaneously, the expression levels of glucose metabolism-related genes GLP1R, Gcg, and GK in colon tissue were significantly reduced, while the expression level of PEPCK, a key rate-limiting enzyme gene for gluconeogenesis, was significantly increased, indicating that a high-fat diet induced damage to the intestinal GLP-1 signaling pathway and abnormal hypergluconeogenesis. After intervention with strains LTJ47 and SLJ451 and their metabiotics, the abnormal expression of the above genes was reversed. The low-dose group only partially upregulated the expression of thermogenesis and glucose metabolism-related genes, while the high-dose group significantly increased the transcriptional levels of these genes and inhibited the expression of gluconeogenesis pathway-related genes. The metabiotic group showed the most significant regulatory effect, significantly upregulating the expression of key genes such as UCP1, PGC-1α, and GLP1R, with transcriptional levels significantly higher than in the NFD group, exhibiting obvious gene activation expression.

[0058] The above results indicate that the *Pediococcus lactis* LTJ47 and *Lactobacillus plantarum* SLJ451 and their metabolites obtained by screening in this invention can effectively alleviate energy and glucose metabolism disorders induced by high-fat diets at the molecular level by activating the dual targets of UCP1 and GLP1R, upregulating the expression of brown adipose-derived thermogenesis-related genes, and improving the expression of colonic glucose metabolism-related genes. They also significantly inhibit weight gain in obese mice, improve dyslipidemia, alleviate hepatic steatosis and chronic inflammation, and have a clear lipid-lowering and weight-loss effect.

[0059] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for screening probiotics that target lipid reduction and weight loss on dual targets, characterized in that, Includes the following steps: Construct a dual-luciferase reporter gene cell screening model driven by the UCP1 promoter and the GLP1R promoter; The fermentation supernatant of the probiotic strain to be tested was co-cultured with the dual-luciferase reporter gene cell screening model, and the relative activities of firefly luciferase and kidney luciferase were detected to screen candidate strains that can simultaneously activate the UCP1 promoter and the GLP1R promoter. The α-glucosidase inhibitory activity of the fermentation supernatant of the candidate strain was determined; Using a blank control without the fermentation supernatant of the probiotics to be tested as the control group, strains that simultaneously meet the requirements of having relative luciferase activities of both the UCP1 promoter and the GLP1R promoter ≥ 2.0 times that of the control group and an α-glucosidase inhibition rate ≥ 60% are selected as the dual-target lipid-lowering and weight-loss probiotics.

2. The screening method according to claim 1, characterized in that, The amplification primer sequences for the UCP1 promoter are shown in SEQ ID NO.1-2; the amplification primer sequences for the GLP1R promoter are shown in SEQ ID NO.3-4.

3. A probiotic strain that helps control body fat and maintain healthy blood sugar levels, characterized in that... The probiotic is Pediococcus lactis ( Pediococcus acidilactici LTJ47; The lactic acid cocci LTJ47 was deposited at the China General Microbiological Culture Collection Center on April 3, 2026, with accession number CGMCC No. 38041.

4. A probiotic strain that helps control body fat and maintain healthy blood sugar levels, characterized in that... The probiotic is *Lactobacillus plantarum* (… Lactiplantibacillus plantarum SLJ451; The Lactobacillus plantarum SLJ451 was deposited at the China General Microbiological Culture Collection Center on April 3, 2026, with accession number CGMCC No. 38029.

5. A probiotic preparation, characterized in that, The probiotic preparation is a live bacteria preparation or a postbiotic preparation; the postbiotic preparation is prepared by inactivation treatment of Pseudococcus lactis LTJ47 as described in claim 3 and / or Lactobacillus plantarum SLJ451 as described in claim 4.

6. The probiotic preparation according to claim 5, characterized in that, The inactivation treatment was performed by boiling at 100°C for 30 minutes.

7. The use of a probiotic preparation according to claim 3, claim 4, or claim 5 or 6 in the preparation of a product that helps control body fat and helps maintain healthy blood sugar levels, characterized in that, The product can simultaneously activate two targets: thermogenesis and glucose metabolism, significantly improving obesity-related glucose and lipid metabolism disorders.

8. The application according to claim 7, characterized in that, The products include health supplements and pharmaceuticals.

9. A product that helps control body fat and helps maintain healthy blood sugar levels, characterized in that, The active ingredients include the probiotics of claim 3, the probiotics of claim 4, or the probiotic preparation of claim 5 or 6.

10. The product according to claim 9, characterized in that, It also includes other pharmaceutically or health-promoting excipients.

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