A type of Lactobacillus plantarum YBE2 and its application

Abstract

This invention discloses a *Lactobacillus plantarum* YBE2 and its applications. The *Lactobacillus plantarum* provided by this invention, which produces a high amount of extracellular polysaccharides primarily composed of rhamnose, can simultaneously degrade different types of bile salts, including specifically hydrolyzing glycocholic acid. It also exhibits cholesterol-lowering and acute inflammation-relieving effects. Furthermore, *Lactobacillus plantarum* YBE2 can reduce fat deposition in the abdomen of caged broilers, increase intramuscular fat, promote fatty acid deposition in muscles, and reduce plasma total bile acids, triglycerides, and low-density lipoprotein cholesterol. This significantly improves lipid metabolism and muscle quality in poultry, and can be widely applied in caged broiler farming to promote healthy poultry farming and improve farming efficiency.

Description

Technical Field

[0001] This invention belongs to the field of probiotic technology, specifically relating to Lactobacillus plantarum YBE2 and its applications. Background Technology

[0002] In recent years, the three-dimensional cage-raising chicken house model has gradually become the mainstream trend in the development of the broiler industry. It has many advantages such as strong environmental controllability, low broiler disease incidence, and good economic benefits. However, it also has some shortcomings and drawbacks. Because the chickens are in a state of limited activity space for a long time, their exercise is reduced and their energy consumption is lowered. When cage-raised broilers consume a lot of high-energy feed, mainly corn, the excessive intake of fat and energy is easy to accumulate in the liver and abdomen. This can lead to excessive accumulation of abdominal and subcutaneous fat, cholesterol metabolism disorder, and negatively affect the health and meat quality of the broilers. At the same time, it restricts the development of the broiler industry.

[0003] Lactic acid bacteria are the most representative genus of probiotics. Besides improving feed flavor and inhibiting pathogens, they also improve the host's gut microbiota, enhance immunity, lower cholesterol, and improve lipid metabolism. Bile salt hydrolase, an intracellular enzyme produced during the growth and reproduction of lactic acid bacteria, hydrolyzes bound glycine bile salts and taurine bile salts into glycine, taurine, and conjugated bile salts, thereby gradually lowering cholesterol levels and effectively reducing liver fat accumulation and blood lipid levels. Extracellular polysaccharides from lactic acid bacteria promote the growth of beneficial bacteria such as Bifidobacteria and have antioxidant, anti-inflammatory, anti-tumor, and cholesterol-lowering effects. Current research on cholesterol-lowering effects of lactic acid bacteria mainly focuses on human food. Therefore, screening functional probiotics for caged broilers is an important way to improve lipid metabolism, regulate gut microbiota, and enhance the immune performance of poultry.

[0004] While *Lactobacillus plantarum* has been reported to have some cholesterol-lowering function, there is currently no *Lactobacillus plantarum* that possesses both high extracellular polysaccharide production and cholesterol-lowering ability. Therefore, based on the principle of microbial adaptability and specificity to their habitats, this study aims to screen functional lactic acid bacteria from the gut of broilers to regulate lipid metabolism in caged broilers, reduce blood lipids, enhance immunity, improve chicken meat quality and gut health, and provide strong support for the high-quality development of caged broilers. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of the embodiments of the present invention and to briefly describe some preferred embodiments.

[0006] The first objective of this invention is to provide a strain of *Lactobacillus plantarum* YBE2, which is a lactic acid bacterium screened from chicken intestines. It is characterized by high production of extracellular polysaccharides, production of bile salt hydrolase, and cholesterol reduction. The *Lactobacillus plantarum* YBE2 strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number M 20241894 and deposit date of September 2, 2024.

[0007] The Lactobacillus plantarum strain YBE2 was isolated from the chicken intestine. After colony morphology observation, Gram staining, and 16S rDNA amplification of the strain's genome, the PCR product was obtained and sequenced for identification. Finally, it was identified as Lactobacillus plantarum (l. plantarum YBE2), and named YBE2.

[0008] The second objective of this invention is to provide the application of the aforementioned *Lactobacillus plantarum* YBE2 in caged broiler farming.

[0009] A third objective of this invention is to provide the application of the aforementioned *Lactobacillus plantarum* YBE2 in the preparation of probiotics that reduce abdominal fat deposition in caged broilers while increasing intramuscular fat content in caged broilers.

[0010] The fourth objective of this invention is to provide the application of *Lactobacillus plantarum* YBE2 in the production of rhamnose.

[0011] The fifth objective of this invention is to provide the application of *Lactobacillus plantarum* YBE2 in the preparation of probiotics that specifically hydrolyze glycocholic acid.

[0012] The sixth objective of this invention is to provide the application of *Lactobacillus plantarum* YBE2 in the preparation of probiotics that lower cholesterol in high-fat mice.

[0013] The seventh objective of this invention is to provide the application of Lactobacillus plantarum YBE2 in the preparation of probiotics with anti-inflammatory and antioxidant functions.

[0014] This invention isolates and screens *Lactobacillus plantarum* YBE2 from chicken intestines, which exhibits cholesterol-lowering properties, high production of bile salt hydrolase, and extracellular polysaccharides. Compared to existing technologies, *Lactobacillus plantarum* YBE2 of this invention also possesses the following unexpected technical effects:

[0015] 1) The present invention contains a high-yield extracellular polysaccharide from Lactobacillus plantarum YBE2, with rhamnose as the main component of the polysaccharide, and the extracellular polysaccharide has specificity.

[0016] 2) The plant lactobacillus YBE2 of the present invention has the ability to hydrolyze four kinds of bile salts, among which the hydrolysis rate of glycocholic acid is as high as 95%, and it has strong bile salt hydrolytic enzyme activity.

[0017] 3) The *Lactobacillus plantarum* YBE2 of this invention significantly reduces abdominal fat deposition in caged broilers, lowers plasma alanine aminotransferase, total bile acids, triglycerides, and low-density lipoprotein cholesterol, while simultaneously increasing intramuscular fat content, promoting fatty acid deposition in muscle, and regulating fat metabolism. *Lactobacillus plantarum* YBE2 improves meat color and enhances muscle quality in broilers.

[0018] 4) The plant lactobacillus YBE2 of the present invention can effectively slow down the weight gain of experimental mice fed a high-fat diet, reduce the serum cholesterol level of high-fat mice, reduce the content of conjugated bile acids, and increase the content of unconjugated bile acids, thus having the effect of lowering cholesterol and blood lipids, and improving liver lesions caused by a high-cholesterol diet.

[0019] 5) The *Lactobacillus plantarum* YBE2 of the present invention can inhibit the expression of pro-inflammatory factors and increase the expression of anti-inflammatory factors in mice in the LPS inflammation model experiment, thus alleviating acute inflammation; it can reduce NO levels and increase the levels of GSH-PX and CAT in the ileal supernatant, thus exhibiting antioxidant capacity.

[0020] In summary, the *Lactobacillus plantarum* strain provided by this invention, which produces a high amount of extracellular polysaccharides, primarily rhamnose, can simultaneously degrade different types of bile salts, including specifically hydrolyzing glycocholic acid. It also exhibits cholesterol-lowering and acute inflammation-relieving effects. Furthermore, *Lactobacillus plantarum* YBE2 can reduce fat deposition in the abdomen of caged broilers, increase intramuscular fat, promote fatty acid deposition in muscles, and reduce plasma total bile acids, triglycerides, and low-density lipoprotein cholesterol. This significantly improves lipid metabolism and muscle quality in poultry, and can be widely applied in caged broiler farming to promote healthy poultry farming and improve farming efficiency.

[0021] The preservation information for the microorganisms involved in this invention is as follows:

[0022] Depository: China Center for Type Culture Collection;

[0023] Address of the depositary institution: Wuhan University, Wuhan, China;

[0024] Classification and nomenclature: Lactobacillus plantarum YBE2;

[0025] Accession number: CCTCC NO: M 20241894;

[0026] Date of preservation: September 2, 2024. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below, wherein:

[0028] Figure 1 The effect of Lactobacillus plantarum YBE2 on intramuscular fat in caged broilers.

[0029] Figure 2 The effect of Lactobacillus plantarum YBE2 on the fatty acid composition of caged broiler muscle.

[0030] Figure 3 This is a pathological image of a mouse liver. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments. These embodiments are intended to facilitate a better understanding of the present invention, but do not limit the scope of the invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods.

[0032] The culture medium used in this invention was purchased from Qingdao High-tech Park Haibo Biotechnology Co., Ltd. Glycocholic acid (GCA), taurocholic acid (TCA), glycodeoxycholic acid (GDCA), and taurodeoxycholic acid (TDCA) were all purchased from Sigma-Aldrich, USA. Other reagents, unless otherwise specified, were obtained commercially.

[0033] Example 1: Screening of a high-extracellular polysaccharide-producing Lactiplantibacillus plantarum YBE2 strain

[0034] (1) Strains Isolation, Purification and Primary Screening

[0035] Lactobacillus was isolated and purified from chicken intestines as follows: 1g of intestinal chyme sample was weighed and added to 99ml of sterile distilled water, shaken on a constant temperature shaker for 1 hour, and then serially diluted 10-fold. 10g samples were then taken from each sample. -3 10 -4 The diluted sample solution was spread onto MRS solid medium and incubated for 48 hours. Then, the culture was removed, and single colonies were picked based on colony size, morphology, and color (medium-sized, raised, slightly whitish, moist, with neat edges, and round in shape, 3.0 mm ± 1 mm in diameter). Catalase and Gram staining were then performed. Catalase-negative and Gram-positive colonies were tentatively identified as *Lactobacillus* and purified twice more by streaking on MRS solid medium.

[0036] (2) 16S rDNA identification

[0037] Lactobacillus strains purified and cultured on solid plates for 24 hours were amplified by colony PCR for 16S rRNA gene sequence identification. Universal primers for bacterial 16S rRNA gene amplification, 27F and 1492R, were used. The primer sequences were: upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′; downstream primer: 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction system consisted of 20 μl: 10 μl PremixTaq, 1 μl of 27F and 1492R (both 20 μM), and sterile distilled water added to a final volume of 20 μl. Colonies were directly added to the reaction system for PCR amplification to obtain the PCR products. The PCR products were sent to Qingke Biotechnology Co., Ltd. for sequencing, and the results showed that five strains were identified as *Lactobacillus plantarum*. The unidentified bacterial strains were stored at -80℃.

[0038] (3) Strain activation and rescreening

[0039] The strain stored at -80℃ was activated by streaking on MRS solid medium and incubated at 37℃ for 24 hours; single colonies grown on the plate were picked and inoculated into 3 mL of MRS liquid medium and incubated overnight at 37℃.

[0040] 2 mL of seed culture was inoculated into 100 mL of MRS liquid medium at a 2% (v / v) ratio for fermentation, and cultured statically at 37°C for 32 h. The fermentation broth was centrifuged (10000×g, 4°C, 15 min), and the supernatant was collected. 80% trichloroacetic acid was added to the supernatant to a final concentration of 4% (w / v), and the mixture was allowed to stand at 4°C for 8 h before centrifugation (10000×g, 4°C, 15 min) to remove protein precipitate. Subsequently, 3 volumes of pre-cooled anhydrous ethanol solution were added to the supernatant, and the mixture was precipitated overnight at 4°C. The precipitate was collected by centrifugation (10000×g, 4°C, 20 min). The precipitate was dissolved in an appropriate volume of deionized water, and the solution was placed in a dialysis bag with a molecular weight cutoff of 14000 Da. Dialysis was performed at 4°C for 2 days, with the medium changed every 8 h. The dialysis solution was freeze-dried for 24 h to obtain the crude EPS sample, which was then weighed.

[0041] Determination of crude EPS concentration using the phenol-sulfuric acid method: Take an appropriate amount of analytical grade glucose, dry it to constant weight, cool it, and prepare an 80 mg / L standard solution. Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the 80 mg / L glucose standard solution into 25 mL colorimetric tubes, add water to make up to 2 mL, then add 2 mL of 6% (v / v) phenol and 10 mL of concentrated sulfuric acid sequentially, mix immediately, let stand at room temperature for 20 min, and measure the absorbance at 490 nm. Plot a standard curve to obtain the regression equation (y = 0.011x - 0.024, R² = 0.9952). Take 1 mL of the test sample to replace the standard sample, add water to make up to 2 mL, repeat the above operation, and measure the absorbance value. Perform three parallel measurements, and substitute the absorbance values ​​into the regression equation to calculate the polysaccharide content. The screening results showed that *Lactobacillus plantarum* 2 had the strongest ability to produce extracellular polysaccharides, and it was named *Lactobacillus plantarum* YBE2. The extracellular polysaccharide production results of the five strains are shown in Table 1.

[0042] Table 15 Extracellular Polysaccharide Production of Lactobacillus plantarum strains

[0043]

[0044] Following the above identification, Lactip / antibaci / / usplantarum YBE2 was deposited at the China Center for Type Culture Collection (CCTCC) on September 2, 2024. The accession number for Lactip / antibaci / / usplantarum YBE2 is: CCTCC NO: M 20241894.

[0045] Example 2: Monosaccharide composition analysis of extracellular polysaccharides from Lactobacillus plantarum YEB2

[0046] Crude extracellular polysaccharides (EPS) from *Lactobacillus plantarum* strains WCFS1 and YBE2 were extracted according to step (3) in Example 1. 4 mg of each crude EPS sample was weighed into a sample bottle, dissolved in a suitable amount of freshly prepared 2 mol / L TFA solution, and incubated in a water bath at 85°C for 3 hours. Simultaneously, 4 mg each of ribose, xylose, arabinose, fucose, rhamnose, mannose, glucose, and galactose standards were weighed, and 1 mL of pyridine and 10 mg of hydroxylamine hydrochloride were added respectively. The mixture was vortexed and incubated in a water bath at 80°C for 2 hours. After the reaction, the mixture was cooled to room temperature, 1 mL of acetic anhydride was added, and the mixture was incubated in a water bath at 80°C for 2 hours. After sample preparation, the samples were cooled, dried, and reconstituted with 1 mL of methanol. The samples were then filtered through a 0.22 μm filter and analyzed by GC-MS. Chromatographic conditions: HP-5 capillary column (30 mm × 0.25 mm, 0.25 μm); carrier gas: helium, flow rate: 1 mL / min; injection volume: 2 μL; split ratio: 1:20; temperature program: initial column temperature 160 °C, increased to 200 °C at 6 °C / min, held for 6 min; vaporization chamber temperature: 250 °C; detector temperature: 240 °C. Methylation determination chromatographic conditions: injection volume 1 μL, split ratio 1:10.

[0047] Table 2 shows that the monosaccharide components of the crude polysaccharides from the two *Lactobacillus plantarum* species include rhamnose, mannose, glucose, and galactose. Among them, glucose (59.44%) was the most abundant in WCFS1, while rhamnose (1.04%) was the least abundant, whereas rhamnose (50.76%) was the most abundant in YBE2, and mannose (7.83%) was the least abundant. These results indicate that the extracellular polysaccharides produced by *Lactobacillus plantarum* YBE2 are specific.

[0048] Table 2 Monosaccharide composition analysis of Lactobacillus plantarum

[0049]

[0050] Example 3: High-performance liquid chromatography analysis of bile salt hydrolase activity in Lactobacillus plantarum YBE2

[0051] The strain stored at -80℃ was activated by streaking on MRS solid medium and incubated statically at 37℃ for 24 h. A single colony grown on the plate was picked and inoculated into 3 mL of MRS liquid medium, and incubated overnight at 37℃. The OD600 value was measured, and the seed culture concentration was diluted to OD600. 600nm =0.1; subsequently, it was incubated statically in a 37℃ incubator for approximately 5 hours until OD... 600 ≈2.5, concentrating the bacterial culture concentration to OD. 600=30. Centrifuge (12000×g, 4℃, 1min), discard the supernatant, and resuspend the bacterial sludge in 0.1M HAc-NaAc buffer. Mix 50μL of bacterial suspension with 50μL of bile salt mixture and react in a 37℃ water bath for 1h. The control sample was prepared by mixing 50μL of HAc-NaAc buffer and 50μL of 20mM bile salt mixture (bile salt mixture: weigh 0.0195g of GCA, 0.0215g of TCA, 0.0189g of GDCA and 0.0209g of TDCA, dissolved in 2mL of pH 5.0 HAc-NaAc buffer). After the reaction was completed, add an equal volume of TCA stop solution to terminate the reaction, mix well and centrifuge (12000×g, 4℃, 10min). The supernatant of the reaction solution was filtered through a 0.45 μm nylon membrane, and the content of different bile salts in the mixture was determined by high performance liquid chromatography (HPLC). The BSH hydrolysis rate was calculated, and the results are shown in Table 3. It can be seen that YBE2 has a certain degree of hydrolytic ability for different bile salts, with a GCA hydrolysis rate of 95.43%, indicating that YBE2 has strong bile salt hydrolytic enzyme activity.

[0052] Table 3 Hydrolysis rates of different bile salts by Lactobacillus plantarum YBE2

[0053]

[0054] Example 4: Application of Lactobacillus plantarum YBE2 in the growth of caged broilers

[0055] Preparation of the bacterial powder: Frozen *Lactobacillus plantarum* YBE2 was inoculated into MRS liquid medium and cultured at 37°C for 24 hours. This process was repeated twice to obtain *Lactobacillus plantarum* YBE2. The activated bacterial strain was then inoculated into MRS liquid medium and cultured at 37°C. The bacterial solution was centrifuged, freeze-dried, pulverized, and mixed with excipients to prepare freeze-dried *Lactobacillus plantarum* YBE2 powder, ensuring a viable count of 1 x 10⁻⁶ cells / mL. 10 cfu / g.

[0056] Preparation of fermented feed: The fermented feed formula is as follows: corn 35%, soybean meal 18%, wheat bran 13%, water 34%. The prepared YBE2 bacterial powder is added at 0.1% (i.e., 1×10⁻⁶). 6 Fermentation inoculum was prepared at an inoculum size of cfu / g. Anaerobic fermentation was adopted. The fermentation substrate and fermentation inoculum were evenly mixed and then placed in a bag equipped with a breathing valve for fermentation for 7 days.

[0057] The experiment selected healthy, disease-free broiler chickens of the same breed, origin, age at introduction, and similar weight, and divided them into two groups with three replicates per group. Each replicate was housed in one chicken house, for a total of six cage-raised broiler houses (each house was a three-tiered cage system, housing 33,000 chickens), totaling 200,000 white-feathered broilers. The groups were designated A1-B1, A2-B2, and A3-B3. Group A was the control group, fed a basal diet; Group B was the experimental group, fed a basal diet plus 5% fermented feed, with YBE2 bacterial powder added to the drinking water at a concentration of 1 x 10^6 microbial powder per milliliter of water. 6 CFU. On day 40 of the trial (market age), one rooster and one hen were selected from the middle row of each chicken house, from the front, middle, and back of the row, and from the top, middle, and bottom of the row. A total of 18 chickens were sampled from each house each time, for a total of 108 chickens, and they were slaughtered. Different markings were made according to the layer, row, and rooster / female.

[0058] Test indicators: 1) Growth performance: Initial weight of experimental chicken chicks was measured. Feed intake and health status of the flock were observed daily, and feed consumption, number of dead chickens and their weights were recorded. Fasted chickens were weighed once every 7 days, and daily weight gain, daily feed intake, feed conversion ratio, and survival rate were calculated for each treatment. 2) Carcass quality: 18 chickens were selected from each replicate based on average weight for slaughter. Live weight was measured, blood was collected from the jugular vein, and the chickens were euthanized by bleeding and wet plucking. Slaughter was carried out in accordance with the "Recommendations on Slaughter Measurement and Determination of Some Anatomical Locations of Commercial Poultry" in the "Compilation of Yellow Chicken Data". All breast muscles, leg muscles, and abdominal fat on the same side were taken to measure breast muscle weight, leg muscle weight, breast muscle percentage, leg muscle percentage, and abdominal fat percentage. 3) Physicochemical indicators of meat quality: 18 healthy fasted chickens close to average weight were taken from each replicate for slaughter. Right breast muscles and leg muscles were taken to measure pH value, meat color, shear force, drip loss, and cooking loss. 4) Determination of fatty acids in chicken meat: The fatty acid content was determined according to GB / T9695-2009. 5) Determination of serum biochemical indicators: After collecting blood from each chicken vein, the serum was separated and the alanine aminotransferase, aspartate aminotransferase, bile acids, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were measured.

[0059] (1) Effects of Lactobacillus plantarum YBE2 and fermented feed on the growth performance of caged broilers

[0060] As shown in Table 4, there were no significant differences in average daily weight gain, daily feed intake, feed conversion ratio, and survival rate between the experimental group and the control group.

[0061] Table 4. Effects of Lactobacillus plantarum YBE2 and fermented feed on the growth performance of caged broilers.

[0062] project control group experimental group Average daily weight gain (g) 68.12±0.80 67.11±2.57 Average daily feed intake (g) 100.99±1.11 101.99±2.22 Meat-to-fat ratio 1.48±0.02 1.52±0.03 Survival rate (%) 94.26±1.67 94.14±1.09

[0063] (2) Effects of Lactobacillus plantarum YBE2 and fermented feed on the slaughter quality of caged broilers

[0064] As shown in Table 5, there were no significant differences in slaughter rate, semi-eviscerated rate, fully eviscerated rate, breast muscle rate, and leg muscle rate between the experimental group and the control group. However, the abdominal fat rate was significantly lower than that of the control group, decreasing by 27.7%. The results indicate that Lactobacillus plantarum YBE2 and fermented feed significantly reduced abdominal fat deposition in caged broilers and regulated fat metabolism.

[0065] Table 5. Effects of Lactobacillus plantarum YBE2 on slaughter performance of caged broilers (%)

[0066] project control group experimental group Slaughter rate 93.33±0.43 93.41±0.07 Semi-cleaned rate 87.93±0.69 87.82±0.21 Full turret clearance 75.16±2.12 75.10±1.23 pectoral muscle rate 20.76±0.18 20.74±0.25 Leg muscle rate 14.18±0.22 14.37±0.174 Abdominal fat percentage 1.41 ± 0.43 b ]] 1.02 ± 0.08 a ]]

[0067] (3) Effects of Lactobacillus plantarum YBE2 and fermented feed on muscle quality of caged broilers

[0068] Meat color is an important indicator for evaluating muscle appearance, mainly expressed by brightness value L*, redness value a*, and yellowness value b. Generally, the smaller the L value, the larger the a value, and the smaller the b value, the better the meat color. Shear force is an objective indicator for evaluating muscle tenderness; the smaller the shear force, the more tender the meat and the better the taste. The water-holding capacity of muscle directly affects the edible quality of the meat, and the less water loss during cooking, the better the water-holding capacity. As shown in Table 6, *Lactobacillus plantarum* YBE2 and fermented feed can significantly reduce the brightness and yellowness values ​​of duck breast muscle, while there are no significant differences in pH, shear force, and cooking loss between the two groups. *Lactobacillus plantarum* YBE2 can improve the meat color and enhance the muscle quality of caged broilers.

[0069] Table 6. Effects of Lactobacillus plantarum YBE2 on the quality of breast muscle in caged broilers.

[0070]

[0071]

[0072] (4) Effects of Lactobacillus plantarum YBE2 and fermented feed on plasma biochemical parameters of caged broilers

[0073] As shown in Table 7, the levels of alanine aminotransferase, total bile acids, triglycerides, and low-density lipoprotein cholesterol in the plasma of caged broilers in the experimental group were significantly lower than those in the control group, and the total cholesterol in the experimental group also decreased to varying degrees.

[0074] Table 7 Effects of Lactobacillus plantarum YBE2 on plasma biochemical parameters of breast muscle in caged broilers

[0075] Blood biochemical indicators control group experimental group Alanine aminotransferase (U / L) 1.51 ± 0.29 b ]] 1.26 ± 0.28 a ]] Aspartate aminotransferase (U / L) 34.14±7.82 33.18±11.64 Total bile acids (μmol / L) 8.44 ± 1.94 b ]] 7.36 ± 2.01 a ]] Triglycerides (mmol / L) 0.57±0.11b 0.47 ± 0.09 a ]] Total cholesterol (mmol / L) 3.79±0.45 3.39±0.32 High-density lipoprotein cholesterol (mmol / L) 2.14±0.26 2.08±0.31 Low-density lipoprotein cholesterol (mmol / L) <![CDATA[1.18±0.18 b ]]> <![CDATA[0.94±0.12 a ]]>

[0076] (5) Effects of Lactobacillus plantarum YBE2 and fermented feed on intramuscular fat and fatty acid composition of caged broilers

[0077] like Figure 1As shown, the muscle fat content of caged broilers in the experimental group was significantly higher than that in the control group, with an intramuscular fat increase of 21.8%. Figure 2 As shown, the levels of 15 fatty acids in the muscle of caged broilers in the experimental group, including lauric acid, arachidic acid, behenic acid, myristic acid, trans oleic acid, erucic acid, γ-linolenic acid, linolenic acid, myristic acid, arachidic acid, stearic acid, hexadecenoic acid, linoleic acid, palmitic acid, and oleic acid, were significantly higher than those in the control group. The levels of saturated fatty acids and unsaturated fatty acids (monounsaturated and polyunsaturated fatty acids) were also significantly higher in the experimental group than in the control group. These results indicate that *Lactobacillus plantarum* YBE2 can regulate fat metabolism in caged broilers and promote fatty acid deposition in muscle.

[0078] Example 5: Application of Lactobacillus plantarum YBE2 in lowering cholesterol in high-fat mice

[0079] To further investigate the cholesterol-lowering effect of Lactobacillus plantarum YBE2 in animals, a high-fat mouse model was selected for the study.

[0080] Eighteen 5-week-old GF-grade male mice were selected and placed under controlled environmental conditions (temperature 25℃, relative humidity 50%-60%, light / dark cycle 12 / 12h, with free access to water and food during the experiment). Animal experiments were conducted in accordance with the guidelines of the Ethics Committee on Institutional Animal Care and Use of Nanjing Normal University.

[0081] Mice were allowed to acclimatize one week before the experiment and were randomly divided into three groups of six mice each.

[0082] (1) Control group: Normal feed (ND);

[0083] (2) Model group: High cholesterol diet (HCD);

[0084] (3) Experimental group: High cholesterol diet (HCD);

[0085] Mice were given free access to food and water during the experiment. Mice were administered 1×10⁻⁶ mg / L via gavage; each mouse in the experimental group received this dose. 9 CFU / mL Lactobacillus plantarum YBE2 was administered to both the control and model groups, along with an equal volume of 0.9% saline, for a total of 6 weeks. Animals in each group were fasted overnight on the day before week 7.

[0086] Detection indicators: 1) Body weight: The initial body weight of the experimental mice was measured. After 6 weeks of experimentation, the final body weight of the experimental mice was measured, and the growth rate and growth rate of the mice were calculated. 2) Serum biochemical indicators: Whole blood was collected from mice using the ocular blood collection method. The whole blood was allowed to stand at room temperature for 30 min, and then centrifuged at 1500 g / min for 15 min. 10 μL of serum sample was taken and the blood biochemistry analyzer of the Experimental Animal Center of Nanjing Medical University was used to detect four biochemical indicators in mouse serum: total cholesterol (CHOL), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C), and triglycerides (TG). 3) Ileal contents bile acids: After the mice were sacrificed, the ileum was immediately removed and rinsed clean. 50 mg of ileal contents was weighed into a 1.5 mL EP tube, and 1 mL of methanol was added to break up the sample. After the sample was fully dissolved, it was allowed to stand at room temperature for 1 h. It was then centrifuged at 12000 rpm at 4℃ for 15 min. The supernatant was filtered through a 0.22 μm filter membrane into a sample vial, and TCA, CA, T-β-MCA, and β-MCA were determined by high-performance liquid chromatography (HPLC). 4) Liver pathological analysis: After euthanasia, the livers of mice were immediately removed and rinsed thoroughly. Appropriate amounts of liver samples were randomly collected from the control group, model group, and experimental group. After washing with sterile PBS, the samples were fixed in 4% paraformaldehyde solution, dehydrated, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under a conventional optical microscope.

[0087] (1) Effects of Lactobacillus plantarum YBE2 on body weight in high-fat mice

[0088] Table 8 shows that during the six-week period, normal mice fed a normal diet increased in weight from an average of 23 grams to 26 grams, a weight gain rate of 13.95%. The other two groups were maintained on a high-cholesterol diet. The model group (34.12%) showed the highest growth rate, followed by the experimental group (23.28%), namely the group receiving *Lactobacillus plantarum* YBE2 via gavage. The results indicate that *Lactobacillus plantarum* YBE2 can reduce weight gain in mice.

[0089] Table 8 Results of mouse weight change detection

[0090]

[0091]

[0092] (2) Effects of Lactobacillus plantarum YBE2 on serum biochemical indicators in high-fat mice

[0093] Table 9 shows that, compared with the control group, a high-fat diet led to varying degrees of increases in serum total cholesterol (CHOL), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) levels in mice, indicating that a high-fat diet causes lipid metabolism disorders. Compared with the model group, after intervention with *Lactobacillus plantarum* YBE2, the levels of CHOL, TG, HDL-C, and LDL-C in the experimental group showed a decreasing trend, with TG and LDL-C showing significant decreases. The results indicate that *Lactobacillus plantarum* YBE2 can reduce serum cholesterol levels in hypercholesterolemia model mice.

[0094] Table 9. Results of serum lipid detection in mice of each group.

[0095]

[0096] (3) Effects of Lactobacillus plantarum YBE2 on bile acids in ileal contents of high-fat mice

[0097] Table 10 shows that, compared with the control group, there were no significant changes in conjugated and unconjugated bile acids in the model group, indicating that a high-cholesterol diet did not significantly affect BSH activity in mice, thus not causing significant changes in bile acids. Compared with the control group, the levels of TCA, CA, T-β-MCA, and β-MCA in the model group did not change significantly; however, the levels of TCA and T-β-MCA, as well as CA and β-MCA, were significantly increased in the experimental group. These results indicate that *Lactobacillus plantarum* YBE2, which specifically hydrolyzes GCA, can hydrolyze some other conjugated bile acids under the influence of intestinal flora, and thus has a cholesterol-lowering function to some extent.

[0098] Table 10. Ileal bile acid content in mice of each group

[0099]

[0100] (4) Effects of Lactobacillus plantarum YBE2 on liver pathology in high-fat mice

[0101] Depend on Figure 3 The results showed that the liver tissue of the control group had normal color, clear structure, and tightly and orderly arranged hepatocytes, with no fatty degeneration. Compared with the control group, the liver tissue of mice fed a high-fat diet showed varying degrees of fatty degeneration, especially in the model group where the fatty degeneration was most severe, with large and densely packed fat vacuoles, along with hepatocyte necrosis and altered nucleus shape. The pathological condition of the experimental group treated with *Lactobacillus plantarum* YBE2 by gavage improved, with a reduction in the volume and number of fat vacuoles and a decrease in the number of necrotic cells. These results indicate that *Lactobacillus plantarum* YBE2 has a certain ameliorative effect on liver pathology induced by a high-cholesterol diet.

[0102] Example 6: Application of Lactobacillus plantarum YBE2 in anti-inflammatory effects in LPS model mice

[0103] To further investigate the effect of Lactobacillus plantarum YBE2 on enhancing the immune function of animals, an intestinal inflammation model induced by LPS in mice was used as a model for the study.

[0104] Eighteen male SPF-grade C57BL mice, aged 5 weeks, were selected and placed under controlled environmental conditions (temperature 25℃, relative humidity 50%-60%, light / dark cycle 12 / 12h, with free access to water and food during the experiment). Animal experiments were conducted in accordance with the guidelines of the Ethics Committee on Institutional Animal Care and Use of Nanjing Normal University.

[0105] Mice were allowed to acclimatize one week before the experiment and were randomly divided into three groups of six mice each.

[0106] (1) Control group: Normal feed (ND);

[0107] (2) Experimental Group A: Normal feed (ND);

[0108] (3) Experimental Group B: Normal feed (ND).

[0109] Mice were given free access to food and water during the experiment. Mice in group B were administered 1×10⁻⁶ gavage. 9 CFU / mL *Lactobacillus plantarum* was administered to both the control and experimental groups (Group A), along with the same volume of 0.9% saline, for a total of 14 days. Animals in all groups were fasted overnight on day 14. Mice were fasted for 12 hours prior to the experiment but allowed free access to water. Except for the control group, all mice were injected intraperitoneally (IP) with 0.2 mL of LPS (5 mg / kg). -1 Induced for 6 hours, then sacrificed and sampled.

[0110] Detection indicators: 1) Serum inflammatory factors: After mouse sacrifice, whole blood was collected using ocular blood collection. The whole blood was allowed to stand at room temperature for 30 min, then centrifuged at 1500 g / min for 15 min. The collected serum samples were analyzed using enzyme-linked immunosorbent assay (ELISA) to measure the levels of IgA, TNF-α, and IL-1β cytokines, following the ELISA kit instructions. 2) Ileal supernatant antioxidant indicators: After mouse sacrifice, the ileum was immediately removed and rinsed thoroughly. An appropriate amount of ileum was weighed, and 9 times the weight of the tissue block was measured in physiological saline. The sample was transferred to a 2 mL centrifuge tube containing 1 g of 0.1 mm glass beads and homogenized using a biological sample homogenizer in an ice-water bath. The program was set to 6.00 m / s, 10 cycles, 30 s intervals, and 30 s intervals. After homogenization, the NO and oxidation indicators (CAT and GSH-PX content) of the ileal sample were measured using the kit.

[0111] (1) Effects of Lactobacillus plantarum YBE2 on serum inflammatory factors in LPS model mice

[0112] Table 11 shows that, compared with the control group, the serum IgA level in experimental group A mice was significantly decreased, while the levels of IL-1β and TNF-α were significantly increased. Compared with experimental group A, the IgA level in experimental group B was significantly increased, while the levels of TNF-α and IL-1β were significantly decreased. These results indicate that *Lactobacillus plantarum* YBE2 can inhibit the secretion of inflammatory cytokines, promote the expression of anti-inflammatory factors, and suppress serum LPS inflammatory responses.

[0113] Table 11 Serum inflammatory factors and ileal supernatant antioxidant markers in each group of mice

[0114]

[0115] (2) Effects of Lactobacillus plantarum YBE2 on antioxidant indices in the ileum supernatant of LPS model mice

[0116] Table 11 shows that, compared with the control group, the NO level in experimental group A was significantly increased, while the levels of GSH-PX and CAT in the ileal supernatant were significantly decreased. Compared with experimental group A, YBE2 in experimental group B significantly reduced the NO level, while significantly increasing the GSH-PX level and CAT level. The results indicate that *Lactobacillus plantarum* YBE2 has antioxidant effects to some extent.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A plant lactobacillus ( Lactiplantibacillus plantarum ), characterized by: The plant Lactobacillus (Lactobacillus plantarum) Lactiplantibacillus plantarum ) is Lactobacillus plantarum YBE2, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC No: M 20241894, and the preservation date is September 2, 2024.

2. The *Lactobacillus plantarum* according to claim 1 ( Lactiplantibacillus plantarum The application of this product in the production of rhamnose and / or in the preparation of probiotic agents for caged broiler farming is characterized by: The plantarum lactiplantibacillus is used for preparing probiotics for reducing abdominal fat deposition of cage-bred broilers while increasing intramuscular fat content of cage-bred broilers.

3. Use according to claim 2, characterized in that: The plantarum lactiplantibacillus can specifically hydrolyze glycocholic acid and has anti-inflammatory and antioxidant functions.

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