Lactobacillus plantarum YBE2 and application thereof

Abstract

The invention discloses lactobacillus plantarum YBE2 and application thereof.According to the lactobacillus plantarum YBE2 for high yield of extracellular polysaccharide mainly containing rhamnose, different cholate types can be degraded at the same time, glycocholic acid is specifically hydrolyzed, and the lactobacillus plantarum YBE2 has the effects of lowering cholesterol and relieving acute inflammation at the same time. Meanwhile, the plant lactobacillus YBE2 can reduce the deposition of fat in the belly of the cage rearing broiler chicken, improve intramuscular fat, promote the deposition of fatty acid in muscles, reduce the total bile acid of plasma, triglyceride and low-density lipoprotein cholesterol, and remarkably improve the lipid metabolism and muscle quality of meat poultry, and can be widely applied to the breeding of the cage rearing broiler chicken, promote the healthy breeding of the meat poultry and improve the quality of the meat poultry. The culture benefit is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to a Lactobacillus plantarum YBE2 and an application thereof. Background Art

[0002] In recent years, the three-dimensional cage chicken house breeding model has gradually become the mainstream trend of the development of the broiler industry. It has many advantages such as strong environmental controllability, low morbidity rate of broilers, and good economic benefits. However, it has certain shortcomings and disadvantages. Because the chickens are in a state of restricted activity space for a long time, the amount of chicken exercise is reduced and the energy consumption is reduced. When caged broilers eat a large amount of high-energy feed mainly composed of corn, the excessive intake of fat and energy is easily deposited around the liver and abdomen, which can easily lead to excessive deposition of abdominal and subcutaneous fat in broilers, and cholesterol metabolism disorders, which have a negative impact on the body health and meat quality of broilers, and at the same time restrict the development of the broiler industry.

[0003] Lactobacillus is the most representative genus among probiotics. In addition to improving feed flavor and inhibiting pathogens, lactobacillus also has physiological functions such as improving host intestinal flora, enhancing immunity, lowering cholesterol, and improving blood lipid metabolism. Bile salt hydrolase is an intracellular enzyme produced during the growth and reproduction of lactobacillus. It hydrolyzes bound glycine bile salts and taurine bile salts into glycine, taurine and conjugated bile salts, thereby gradually reducing cholesterol levels in the body and effectively reducing fat accumulation in the liver and blood lipid levels. The extracellular polysaccharides of lactobacillus have the effects of promoting the growth of probiotics such as bifidobacteria, anti-oxidation, anti-inflammatory, anti-tumor, and lowering cholesterol. At present, the research on cholesterol-lowering by lactobacillus is mainly focused on human food. Therefore, screening functional probiotics for caged broilers is an important way to improve lipid metabolism, regulate intestinal flora, and enhance the immune performance of meat poultry.

[0004] The Lactobacillus plantarum that has been reported to have certain cholesterol-lowering functions, but there is no Lactobacillus plantarum that has both high production of extracellular polysaccharides and cholesterol-lowering capabilities. Therefore, based on the principle of microbial adaptability and specificity to the habitat, functional lactic acid bacteria are screened from the intestines of broiler chickens to regulate lipid metabolism in caged broilers, reduce blood lipids, enhance immunity, improve chicken quality and intestinal 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 summarize some aspects of embodiments of the present invention and to briefly introduce some preferred embodiments.

[0006] The first object of the present invention is to provide a strain of Lactobacillus plantarum (lactiplantibacillus plantarum) YBE2, which is screened from the chicken intestine and has the characteristics of high production of extracellular polysaccharides, bile salt hydrolase production and cholesterol-lowering lactic acid bacteria. The Lactobacillus plantarum YBE2 strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M 20241894 and a deposit date of September 2, 2024.

[0007] The Lactobacillus plantarum YBE2 strain was isolated from the chicken intestine. Through colony morphology observation, Gram staining and 16S rDNA amplification of the strain's genome, the PCR product was obtained for sequencing and identification, and it was finally determined to be Lactobacillus plantarum (L. plantarum YBE2), and named and numbered YBE2.

[0008] The second object of the present invention is to provide the application of Lactobacillus plantarum YBE2 in caged broiler breeding and production.

[0009] The third object of the present invention is to provide the use of the Lactobacillus plantarum YBE2 in the preparation of probiotics that reduce abdominal fat deposition in caged broilers while increasing the intramuscular fat content of caged broilers.

[0010] The fourth object of the present invention is to provide the use of Lactobacillus plantarum YBE2 in producing rhamnose.

[0011] The fifth object of the present invention is to provide the use of Lactobacillus plantarum YBE2 in the preparation of probiotics that specifically hydrolyze glycocholic acid.

[0012] The sixth objective of the present invention is to provide the use of Lactobacillus plantarum YBE2 in the preparation of probiotics for reducing cholesterol in high-fat mice.

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

[0014] The present invention isolates and screens from chicken intestines to obtain Lactobacillus plantarum YBE2 that reduces cholesterol and produces high levels of bile salt hydrolase and extracellular polysaccharides. Compared with the prior art, the Lactobacillus plantarum YBE2 of the present invention also has the following unexpected technical effects:

[0015] 1) The Lactobacillus plantarum YBE2 of the present invention produces a high amount of extracellular polysaccharides, wherein rhamnose is the main component of the polysaccharides, and the extracellular polysaccharides are specific.

[0016] 2) The Lactobacillus plantarum YBE2 of the present invention has the ability to hydrolyze four bile salts, among which the hydrolysis rate of glycocholic acid is as high as 95%, and has strong bile salt hydrolase activity.

[0017] 3) The plant lactobacillus YBE2 of the present invention significantly reduces abdominal fat deposition in caged broilers, reduces plasma alanine aminotransferase, total bile acid, triglycerides, and low-density lipoprotein cholesterol, and at the same time increases intramuscular fat content, promotes fatty acid deposition in muscle, and regulates fat metabolism. Plant lactobacillus YBE2 improves the meat color of broilers and improves muscle quality.

[0018] 4) The Lactobacillus plantarum YBE2 of the present invention can effectively slow down the weight gain of experimental mice fed with a high-fat diet, reduce the serum cholesterol level of high-fat mice, reduce the content of conjugated bile acid, and increase the content of unconjugated bile acid, thus having the effect of lowering cholesterol and blood lipids, and at the same time 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 in mice in the LPS inflammation model experiment, increase the expression of anti-inflammatory factors, and has the effect of alleviating acute inflammation; it can reduce the NO level, increase the GSH-PX and CAT levels in the ileum supernatant, and has antioxidant capacity.

[0020] In summary, the plant lactobacillus with high yield of rhamnose-based extracellular polysaccharides provided by the present invention can degrade different types of bile salts at the same time, specifically hydrolyze glycocholic acid, and has the effects of lowering cholesterol and relieving acute inflammation. At the same time, plant lactobacillus YBE2 can reduce the deposition of fat in the abdomen of caged broilers, increase intramuscular fat, promote the deposition of fatty acids in muscles, reduce plasma total bile acid, triglycerides, and low-density lipoprotein cholesterol, significantly improve meat poultry lipid metabolism and muscle quality, and can be widely used in caged broiler farming, promote healthy farming of meat poultry, and improve farming efficiency.

[0021] The deposit information of the microorganism involved in the present invention is as follows:

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

[0023] Address of depository: Wuhan University, Wuhan, China;

[0024] Classification name: lactiplantibacillus plantarum YBE2;

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

[0026] Date of preservation: September 2, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the following briefly introduces the drawings required for describing the embodiment, wherein:

[0028] Figure 1 To investigate the effect of Lactobacillus plantarum YBE2 on intramuscular fat in caged broiler chickens.

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

[0030] Figure 3 This is a picture of mouse liver pathology. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the examples, which are provided for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified.

[0032] The culture medium used in the present invention was purchased from Qingdao Hi-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, and other reagents, unless otherwise specified, were obtained from commercial sources.

[0033] Example 1: Screening of a strain of Lactiplantibacillus plantarum YBE2 that produces high levels of exopolysaccharides

[0034] (1) Strain isolation, purification and initial screening

[0035] Lactobacillus was isolated and purified from the chicken intestine as follows: 1 g of intestinal chyme sample was weighed and added to 99 ml of sterilized distilled water, shaken on a constant temperature shaker for 1 hour, and then diluted 10 times in a gradient, and 10 -3 , 10 -4 The sample dilution was spread on MRS solid medium and cultured for 48 hours, then taken out and single colonies grown on MRS were picked according to the size, shape and color of the colonies (the colonies were medium-sized, convex, slightly white, moist, with neat edges, round colonies, and a diameter of 3.0 mm ± 1 mm) for catalase test and Gram staining. Catalase-negative and Gram-positive strains were tentatively identified as lactobacilli and continued to be streaked and purified twice on MRS solid medium.

[0036] (2) 16S rDNA identification

[0037] The lactobacillus strains purified and cultured on the solid plate for 24 hours were amplified by colony PCR to identify the 16SrRNA gene sequence. The primers used were the universal primers for bacterial 16S rRNA gene amplification: 27F and 1492R. The primer sequences were: upstream primer: 5′-AGAGTTTGATCCTGGCTCAG-3′; downstream primer: 5′-GGTTACCTTGTTACGACTT-3′. The PCR reaction system was 20μl: PremixTaq 10μl, 27F and 1492R (both 20μM) 1μl, supplemented with sterile distilled water to 20μl, and the colonies were directly added to the reaction system for PCR amplification to obtain PCR products. The PCR products were sent to Qingke Bioengineering Co., Ltd. for sequencing, and the results showed that 5 strains were plant lactobacillus. The undetermined strains were stored in a -80℃ refrigerator.

[0038] (3) Strain activation and rescreening

[0039] The strains stored at -80°C were streaked on MRS solid medium for activation and cultured at 37°C for 24 h; a single colony grown on the plate was picked and inoculated into 3 mL of MRS liquid medium and cultured at 37°C overnight.

[0040] 2mL of seed solution was inoculated into 100mL of MRS liquid medium at a volume ratio of 2% for fermentation, and the culture was stopped at 37℃ for 32h. The fermentation broth was centrifuged (10000×g, 4℃, 15min), the supernatant was collected, 80% trichloroacetic acid was added to the supernatant to a final concentration of 4% (w / v), and the protein precipitate was removed by centrifugation (10000×g, 4℃, 15min) after standing for 8h at 4℃. Subsequently, 3 volumes of pre-cooled anhydrous ethanol solution were added to the supernatant, alcohol precipitation was performed overnight at 4℃, and the precipitate was collected by centrifugation (10000×g, 4℃, 20min). The precipitate was dissolved with an appropriate volume of deionized water, and the dissolved solution was placed in a dialysis bag with a molecular weight cutoff of 14000Da, dialyzed at 4℃ for 2d, and the solution was changed every 8h. After dialysis, the solution was freeze-dried for 24h, and the crude EPS sample was finally obtained and its mass was weighed.

[0041] Phenol-sulfuric acid method to determine the mass concentration of crude EPS: Take an appropriate amount of analytical pure glucose and dry it to constant weight, and prepare a standard solution of 80 mg / L after cooling. Take 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 80 mg / L glucose standard solution in a 25 mL colorimetric tube, add water to make up 2 mL, add 6% (v / v) phenol 2 mL and concentrated sulfuric acid 10 mL in sequence, mix immediately, let stand at room temperature for 20 minutes, and measure the absorbance at 490 nm. Draw a standard curve and obtain a regression equation (y = 0.011x-0.024, R2 = 0.9952). Take 1 mL of the sample to be tested instead of the standard sample, add water to make up 2 mL, repeat the above operation, measure the absorbance value, parallel 3 times, and substitute the absorbance value into the regression equation to obtain the polysaccharide content. The screening results showed that Lactobacillus plantarum 2 had the strongest ability to produce extracellular polysaccharides, so it was named Lactobacillus plantarum YBE2. The results of the extracellular polysaccharide production of the five strains are shown in Table 1.

[0042] Table 1 Extracellular polysaccharide production of 5 strains of Lactobacillus plantarum

[0043]

[0044] After the above identification, Lactobacillus plantarum YBE2 was deposited in the China Center for Type Culture Collection (CCTCC) on September 2, 2024, and the preservation number of Lactobacillus plantarum YBE2 (Lactip / antibaci / / usplantarum YBE2) is: CCTCC NO: M 20241894.

[0045] Example 2: Analysis of Monosaccharide Composition of Exopolysaccharides of Lactobacillus plantarum YEB2

[0046] Extract the crude extracellular polysaccharide of Lactobacillus plantarum WCFS1 and YBE2 strains with reference to step (3) in Example 1. Weigh 4 mg of each crude EPS sample into a sample bottle, add an appropriate amount of freshly prepared 2 mol / L TFA solution to dissolve, and bathe in 85 ° C for 3 h. At the same time, weigh 4 mg of ribose, xylose, arabinose, fucose, rhamnose, mannose, glucose and galactose standards, add 1 mL of pyridine and 10 mg of hydroxylamine hydrochloride, vortex mix, bathe in 80 ° C for 2 h, cool to room temperature after the reaction, add 1 mL of acetic anhydride, and bathe in 80 ° C for 2 h. After the sample is prepared, cool, blow dry, and re-dissolve in 1 mL of methanol. GC-MS analysis after filtration through a 0.22 μm filter membrane. Chromatographic conditions: HP-5 capillary column (30 mm × 0.25 mm, 0.25 μm); carrier gas is helium, flow rate is 1 mL / min; injection volume is 2 μL; split ratio is 1:20; program temperature rise: column initial temperature is 160°C, increased to 200°C at 6°C / min, and maintained for 6 min; vaporization chamber temperature is 250°C; detector temperature is 240°C. The chromatographic conditions for methylation determination are injection volume 1 μL, split ratio 1:10.

[0047] As shown in Table 2, the monosaccharide components of the crude polysaccharides of the two Lactobacillus plantarum strains include rhamnose, mannose, glucose and galactose. Among them, glucose (59.44%) accounts for the largest proportion in WCFS1, and rhamnose (1.04%) accounts for the smallest proportion, while rhamnose (50.76%) accounts for the largest proportion in YBE2, and mannose (7.83%) accounts for the smallest proportion. The results show that the exopolysaccharides produced by Lactobacillus plantarum YBE2 are specific.

[0048] Table 2 Analysis of monosaccharide composition of Lactobacillus plantarum

[0049]

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

[0051] The strain stored at -80℃ was streaked on MRS solid medium for activation and incubated at 37℃ for 24h; a single colony grown on the plate was picked and inoculated into 3mL MRS liquid medium and incubated at 37℃ overnight. The OD600 value was measured and the seed solution was diluted to OD 600nm =0.1; then, the cells were cultured in a 37°C constant temperature incubator for about 5 h until OD 600 ≈2.5, concentrate the bacterial solution to OD 600=30. Centrifuge (12000×g, 4℃, 1min), remove the supernatant, and resuspend the bacterial sludge with 0.1M HAc-NaAc buffer. Mix 50μL of bacterial suspension and 50μL of bile salt mixture, and react in a 37℃ water bath for 1h. The control group sample is mixed with 50μL HAc-NaAc buffer and 50μL 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 is completed, add an equal volume of TCA stop solution to terminate the reaction, mix and centrifuge (12000×g, 4℃, 10min). The supernatant of the reaction solution was filtered through a 0.45 μm nylon membrane, and the contents of different bile salts in the mixed solution were determined by high performance liquid chromatography, and the BSH hydrolysis rate was calculated. The results are shown in Table 3. It can be seen that YBE2 has a certain degree of hydrolysis ability for different bile salts, among which the hydrolysis rate for GCA is 95.43%, indicating that YBE2 has strong bile salt hydrolase activity.

[0052] Table 3 Hydrolysis rate 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 bacterial powder: inoculate the frozen Lactobacillus plantarum YBE2 into MRS liquid culture medium, culture at 37°C for 24h, and subculture twice to obtain the Lactobacillus plantarum YBE2. Inoculate the activated strain into MRS liquid culture medium and culture at 37°C, centrifuge the bacterial solution, freeze-dry, pulverize, and mix the auxiliary materials to prepare the lyophilized powder of Lactobacillus plantarum YBE2, so that the number of viable bacteria in the lyophilized powder of lactic acid bacteria reaches 1x10 10 cfu / g.

[0056] Preparation of fermented feed: The formula of fermented feed is 35% corn, 18% soybean meal, 13% bran, and 34% water. The YBE2 bacterial powder prepared above was added at 0.1% (i.e. 1×10 6 cfu / g) to prepare the fermentation agent. Anaerobic fermentation was adopted, and the fermentation substrate and the fermentation bacteria were evenly mixed, and then put into a bag with a breathing valve for fermentation, and the fermentation time was 7 days.

[0057] The experiment selected healthy, disease-free broilers of the same breed, source, age and weight, and divided them into 2 groups, with 3 replicates in each group and 1 chicken house in each replicate, a total of 6 caged broilers (each broiler house is a three-layer cage model, 33,000 broilers), a total of 200,000 white-feathered broilers. The groups are A1-B1, A2-B2, and A3-B3. Group A: control group, fed with a basic diet; Group B: experimental group, basic diet + 5% fermented feed, and YBE2 bacterial powder added to drinking water, 1*10 per ml of water 6 CFU. On the 40th day of the experiment (marketing age), the middle row of each chicken house was selected, and one rooster and one hen were selected from the front, middle, back, upper, middle, and lower of this row, respectively. 18 chickens were sampled each time from each house, and a total of 108 chickens were slaughtered and marked according to different layers, rows, and genders.

[0058] Test indicators: 1) Growth performance: weigh the initial weight of the experimental chicks, observe the feeding and health of the flock every day, record the feed consumption, number of dead chickens and their weight, weigh them once every 7 days on an empty stomach, and calculate the daily weight gain, daily feed intake, feed-to-meat ratio and survival rate of each experimental chicken. 2) Carcass quality: select 18 chickens from each repetition according to the average weight for slaughter, weigh the live weight, collect blood from the jugular vein, bleed and kill, and wet pluck the feathers. Slaughter is carried out in accordance with the "Recommendations on the Determination and Determination of Some Anatomical Parts of Commercial Meat Poultry Slaughter" in the "Yellow Chicken Data Compilation". Take all the breast muscles, leg muscles and abdominal fat on the same side to measure the breast muscle weight, leg muscle weight, breast muscle rate, leg muscle rate and abdominal fat rate respectively. 3) Meat quality physical and chemical indicators: 18 healthy empty-stomach chickens with a weight close to the average weight are slaughtered in each repetition, and the right breast muscles and leg muscles are taken to measure the pH value, meat color, shear force, drip loss and cooking loss. 4) Chicken fatty acid determination: fatty acid content was determined according to GB / T9695-2009. 5) Serum biochemical index determination: venous blood was collected from each chicken to separate serum and determine alanine aminotransferase, aspartate aminotransferase, bile acid, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides.

[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 the average daily weight gain, daily feed intake, feed-to-meat ratio and survival rate of caged broilers in the experimental group and the control group.

[0061] Table 4 Effects of Lactobacillus plantarum YBE2 and fermented feed on 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 Feed to meat 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 the slaughter rate, half-evisceration rate, full-evisceration rate, breast muscle rate and leg muscle rate of caged broilers in the experimental group compared with those in the control group, and the abdominal fat rate was significantly lower than that in the control group, with a decrease of 27.7%. The results showed that Lactobacillus plantarum YBE2 and fermented feed significantly reduced abdominal fat deposition in caged broilers and regulated fat metabolism.

[0065] Table 5 Effect 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 Half-eviscerated rate 87.93±0.69 87.82±0.21 Full evisceration rate 75.16±2.12 75.10±1.23 Chest muscle rate 20.76±0.18 20.74±0.25 Leg muscle rate 14.18±0.22 14.37±0.174 Abdominal fat percentage <![CDATA[1.41±0.43 b ]]> <![CDATA[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, which is 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 retention capacity of muscle can directly affect the edible quality of meat, and the smaller the cooking loss, the better the water retention capacity of the muscle. As shown in Table 6, Lactobacillus plantarum YBE2 and fermented feed can significantly reduce the brightness value and yellowness value of duck breast muscle. There is no significant difference in pH, shear force and cooking loss between the two groups. Lactobacillus plantarum YBE2 can improve the meat color and muscle quality of caged broilers.

[0069] Table 6 Effects of Lactobacillus plantarum YBE2 on breast muscle quality of caged broiler chickens

[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 plasma alanine aminotransferase, total bile acid, triglyceride, and low-density lipoprotein cholesterol of caged broiler chickens 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 indexes Control group Experimental Group Alanine aminotransferase (U / L) <![CDATA[1.51±0.29 b ]]> <![CDATA[1.26±0.28 a ]]> Aspartate aminotransferase (U / L) 34.14±7.82 33.18±11.64 Total bile acid (μmol / L) <![CDATA[8.44±1.94 b ]]> <![CDATA[7.36±2.01 a ]]> Triglycerides (mmol / L) 0.57±0.11b <![CDATA[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 muscle fatty acid composition of caged broilers

[0077] like Figure 1As shown in the figure, the muscle fat of caged broilers in the experimental group was significantly higher than that in the control group, and the intramuscular fat increased by 21.8%. Figure 2 As shown, the 15 fatty acids in the muscle of caged broilers in the experimental group, including lauric acid, arachidic acid, behenic acid, myristic acid, elaidic 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, while saturated fatty acids and unsaturated fatty acids (monounsaturated fatty acids and polyunsaturated fatty acids) were also significantly higher than those in the control group. The results showed that Lactobacillus plantarum YBE2 can regulate the fat metabolism of caged broilers and promote the deposition of fatty acids in the muscles.

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

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

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

[0081] The mice were acclimated 1 week before the experiment and randomly divided into 3 groups with 6 mice in each group.

[0082] (1) Control group: normal diet (ND);

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

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

[0085] During the experiment, the mice were given free access to food and water. 9 CFU / mL Lactobacillus plantarum YBE2, the control group and the model group were given the same volume of 0.9% saline for 6 weeks. The animals in each group were fasted overnight on the day before the 7th week.

[0086] Detection indicators: 1) Body weight: The experimental mice were weighed at the initial weight. After the 6-week experiment, the experimental mice were weighed at the final weight to calculate the mouse growth and growth rate. 2) Serum biochemical indicators: The whole blood of the mice was collected by eyeball blood collection. The whole blood was allowed to stand at room temperature for 30 minutes and centrifuged at 1500g / min for 15 minutes. Take 10μL of serum sample and use the blood biochemical instrument of the Medical Experimental Animal Center of Nanjing Medical University to detect the four biochemical indicators of total cholesterol (CHOL), high-density lipoprotein (HDL-C), low-density lipoprotein (LDL-C) and triglycerides (TG) in the mouse serum. 3) Bile acid in ileum contents: After the mice were killed, the ileum was immediately removed and rinsed. Weigh 50mg of ileum contents in a 1.5mL EP tube and add 1mL of methanol to break the sample. After the sample is fully dissolved, let it stand at room temperature for 1h. Centrifuge at 12000rpm at 4℃ for 15min. The supernatant was filtered through a 0.22 μm filter membrane into a sample injection bottle, and TCA, CA, T-β-MCA, and β-MCA were determined by high performance liquid chromatography; 4) Liver pathology analysis: After the mice were killed, the liver was immediately removed and rinsed. An appropriate amount of liver samples from the control group, model group, and experimental group were randomly collected, washed with sterile PBS, and fixed in a 4% paraformaldehyde solution, dehydrated, paraffin-embedded, sliced, and HE stained. The results were observed under an ordinary optical microscope.

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

[0088] As shown in Table 8, during the six-week period, the weight of normal mice fed with ordinary feed increased from 23 grams to 26 grams on average, with a weight gain rate of 13.95%. The other two groups maintained high cholesterol feed. The model group (34.12%) had the highest growth rate, followed by the experimental group (23.28%), i.e. the group gavaged with Lactobacillus plantarum YBE2. The results showed that Lactobacillus plantarum YBE2 can reduce the weight gain of mice.

[0089] Table 8 Results of mouse weight change detection

[0090]

[0091]

[0092] (2) Effects of Lactobacillus plantarum YBE2 on serum biochemical parameters in hyperlipidemia mice

[0093] As shown in Table 9, compared with the control group, the high-fat diet caused the levels of total cholesterol (CHOL), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) in the serum of mice to increase to varying degrees, indicating that a high-fat diet can lead to lipid metabolism disorders. Compared with the model group, after the intervention of Lactobacillus plantarum YBE2, the levels of CHOL, TG, HDL-C and LDL-C in the experimental group showed a downward trend, among which TG and LDL-C decreased significantly. The results show that Lactobacillus plantarum YBE2 can reduce the serum cholesterol level of hypercholesterolemia model mice.

[0094] Table 9 Results of blood lipid test in serum of mice in each group

[0095]

[0096] (3) Effect of Lactobacillus plantarum YBE2 on bile acids in ileum of high-fat mice

[0097] As shown in Table 10, compared with the control group, there was no significant change in the conjugated bile acid and unconjugated bile acid in the model group, indicating that the high cholesterol diet did not significantly affect the BSH activity in mice, and thus did not cause significant changes in bile acid. Compared with the control group, the content of TCA, CA, T-β-MCA and β-MCA in the model group did not change significantly; but the content of TCA and T-β-MCA in the experimental group increased significantly, and the content of CA and β-MCA increased. The results show that Lactobacillus plantarum YBE2, which specifically hydrolyzes GCA, can hydrolyze some other conjugated bile acids under the action of intestinal flora, and has the function of lowering cholesterol to a certain extent.

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

[0099]

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

[0101] Depend on Figure 3 It can be seen that the liver tissue of the control group had normal color, clear structure, and the liver cells were arranged tightly and orderly, without fatty degeneration. Compared with the control group, the liver tissue of mice fed a high-fat diet showed different degrees of fatty degeneration, especially the model group, where the fatty degeneration of the liver tissue was the most serious, with large and dense fat vacuoles, and liver cell necrosis and changes in the shape of the cell nucleus. The pathological condition of the experimental group gavaged with Lactobacillus plantarum YBE2 improved, as shown by a decrease in the volume and number of fat vacuoles and a decrease in the number of necrotic cells. The results showed that Lactobacillus plantarum YBE2 had a certain improvement effect on the liver pathology caused by a high cholesterol diet.

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

[0103] In order to further study the effect of Lactobacillus plantarum YBE2 on improving animal immune performance, a study was conducted using LPS-induced intestinal inflammation in mice as a model.

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

[0105] The mice were acclimated 1 week before the experiment and randomly divided into 3 groups with 6 mice in each group.

[0106] (1) Control group: normal diet (ND);

[0107] (2) Experimental group A: normal diet (ND);

[0108] (3) Experimental group B: normal diet (ND).

[0109] During the experiment, mice were given oral administration, and each mouse in group B was given 1×10 9 CFU / mL plantarum Lactobacillus, the control group and experimental group A were given the same volume of 0.9% saline for 14 days. All animals in each group were fasted overnight on the 14th day. Mice were fasted for 12 hours before the experiment, but not water. All mice except the control group were injected with 0.2mL LPS (5mg kg -1 ) were induced for 6 hours and then the mice were killed and sampled.

[0110] Detection indicators: 1) Serum inflammatory factors: After the mice were killed, the whole blood of the mice was collected by eyeball blood collection. The whole blood was allowed to stand at room temperature for 30 minutes and centrifuged at a speed of 1500g / min for 15 minutes. The collected serum samples were taken and the levels of IgA, TNF-α and 1L-1β cytokines were measured by enzyme-linked immunosorbent assay according to the instructions of the ELISA kit. 2) Ileal supernatant antioxidant index: After the mice were killed, the ileum was immediately removed and rinsed. Weigh an appropriate amount of ileum and measure 9 times the weight of the tissue block with physiological saline. The sample was transferred to a 2mL centrifuge tube containing 1g 0.1mm glass beads, and homogenized using a biological sample homogenizer under ice water bath conditions. The program was set to 6.00m / s, 10cycles, running for 30s, and an interval of 30s. After the crushing was completed, the NO index and oxidation index (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] As shown in Table 11, compared with the blank group, the IgA level in the serum of mice in the experimental group A was significantly reduced, and the levels of IL-1β and TNF-α were significantly increased. Compared with the experimental group A, the IgA in the experimental group B was significantly increased, and TNF-α and 1L-1β were significantly reduced. The results showed that Lactobacillus plantarum YBE2 can inhibit the secretion of inflammatory cytokines, promote the expression of anti-inflammatory factors, and inhibit the serum LPS inflammatory response.

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

[0114]

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

[0116] As shown in Table 11, compared with the blank group, the NO level in the experimental group A was significantly increased, and the GSH-PX and CAT levels in the ileum supernatant were significantly decreased. Compared with the experimental group A, YBE2 in the experimental group B significantly reduced the NO level, while the GSH-PX level was significantly increased, and the CAT level was increased. The results show that Lactobacillus plantarum YBE2 has an antioxidant effect to a certain extent.

[0117] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A Lactobacillus plantarum, characterized in that: Lactobacillus plantarum YBE2 is deposited in the China Center for Type Culture Collection, with the deposit number being CCTCC No: M 20241894 and the deposit date being September 2, 2024.

2. A probiotic preparation containing the Lactobacillus plantarum according to claim 1.

3. Application of Lactiplantibacillus plantarum according to claim 1 in caged broiler breeding and production.

4. Use of the Lactiplantibacillus plantarum according to claim 1 in the preparation of probiotics for reducing abdominal fat deposition in caged broilers while increasing the intramuscular fat content of caged broilers.

5. Use of Lactiplantibacillus plantarum according to claim 1 in producing rhamnose.

6. Use of the plant lactobacillus (Lactiplantibacillus plantarum) according to claim 1 in preparing a probiotic that specifically hydrolyzes glycocholic acid.

7. Use of the Lactiplantibacillus plantarum according to claim 1 in preparing probiotics for reducing cholesterol in hyperlipidemia mice.

8. Use of the plant lactobacillus (Lactiplantibacillus plantarum) according to claim 1 in the preparation of probiotics with anti-inflammatory and antioxidant functions.

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