Bifidobacterium animalis producing bile salt hydrolase and its use in neonatal jaundice

Abstract

The application discloses an animal bifidobacterium producing bile salt hydrolase and application thereof in neonatal jaundice, and belongs to the technical field of microorganisms. The animal bifidobacterium lactis WXM screened by the application has the effect of relieving neonatal jaundice, can significantly improve weight loss of a jaundice individual in an early life stage, improve abnormal increase of a bilirubin level in serum, improve liver damage, improve the level of an enzyme related to liver function, the expression level of a related inflammatory factor in the liver and the expression level of an enzyme UGT1A1 related to bilirubin metabolism, and has the effect of relieving neonatal jaundice. The strain of the application has high safety and has wide application value in the fields of food, functional food, health products, medicine and the like.

Description

TECHNICAL FIELD

[0001] The present application relates to a bile salt hydrolase-producing animal Bifidobacterium and its application in neonatal jaundice, belonging to the technical field of microorganisms. BACKGROUND

[0002] Neonatal jaundice (NJ) is a common disease in the neonatal period, about 60% of full-term infants and 80% of premature infants will suffer from this disease in the first week after birth, the main clinical manifestations are the yellowing of the baby's skin and sclera. The most common form of neonatal jaundice is the increase of unconjugated bilirubin, which cannot be normally excreted and accumulates too much, which can develop into hyperbilirubinemia in severe cases, and lead to acute bilirubin encephalopathy and kernicterus, accompanied by significant risk of neonatal death and long-term neurodevelopmental disorders. The main treatment for neonatal jaundice in clinical practice is to reduce the production of bilirubin, speed up the transport of bilirubin in the body, and accelerate its metabolism and excretion. Currently, blue light therapy, drug therapy and other treatments are selected in clinical practice, but some views suggest that long-term blue light therapy may increase the risk of adverse reactions, such as anemia, fever, diarrhea, etc. Therefore, seeking safe and effective means to prevent and treat neonatal jaundice has become a hot spot of concern.

[0003] Probiotics are a class of active microorganisms that are beneficial to the health of the body, and can produce regulatory effects by stimulating specific and non-specific immune functions of the body and improving the bacterial flora on the mucosal surface of the host's intestine. It has been reported that certain probiotics play a very important auxiliary role in the treatment of jaundice, which is conducive to the subsidence of jaundice. Animal Bifidobacterium BB12 can colonize in the intestines of children with jaundice, change the intestinal flora, enrich the number of Bifidobacterium, and affect the metabolic pathways of neonates, which is speculated to be achieved through the bile acid metabolic pathway, which is a potential target for the treatment of neonatal jaundice.

[0004] Bifidobacterium is a kind of probiotics, which has been used in fermented dairy products for a long time, and some strains are generally considered to be "safe", which has promoted the application of Bifidobacterium as probiotic preparations. Animal Bifidobacterium is a kind of Bifidobacterium, which is the main dominant bacteria in the intestinal tract of mother and infant. Studies have found that the characteristics of the intestinal flora of neonatal jaundice patients are the decrease of the abundance of Bifidobacterium, and the decrease of the number of Bifidobacterium is related to the increase of bilirubin and the abnormality of intestinal flora metabolic pathways. Therefore, if Bifidobacterium with high production of bile salt hydrolase can be used to prevent and treat neonatal jaundice, accelerate the metabolism of bilirubin and bile acid, and then subsides jaundice, it will bring great convenience to the treatment of neonatal jaundice. SUMMARY

[0005] The present application provides an animal Bifidobacterium lactis (Bifidobacterium animalis subsp. lactis) with the characteristics of producing bile salt hydrolase and being able to prevent and / or relieve neonatal jaundice. Bifidobacterium animalis lactis subsp.Bifidobacterium animalis lactis ) WXM, which was preserved in Guangdong Microbial Culture Collection Center on September 13, 2023, with a preservation number of GDMCC No: 63806 and a preservation address of No. 59 Building, 5th Floor, Guangzhou Military Middle Road 100 Courtyard.

[0006] The present application provides a microbial preparation containing the animal Bifidobacterium lactis WXM.

[0007] In an embodiment, the viable count of the animal Bifidobacterium lactis WXM in the microbial preparation is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0008] In an embodiment, the microbial preparation is a liquid preparation or a solid preparation.

[0009] In an embodiment, the preparation method of the microbial preparation comprises: culturing the animal Bifidobacterium lactis WXM in MRS medium, the temperature of the culture being 35-37℃, and the culture time being 36-48 h.

[0010] The present application provides a medicine containing the animal Bifidobacterium lactis WXM.

[0011] In an embodiment, the form of the medicine includes but is not limited to: probiotic capsules, freeze-dried powder, tablets.

[0012] In an embodiment, the viable count of the animal Bifidobacterium lactis WXM in the medicine is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0013] In an embodiment, the medicine contains a pharmaceutically acceptable carrier.

[0014] The present application also provides the use of the animal Bifidobacterium lactis WXM in the preparation of a medicine for preventing and / or relieving neonatal jaundice.

[0015] In an embodiment, the medicine has at least one of the following functions:

[0016] (1) significantly improving the weight loss of individuals with early-life jaundice, having a protective effect;

[0017] (2) significantly improving the abnormal increase of bilirubin level in serum of individuals with early-life jaundice; the bilirubin level includes direct bilirubin level and total bilirubin level;

[0018] (3) significantly improving the liver damage of individuals with early-life jaundice, having a protective effect on the liver;

[0019] (4) Significantly improve the level of enzymes related to liver function in early life jaundice individuals, including alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, have the effect of protecting liver function;

[0020] (5) Significantly improve the expression level of related inflammatory factors in the liver of early life jaundice individuals, effectively alleviate liver inflammation;

[0021] (6) Effectively improve the expression level of UGT1A1 enzyme related to bilirubin metabolism in early life jaundice individuals, and have the effect of relieving neonatal jaundice.

[0022] Beneficial effects:

[0023] The present application screens the animal bifidobacterium lactis subsp. Bifidobacterium animalis lactis Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) WXM, which has the effect of relieving neonatal jaundice, specifically embodied in:

[0024] (1) Significantly improve the weight loss of neonatal rat jaundice induced by phenylhydrazine hydrochloride, and have the effect of protection;

[0025] (2) Significantly reduce the increase of bilirubin level in serum of neonatal rat jaundice induced by phenylhydrazine hydrochloride, including direct bilirubin and total bilirubin level;

[0026] (3) Significantly improve the liver damage of neonatal rat jaundice induced by phenylhydrazine hydrochloride, and have the effect of protecting liver;

[0027] (4) Significantly reduce the level of enzymes related to liver function in the liver of neonatal rat jaundice induced by phenylhydrazine hydrochloride, including alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, and have the effect of protecting liver function;

[0028] (5) Significantly reduce the expression level of related inflammatory factors in the liver of neonatal rat jaundice induced by phenylhydrazine hydrochloride, effectively alleviate liver inflammation;

[0029] (6) Effectively improve the expression level of UGT1A1 enzyme related to bilirubin metabolism, and have the effect of relieving neonatal jaundice.

[0030] Biological material preservation

[0031] Animal bifidobacterium lactis subsp. Bifidobacterium animalis lactis subsp. Bifidobacterium animalis lactis ) WXM, taxonomically named Figure 1 subsp Figure 2Bifidobacterium animalis lactis WXM was preserved in Guangdong Microbial Culture Collection Center on September 13, 2023, with a preservation number of GDMCC No: 63806 and a preservation address of No. 59 Building, 5th Floor, Guangzhou Institute, 100 Middle Martyrs Road, Guangzhou. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 3 Figure 2 is a diagram of the precipitation circle of each strain on the MRS solid medium plate containing different bile salts, wherein the left graph is a result diagram of the precipitation circle of each strain on the MRS solid medium plate containing 5 mM GDCA, and the right graph is a result diagram of the precipitation circle of each strain on the MRS solid agar medium plate containing 0.5% TDCA.

[0033] Figure 4 Figure 4 is the body weight change of each experimental group during gavage and modeling.

[0034] Figure 5 Figure 6 is the apparent results of Bifidobacterium animalis lactis WXM in reducing jaundice in newborn rats, including serum photos of each experimental group, total bilirubin levels of each experimental group, and direct bilirubin levels of each experimental group.

[0035] Figure 6 Figure 7 is the liver function results of Bifidobacterium animalis lactis WXM in relieving jaundice in newborn rats, including alanine aminotransferase levels of each experimental group, aspartate aminotransferase levels of each experimental group, and alkaline phosphatase levels of each experimental group.

[0036] Figure 7 Figure 8 is the liver photos and liver tissue sections and pathological analysis results of each experimental group.

[0037] rpm Figure 9 is the inflammatory factor TNF-α / β-actin mRNA relative expression level, IL-6 / β-actin mRNA relative expression level, and IL-1β / β-actin mRNA relative expression level of liver tissue of each experimental group.

[0038] rpm Figure 10 is the UGT1A1 enzyme expression level of each experimental group. DETAILED DESCRIPTION

[0039] The present application is further illustrated by the following description of the drawings and specific examples. These examples are intended to be illustrative only and are not intended to limit the scope of the present application. Unless otherwise indicated, the experimental methods in the following example were conducted in accordance with conventional conditions in the art or in accordance with the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meaning as familiar to those skilled in the art.

[0040] Bifidobacterium animalis lactis WXM, Bifidobacterium breve MYQQ-7 and Bifidobacterium breve MYQQ-10 are isolated from the feces of healthy infants fed with pure breast milk in Wuxi, Jiangsu Province.

[0041] The experimental animals involved in the following examples are specific pathogen free (SPF) Sprague dawley (SD) newborn mice (four days old), purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.; the biochemical analyzer involved in the following examples is purchased from Zhongyuanhuiji Biotechnology Co., Ltd.; the anhydrous ethanol and dimethylbenzene involved in the following examples are purchased from National Pharmaceutical Group Chemical Reagent Co., Ltd.; the HE staining solution involved in the following examples is purchased from Wuhan Saivier Biological Technology Co., Ltd.; the reverse transcription kit and SYBR involved in the following examples are purchased from Shanghai Yikesheng Biological Technology Co., Ltd.; the enzyme linked immunosorbent assay (ELISA) kit involved in the following examples is purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

[0042] The culture medium involved in the following examples is as follows:

[0043] MRS liquid medium: tryptone 10 g / L, beef infusion powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, potassium phosphate dibasic 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L, 0.5 g / L L-cysteine hydrochloride, pH 5.7±0.2, sterilized at 121 ℃ for 15 min.

[0044] MRS solid medium: tryptone 10 g / L, beef infusion powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, potassium phosphate dibasic 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 mL / L, 0.5 g / L L-cysteine hydrochloride, 18 g / L agar powder, pH 5.7±0.2, sterilized at 121 ℃ for 15 min.

[0045] Example 1 Qualitative screening of bile salt hydrolase-producing bifidobacteria

[0046] (1) Strain activation

[0047] The strains preserved in -80 ℃ glycerol tubes were inoculated into test tubes containing 5 mL of MRS medium at an inoculation amount of 1%-2%, and incubated at 37 ℃ anaerobically until the stationary phase.

[0048] (2) Expansion culture

[0049] The activated strains in (1) were inoculated into MRS medium containing 20 mL of MRS medium at an inoculation amount of 1%-2% for expansion culture, and incubated at 37 ℃ anaerobically until the stationary phase.

[0050] (3) Screening

[0051] 11,000 Figure 1 Centrifuged for 2 min, the bacterial cells were collected and resuspended twice with 0.1 M sodium phosphate buffer (pH=7.0), and then disrupted by an ultrasonic cell disruptor for 20 min (working time: interval time=2:3) at an amplitude of 20%, 11,000 Centrifuged for 5 min-10 min, the supernatant was collected. 10 μL of the supernatant was spotted onto MRS solid agar medium plates containing 0.37 g / L of calcium chloride and 5 g / L of sodium taurine deoxycholate (TDCA) and MRS solid agar medium plates containing 0.37 g / L of calcium chloride and 5 mM of sodium glycocholate (GDCA), respectively; TDCA and GDCA were sterilized by filtration and needed to be added after the sterilized medium was slightly cooled; MRS solid agar medium plates without added bile salts were used as negative controls, and incubated at 37 ℃ anaerobically for about 48 h.

[0052] rpm The results are shown in Table 1.

[0053] The results are shown in Table 1. rpm WXM, MYQQ-7 and BB12 had very obvious precipitation rings on MRS solid agar medium plates containing 5 mM GDCA, and MYQQ-10 had a relatively obvious precipitation ring on MRS solid agar medium plates containing 5 mM GDCA; WXM, MYQQ-7 and BB12 had very obvious precipitation rings on MRS solid agar medium plates containing 0.5% TDCA, and MYQQ-10 had no precipitation ring on MRS solid agar medium plates containing 5 mM GDCA. The precipitation ring production of all the bacteria is shown in Table 1.

[0054] Table 1 Qualitative screening of bile salt hydrolase-producing Bifidobacterium

[0055]

[0056] Note: +, there is a precipitation ring around the bacterial cells on the plate; -, there is no precipitation ring around the bacterial cells on the plate.

[0057] Quantitative screening of Bifidobacterium breve producing bile salt hydrolase

[0058] (1) Indantrione color developing solution

[0059] Dissolve indantrione in 0.5 M citric acid buffer (pH=5.5) at a ratio of 1%, and add 0.5 mL of the above mixture to 1.2 mL of glycerol and 0.2 mL of 0.5 M citric acid buffer (pH=5.5).

[0060] (2) Enzyme activity determination method

[0061] (1) Strain activation

[0062] Take the strain preserved in a glycerol tube at -80 ℃ and inoculate it into a test tube containing 5 mL of MRS medium at an inoculation amount of 1%-2%, and incubate it at 37 ℃ under anaerobic conditions until it reaches the stationary phase.

[0063] (2) Expansion culture

[0064] Inoculate the strain activated in (1) into MRS medium containing 20 mL of MRS medium at an inoculation amount of 1%-2% for expansion culture, and incubate it at 37 ℃ under anaerobic conditions until it reaches the stationary phase.

[0065] (3) Enzyme activity determination

[0066] Centrifuge the above bacterial solution for 10 min (1,000 g at 4 ℃), collect the bacterial cells, wash and centrifuge them twice with 0.1 M phosphate buffer (pH=7.0), and adjust the bacterial solution concentration to an absorbance value of 3 at 600 nm. Take 1 mL of the above cell suspension, ultrasonically break it for 3 min (working time: interval time=2:3), and immediately centrifuge it for 10 min (1,000 g at 4 ℃) to remove cell fragments, thereby obtaining a cell-free extract (CFE). Take 0.1 mL of the supernatant, add 1.8 mL of 0.1 M phosphate buffer (pH=6.0) and 0.1 mL of bound bile salt (200 mM) to mix, and incubate it at 37 ℃ for 30 min. Take 0.5 mL of the above reaction solution, add 0.5 mL of 15% trichloroacetic acid (w / t) to terminate the reaction, mix well, centrifuge it for 10 min (12,000 rpm at 4 ℃), and take the supernatant. Mix 0.1 mL of the supernatant with 1.9 mL of indantrione color developing solution, shake well, and boil in a water bath for 14 min. After cooling for 3 min, measure the absorbance value at 570 nm. The standard curve was prepared using glycine or taurine, as shown in Table 2.

[0067] Table 2 Amino acid standard curve

[0068]

[0069] Definition of BSH enzyme activity:

[0070] The BSH (total) enzyme activity (TA) is defined as the amount of substance of amino acids produced by the crude enzyme in unit time and unit volume, unit: μmol (min mL) -1 .

[0071] The calculation formula TA= Caa;

[0072] The BSH specific enzyme activity (SA) is defined as the amount of substance of amino acids produced by the crude enzyme in unit time and unit mass of total protein, unit: μmol (min mg) -1 .

[0073] ;

[0074] In the above formula, Caa represents the amino acid concentration, and Cp represents the protein concentration.

[0075] The content of total protein in the crude enzyme was determined by the classic Coomassie brilliant blue staining method.

[0076] 1) Prepare the bovine serum albumin (BSA) standard curve, as shown in Table 3;

[0077] 2) For each 250 μL standard curve sample or supernatant to be tested, add 2.5 mL of reaction solution, mix thoroughly, and let stand at room temperature for 5 min, then measure the absorbance value at 595 nm, and calculate the total protein concentration Cp.

[0078] Table 3 BSA protein standard curve

[0079]

[0080] Table 4 Total enzyme activity and specific enzyme activity determination results of all strains on two substrates

[0081]

[0082] As can be seen from Table 4, WXM has bile salt hydrolase activity on two substrates, and the total enzyme activity of WXM on GDCA is the highest, and the total enzyme activity on TCDA is also relatively high. Therefore, WXM is finally selected for subsequent animal experiments.

[0083] Example 3 Animal Bifidobacterium lactis WXM relieves jaundice in newborn rats

[0084] (1) Preparation of bacterial suspension

[0085] The strains preserved in -80 ℃ glycerol tubes were inoculated into test tubes containing 5 mL of MRS medium at an inoculation amount of 1%-2%, and incubated at 37 ℃ under anaerobic static culture to the stationary phase. After the incubation was completed, 8000 Figure 1 rpm centrifugation for 20 min to obtain bacterial slurry, which was washed twice with sterile normal saline and then resuspended to a final concentration of 1×10 9 CFU / mL to prepare animal Bifidobacterium lactis WXM and BB12 bacterial suspensions.

[0086] (2) Healthy SD rat pregnant mice and newborn mice (intervention started from the fourth day after birth) were selected, and normal breast milk feeding was performed.

[0087] (3) Modeling

[0088] Phenylhydrazine hydrochloride was injected intraperitoneally, and the dose was determined according to the body weight of the newborn mice, at a dose of 50 mg / kg, and intraperitoneal injection was performed twice continuously. The last injection was performed 24 h before the mice were sacrificed, and the samples were taken.

[0089] (4) Grouping and treatment of newborn SD rat newborn mice

[0090] The newborn SD rat newborn mice 4 days after birth were randomly divided into four groups (6 mice in each group), namely: control group, model group, animal Bifidobacterium lactis WXM group (WXM group), and animal Bifidobacterium BB12 group (BB12 group). 120 μL of animal Bifidobacterium lactis WXM bacterial suspension (1×10 9 CFU / mL) or BB12 bacterial suspension (1×10 9 CFU / mL) was administered orally for 10 consecutive days. On the 15th day, phenylhydrazine hydrochloride (50 mg / kg) was injected intraperitoneally for two consecutive days, and the mice were sacrificed 24 h after the last injection, and the samples were taken. The control group was administered with an equal amount of sterile normal saline.

[0091] (5) During the oral administration and modeling of the SD rat newborn mice, the body weight of the newborn mice was measured every day. 24 h after the second intraperitoneal injection of phenylhydrazine hydrochloride, the plasma was taken, 5000 Figure 2 rpm centrifugation for 20 min, the serum was taken and photographed, and the serum bilirubin (total bilirubin, direct bilirubin) level was determined by a biochemical analyzer. The determination results are shown in Figure 2 and Figure 3 .

[0092] From Figure 4The weight change trend shows that during the gavage of the young mice, the weight of the young mice in each group increased well. During the modeling period, the weight gain rate of the model group of young mice decreased rapidly, and even showed negative growth; while the weight gain rate of the gavage bacteria group of young mice decreased slowly, and the BB12 group also showed negative growth phenomenon, but compared with the control bacteria group BB12, the weight of the WXM group of young mice still maintained a slow growth. It is speculated that the administration of Bifidobacterium animalis lactis WXM to neonatal jaundice mice can effectively reduce the weight loss induced by phenylhydrazine hydrochloride.

[0093] By Figure 6 The serum photograph shows that the plasma hemolysis induced by phenylhydrazine hydrochloride is more serious, and this phenomenon is improved after the administration of Bifidobacterium animalis lactis WXM and BB12 to neonatal jaundice mice. The average level of total bilirubin and the average level of direct bilirubin in the BB12 group were 35.68 μmol / L and 25.04 μmol / L, respectively, and the average level of total bilirubin and the average level of direct bilirubin in the WXM group were 28.09 μmol / L and 18.48 μmol / L, respectively. From the trend of total bilirubin level and direct bilirubin level, it can be seen that the bilirubin level induced by phenylhydrazine hydrochloride rises sharply, while the gavage bacteria group can effectively improve this change, and for the WXM group, this improvement effect is more obvious than the BB12 group. It can be inferred that the administration of Bifidobacterium animalis lactis WXM to neonatal jaundice mice can effectively improve the plasma hemolysis and the increase of bilirubin level in serum induced by phenylhydrazine hydrochloride.

[0094] Example 4 Bifidobacterium animalis lactis WXM reduces liver damage of neonatal jaundice rats

[0095] The method for establishing an animal model is referred to Example 3. After taking the serum of the young mice, the biochemical analyzer is used to determine the levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase in the serum; the liver tissue is taken and photographed, and H&E staining is used to observe the damage of the liver tissue of the neonatal SD rats,

[0096] The H&E staining method is as follows: the green bean size liver tissue is fixed in 4% paraformaldehyde solution for more than 24 h, then dehydrated, immersed in wax and embedded for sectioning, the paraffin section is deparaffinized to water, then stained with hematoxylin and eosin, photographed under a microscope and analyzed by SlideViewer.

[0097] Alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase are one of the most commonly used detection indexes for liver function examination, and are also important auxiliary diagnosis and differential indexes for various liver diseases. From the trend of the levels of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, Figure 7It can be seen that after phenylhydrazine hydrochloride induction, liver function is impaired, and alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase levels are increased (42.27 U / L, 750.57 U / L, 551.11 U / L, respectively), while the bacterial group can reduce the level, and the results show that the WXM group (22.84 U / L, 177.81 U / L, 429.99 U / L, respectively) is more effective than the BB12 group (31.14 U / L, 333.93 U / L, 445.21 U / L, respectively). It shows that the animal Bifidobacterium lactis WXM has a certain protective effect on liver function damage after intervention.

[0098] According to the results of H&E staining, the liver tissue of the blank group of young mice is arranged closely and neatly, the nucleus is complete, and it is large and round, without obvious liver cell damage and no obvious inflammatory cells. Compared with the blank group, the liver tissue of the phenylhydrazine hydrochloride group of young mice is arranged loosely, the liver tissue structure is damaged, there are obvious liver cell necrosis foci, vacuole-like deformation, and inflammatory cell infiltration, indicating that the young mice have severe liver tissue damage after modeling. After the bacteria are poured, it can be found that the degree of loose arrangement of liver tissue structure is reduced, liver cell necrosis foci are reduced, vacuole-like deformation is reduced, and there are a small amount of inflammatory cell infiltration, and the difference between the WXM group and the BB12 group is also obvious. It shows that the animal Bifidobacterium lactis WXM has a certain improvement effect on the pathological changes of liver tissue after intervention.

[0099] Example 5 Animal Bifidobacterium lactis WXM reduces liver inflammation of neonatal rat jaundice

[0100] The method for establishing an animal model is as described in Example 3. After the mice are sacrificed, the liver tissue is taken and stored at -80°C. A certain amount of liver tissue is taken in a 1.5 mL EP tube containing 1 mL TRIzol and three magnetic beads, and the tissue is homogenized. The total RNA is extracted by a conventional method. The RNA concentration is adjusted to 500 ng / μL, and the purity (A260 / A280) is in the range of 1.8-2.2. The cDNA is synthesized by reverse transcription, and the real-time quantitative PCR reaction is performed. The sample is mixed with the fluorescent dye SYBR Green super mix, the PCR system is 10 μL SYBR, 2 μL cDNA, 1 μL forward and reverse primers, and ddH2O is added to a total volume of 20 μL. Three parallel holes are set for each sample, and the average value is taken. The housekeeping gene β-actin is used as an internal reference. The relative expression level of different target genes is calculated according to the following formula:

[0101] .

[0102] Table 5 qPCR primer sequences

[0103]

[0104] Results as ​ showed that compared with the blank group, the model group enhanced the expression of proinflammatory factors (TNF-α, IL-6, IL-1β), while the bacteria group could significantly reduce the mRNA levels of these proinflammatory factors, among which the WXM group and the BB12 group had significant differences in reducing the expression levels of TNF-α and IL-6 mRNA (p<0.01). It showed that the animal Bifidobacterium lactis WXM had a certain improvement effect on liver tissue inflammation after intervention.

[0105] Example 6 Animal Bifidobacterium lactis WXM improves the expression level of bilirubin metabolism related enzyme

[0106] After the mice were sacrificed, the liver tissue was taken and stored at -80 ℃. A certain amount of liver tissue was weighed, a certain amount of PBS (pH 7.2-7.4) (w / v=1:9) was added, the tissue was homogenized and centrifuged for 20 min (2000-3000 rpm). The supernatant was carefully collected. One part was detected after aliquot, and the rest was frozen for later use. The sample was added to the bottom of the enzyme-labeled plate (the final dilution of the sample was 5 times), and after adding the enzyme-labeled reagent, the plate was sealed and incubated at 37 ℃ for 60 min. Wash several times with washing solution, then add color developing agent, develop color at 37 ℃ for 15 min, finally add stop solution to stop the reaction, and measure the absorbance at 450 nm.

[0107] UGT1A1 enzyme plays a crucial role in the process of bilirubin metabolism, and its main function is to convert unconjugated bilirubin in the liver into conjugated bilirubin, which is then excreted from the liver through transporters, thereby reducing the level of bilirubin in the liver. As ​ showed that compared with the blank group, the expression level of UGT1A1 enzyme induced by phenylhydrazine hydrochloride was reduced to 1.15 on average, which to some extent hindered the normal metabolism of bilirubin, making bilirubin unable to be excreted outside the body, and thus exacerbating neonatal jaundice. After WXM bacteria treatment, it could significantly improve this phenomenon, and the average expression level of UGT1A1 enzyme was significantly increased to 1.42, enhancing the conjugation of bilirubin and promoting the excretion of bilirubin, which had a significant subsiding effect on neonatal jaundice.

[0108] As can be known from the above examples, the high-yield bile salt hydrolase animal Bifidobacterium lactis WXM provided by the application has better effects of relieving neonatal jaundice, and the effects are shown as follows: effectively reducing the body weight loss induced by phenylhydrazine hydrochloride; effectively improving the plasma hemolysis and the increase of the bilirubin level in serum induced by phenylhydrazine hydrochloride; having a certain protective effect on liver function damage, a certain improvement effect on liver tissue pathological change, and a certain improvement effect on liver tissue inflammation; significantly increasing the expression level of UGT1A1 enzyme, enhancing the conjugation of bilirubin, and promoting the excretion of bilirubin, which has a significant subsiding effect on neonatal jaundice.

[0109] Although the application has been disclosed with the preferred embodiments as above, it is not intended to limit the application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the application, so the protection scope of the application should be defined by the claims.

Claims

1. A strain of Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalis subsp. lactis WXM was deposited at the Guangdong Provincial Center for Microbial Culture Collection on September 13, 2023, with accession number GDMCC No: 63806.

2. A microbial preparation comprising the animal bifidobacterium WXM of claim 1.

3. The microbial preparation according to claim 2, characterized in that, The microbial preparation is a liquid preparation or a solid preparation.

4. The microbial preparation according to claim 2 or 3, characterized in that, The viable cell number of Bifidobacterium animalis lactis WXM in the microbial preparation is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

5. A medicament comprising the animal bifidobacterium WXM of claim 1.

6. The medicament according to claim 5, characterized in that, The form of the medicament includes: probiotic capsules, lyophilized powder or tablets.

7. The medicament according to claim 5 or 6, characterized in that, The viable cell number of Bifidobacterium animalis ssp. lactis WXM in the medicine is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

8. The medicament according to any one of claims 5 to 7, characterized in that, The medicament comprises a pharmaceutically acceptable carrier.

9. Use of the animal bifidobacterium WXM of claim 1 in the preparation of a medicament for preventing and / or alleviating neonatal jaundice.

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