Probiotic preparation for improving abnormal bile diseases of newborns

Abstract

The invention relates to the technical field of probiotics, and discloses a probiotic preparation for improving abnormal bile diseases of newborns. According to the probiotic preparation provided by the invention, lactobacillus fermentum CECT5716, bifidobacterium breve M-16V and bifidobacterium longum subsp. Longum BB536 in the list of strains for infants are selected as a product formula, and the probiotic preparation can be used for effectively improving abnormal bile diseases of newborns, including jaundice, hyperbilirubinemia and cholestasis, and can be used for preventing and treating the abnormal bile diseases of the newborns, such as jaundice, hyperbilirubinemia and cholestasis. The problems that in the prior art, safety is insufficient, pertinence is insufficient, and clinical requirements of complex illness conditions cannot be met are effectively solved.

Description

Technical Field

[0001] This invention relates to the field of probiotic technology, and more specifically to a probiotic preparation for improving abnormal bile disorders in newborns. Background Technology

[0002] Neonatal jaundice, hyperbilirubinemia, and cholestasis are common clinical problems in the neonatal period. While related, they have different emphases. Neonatal jaundice is a symptom, its root cause being abnormal bilirubin metabolism leading to elevated serum bilirubin concentrations (visible yellowing at 3-5 mg / dL or 50-85 μmol / L), which in turn causes bilirubin deposition in tissues, manifesting as yellowing of the skin and sclera. Approximately 60% of full-term infants and 80% of premature infants experience jaundice in the first week after birth. Hyperbilirubinemia is the pathological basis of jaundice (i.e., elevated bilirubin levels in the blood). Based on the clinical and prognostic characteristics of hyperbilirubinemia, neonatal jaundice can be divided into physiological jaundice and pathological jaundice. Physiological jaundice usually appears within 2-3 days after birth and subsides within 7-10 days. In premature infants, it appears earlier (1-2 days) and subsides slightly longer (2-4 weeks), and the newborn is generally in good condition. Pathological jaundice can be caused by a variety of factors, including hemolysis, infection, biliary tract disease and metabolic abnormalities. It is characterized by early onset (within 24 hours), rapid progression (daily bilirubin increase exceeding 85 μmol / L or 5 mg / dL), high levels (more than 221 μmol / L or 12.9 mg / dL in full-term infants, and more than 257 μmol / L or 15 mg / dL in preterm infants), or long duration (more than 2 weeks in full-term infants and more than 4 weeks in preterm infants), or recurrence after fading.

[0003] The occurrence of neonatal jaundice depends on the concentration of intermediate bilirubin in the blood: when the bilirubin level exceeds 50-85 μmol / L (3-5 mg / dL), visible yellowing of the skin and mucous membranes will appear (due to bilirubin deposition in tissues). If the bilirubin level in hyperbilirubinemia is below this threshold, it will only manifest as abnormal laboratory tests (elevated blood bilirubin) without visible clinical signs of jaundice (clinically termed "asymptomatic hyperbilirubinemia"). If bilirubin continues to rise above the threshold, it will develop into clinical jaundice; in severe cases (such as unconjugated bilirubin >342 μmol / L or 20 mg / dL; the threshold is even lower in premature infants, usually >257 μmol / L or 15 mg / dL), if not intervened in time, it may lead to kernicterus (bilirubin encephalopathy). If serum total bile acids (TBA) are significantly elevated, hyperbilirubinemia may or may not be present. When TBA is elevated but bilirubin does not exceed the above threshold (without jaundice), this condition is often seen in the early stage of neonatal cholestatic disease or specific types of hepatobiliary metabolic abnormalities. The core reason is the degree and time difference of abnormality of different metabolites (bile acids, bilirubin) (e.g., in early cholestasis, bile acid excretion is impaired first, while bilirubin metabolism has not yet been significantly affected).

[0004] Neonatal cholestasis is a disease state caused by impaired bile formation or excretion, leading to the retention of direct bilirubin in the body. Its core manifestations are persistent jaundice (>2 weeks in full-term infants, >4 weeks in premature infants, with elevated direct bilirubin predominating, and conjugated bilirubin accounting for >20% of total bilirubin), clay-colored stools, dark brown urine, and hepatosplenomegaly. The etiology of neonatal cholestasis is complex and can be divided into intrahepatic and extrahepatic types. Extrahepatic cholestasis is mainly caused by biliary atresia. Biliary atresia refers to progressive fibrosis and obstruction of the extrahepatic bile ducts, preventing normal bile drainage. If left untreated, it can lead to liver fibrosis, cirrhosis, and even death. Intrahepatic cholestasis may be related to genetic, infectious, and metabolic factors. Bile acids are products of cholesterol formation; in a state of cholestasis, serum bile acid levels also increase. Experiments have already verified this. A total of 50 jaundiced newborns were selected, and the total bilirubin (TBIL) in their bodies and the total bile acid (TBA) in their serum were tested. The concentrations of the two indicators were analyzed, and it was found that the TBA content in the bodies of the newborns was significantly elevated, and the level of the indicators was highly correlated with the severity of jaundice.

[0005] Probiotics can improve bile acid and bilirubin metabolism in newborns through multiple mechanisms, including regulating gut microbiota structure, lowering intestinal pH, inhibiting β-glucuronidase activity, reducing bilirubin enterohepatic circulation, promoting intestinal motility and bilirubin excretion, enhancing liver enzyme activity, regulating bile salt synthesis-related genes and pathways, and exerting anti-inflammatory and mucosal protective effects. Therefore, they have potential value in intervening in related neonatal diseases. However, the current clinical application of probiotics in intervening in related neonatal diseases still has the following obvious limitations: First, the scope of application is relatively narrow. Most studies and practices only focus on the treatment or adjunctive treatment of neonatal jaundice with Bacillus subtilis and live bacteria, without fully covering a wider range of bile metabolism disorders such as hyperbilirubinemia and cholestasis, resulting in insufficient treatment coverage. Second, safety and specificity need to be improved. The strains currently used, such as some Bifidobacteria and Lactobacillus strains, have unclear origins, and are not derived from humans. Some may carry toxin-producing genes and transferable plasmids, posing potential safety risks. Moreover, existing probiotic preparations are difficult to adapt to the complex and intertwined clinical characteristics of neonatal diseases, and cannot form a systematic and comprehensive intervention strategy for the three interrelated pathological states of jaundice, hyperbilirubinemia, and cholestasis. Summary of the Invention

[0006] This invention provides a probiotic preparation, the product formula of which is selected from *Lactobacillus fermentum* (*Lactobacillus fermentum*) listed in Announcement No. 6 of 2016, "List of Bacterial Strains that Can Be Used in Infants and Young Children". Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp . long BB536, this probiotic preparation can effectively improve abnormal bile conditions in newborns, including jaundice, hyperbilirubinemia, and cholestasis, effectively solving the clinical needs problems of insufficient safety, insufficient targeting, and inability to be applied to complex conditions in existing technologies.

[0007] On one hand, the present invention provides a probiotic preparation for improving abnormal bile disorders in newborns, the probiotic preparation comprising *Lactobacillus fermentum* (… Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium long subsp long BB536.

[0008] Furthermore, the neonatal abnormal bile disorders include jaundice, hyperbilirubinemia, and / or cholestasis.

[0009] Furthermore, in the probiotic preparation, *Lactobacillus fermentum* (… Limosilactobacillus leaven CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long The live bacteria ratio of BB536 is 2-8:1-5:1-5.

[0010] Furthermore, the probiotic preparation contains *Lactobacillus fermentum* (…). Limosilactobacillus leaven CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V or Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long The total number of live bacteria in strain BB536 is not less than 10 billion CFU.

[0011] Furthermore, the probiotic preparations also contain fructooligosaccharides and / or resistant dextrin.

[0012] Furthermore, the probiotic preparation is administered orally.

[0013] Furthermore, the dosage form of the probiotic preparation includes capsules, tablets, or granules.

[0014] Furthermore, the probiotic preparation also contains food-acceptable adjuvants, additives, nutritional fortifiers, and / or raw materials.

[0015] On the other hand, the present invention provides the use of the above-mentioned probiotic preparation in the preparation of a medicament for the prevention and / or improvement of abnormal bile disorders in newborns.

[0016] Furthermore, the neonatal abnormal bile disorders include jaundice, hyperbilirubinemia, and / or cholestasis.

[0017] Furthermore, the prevention and / or improvement of jaundice includes reducing total bilirubin (TBIL) in the blood and total bile acids (TBA) in the serum. Furthermore, the prevention and / or improvement of hyperbilirubinemia includes reducing total bilirubin (TBIL) in the blood and total bile acids (TBA) in the serum. Furthermore, the prevention and / or improvement of cholestasis includes reducing total bilirubin (TBIL), direct bilirubin (DBIL), and serum total bile acids (TBA) in the blood.

[0018] The technical solution of this invention has the following advantages: 1. The fermenting *Lactobacillus mucinus* selected in this invention (… Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium long subsp long BB536 strains were selected from the strains published in Announcement No. 6 of 2016, "List of Strains that Can Be Used in Infants and Young Children". They were all isolated from healthy breast milk or infant intestines, conformed to the characteristics of the native flora of infant intestines, and passed the GRAS certification of the US FDA and the QPS certification of the EU. They were well tolerated and were more suitable for the intestinal physiological environment of newborns.

[0019] 2. Addressing the core issue of reduced Bifidobacterium abundance and associated elevated bilirubin and abnormal gut microbiota metabolism in neonatal jaundice patients, the formula contains Bifidobacterium shortum (… Bifidobacterium breve M-16V, Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long BB536 can directly supplement the dominant probiotic strains in an infant's gut; fermented Lactobacillus mucinus (Limosilactobacillus fermentum CECT5716, derived from breast milk, not only promotes the proliferation of Bifidobacteria, but studies have also found that this strain can alleviate intestinal inflammation, reduce harmful intestinal bacteria, enhance intestinal mucosal integrity, improve the intestinal barrier, regulate lipid metabolism, and optimize the microbiota structure, thus also having a positive effect on total bile acids (TBA) and total bilirubin (TBIL). The combined effect of these three factors synergistically repairs intestinal microbiota imbalance, thereby reducing the activity of β-glucuronidase produced by harmful intestinal bacteria, reducing enterohepatic circulation of bilirubin, and fundamentally improving jaundice-related metabolic disorders.

[0020] 3. Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long Both BB536 strains originate from the gut microbiota of infants in East Asia and can colonize the large intestine. Their bacterial surface is rich in serine protease inhibitors (serpin). Through serpin-mediated protease inhibition, they reduce intestinal inflammation and barrier damage, prevent liver inflammation caused by endotoxin translocation, support liver function recovery, and promote bilirubin metabolism and excretion. Furthermore, both strains produce high levels of bile salt hydrolases, which can hydrolyze conjugated bile acids to generate free bile acids (such as chenodeoxycholic acid), activate FXR / PXR nuclear receptors, upregulate UGT1A1 expression, promote bilirubin binding and excretion, and reduce the accumulation of toxic bile acids (such as lithocholic acid) in the intestine, alleviating bile acid-mediated hepatocellular damage, thereby improving liver metabolism. *Lactobacillus fermentans* (… Limosilactobacillus fermentum CECT5716 is derived from breast milk and can colonize the small intestine. This strain can also improve intestinal barrier function, inhibit harmful bacteria, protect the liver, improve lipid metabolism, produce short-chain fatty acids, and regulate immunity. The three strains form a complete intestinal coverage, synergistically optimizing the liver and intestinal metabolic environment and improving the efficiency of bilirubin and bile acid metabolism.

[0021] 4. Through the synergistic effect of multiple strains, this invention can not only effectively improve neonatal jaundice, but also specifically intervene in hyperbilirubinemia and cholestasis. It solves the problem that existing technologies only target a single disease and have limited applicable populations, and can meet the clinical needs of newborns with complex conditions, thus having a wider range of applications. Attached Figure Description

[0022] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0023] Figure 1 This is a graph showing the results of detecting total bile acids (TBA) in different experimental groups; Figure 2 This is a graph showing the results of detecting serum total bilirubin (TBIL) in different experimental groups. Detailed Implementation

[0024] The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0025] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0026] The fermented Lactobacillus mucinus used in the following examples ( Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V, Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long BB536 was purchased from Baolingbao Biotechnology Co., Ltd. The fructooligosaccharides and resistant dextrin used in the following examples were all commercially available.

[0027] Example 1: Screening for probiotics that can prevent and treat jaundice 1. Experimental Objective Currently, in the use of probiotics as an adjunct treatment for jaundice and related diseases, the main probiotics used include Bifidobacterium animalis subsp. lactis BB-12 (… Bifidobacterium animalum subsp milkBB-12) and Lactobacillus rhamnosus GG ( Lactobacillus rhamnosus This experiment screened strains from a list of strains used in infants and young children that could be used as an adjunct treatment for jaundice and related diseases, aiming to identify more effective and safer probiotic strains for neonatal jaundice-related conditions. This experiment also investigated the intervention effects of different probiotic strains on pathological jaundice in newborn rats, analyzing the influence of different strains on serum total bilirubin and total bile acids, providing a basis for screening strains for adjunctive treatment of jaundice and related diseases.

[0028] 2. Experimental Materials Selected bacterial strains for infant formula: Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp . long BB536, Bifidobacterium longum infantile subspecies M-63 ( Bifidobacterium longum subsp child M-63), Bifidobacterium breve ( Bifidobacterium breve M-16V, Bifidobacterium animalis subsp. lactis BB-12 ( Bifidobacterium animalum subsp milk BB-12), Lactobacillus rhamnosus GG ( Lactobacillus rhamnosus GG) and Lactobacillus fermentum (GG) Limosilactobacillus leaven CECT5716 should be diluted to the required concentration with sterile saline or artificial milk before the experiment (prepare fresh for immediate use).

[0029] Experimental animals: SPF-grade SD newborn rats (all male, 4 days old). Modeling agent: phenylhydrazine hydrochloride; Modeling principle: Phenylaspine hydrochloride is a potent hemolytic agent whose mechanism of action is mainly through oxidative stress damage to red blood cells. After entering the body, phenylhydrazine metabolizes and produces free radicals, which attack lipids and proteins on the red blood cell membrane, leading to damage to the structural integrity of the red blood cell membrane and accelerating red blood cell destruction, thus causing hemolytic anemia. The massive destruction of red blood cells releases bilirubin, exceeding the liver's processing capacity, resulting in elevated serum bilirubin levels, ultimately manifesting as jaundice.

[0030] 3. Experimental Grouping To establish a neonatal rat jaundice model, 4-day-old SD rats were randomly divided into 8 groups, including a control group, a model group, a GG experimental group, a CECT5716 experimental group, an M-16V experimental group, a BB536 experimental group, an M-63 experimental group, and a BB-12 experimental group. Each group contained 6 SD rats.

[0031] From day 4 to 16 after birth, the rats in the experimental group were administered Bifidobacterium longum subsp. longum via gavage. Bifidobacterium longum subsp long BB536, Bifidobacterium longum infantile subspecies M-63 ( Bifidobacterium longum subsp child M-63), Bifidobacterium breve ( Bifidobacterium breve M-16V, Bifidobacterium animalis subsp. lactis BB-12 ( Bifidobacterium animalum subsp milk BB-12), Lactobacillus rhamnosus GG ( Lactobacillus rhamnosus GG) and Lactobacillus fermentum (GG) Limosilactobacillus fermentum The live bacteria of CECT5716 were administered orally at a dose of 1×10⁻⁶. 9 CFU was administered by resuspending the bacterial powder in 0.1 mL of physiological saline to form a bacterial solution, and then administered by gavage once a day. The control group and the model group were administered the same volume of physiological saline by gavage.

[0032] After 14 days of administration of bacterial solution or physiological saline, a neonatal rat jaundice model was established. Rats in the model group and experimental group were injected intraperitoneally with 50 mg / kg phenylhydrazine hydrochloride for two consecutive days. 24 hours after the second injection, the rats were sacrificed and their blood was collected to detect blood indicators.

[0033] 4. Blood index testing Fresh blood was collected, allowed to stand at room temperature (25℃) for 0.5 h, and centrifuged at 3000 r / min for 20 min to collect the supernatant. Serum total bilirubin (TBIL) and total bile acids (TBA) were measured using a fully automated biochemical analyzer.

[0034] Serum total bilirubin (TBIL) and total bile acids (TBA) are core biochemical indicators for assessing liver function, biliary patency, and jaundice-related diseases. They reflect the etiology and severity of jaundice from different perspectives. Total bilirubin (TBIL) is the total amount of bilirubin in serum, and elevated levels are usually a direct manifestation of jaundice. Total bile acids (TBA) are steroidal acids synthesized from cholesterol in the liver. They emulsify fats and promote the absorption of fat-soluble vitamins in bile, and elevated TBA levels usually directly reflect impaired liver clearance or bile excretion disorders. Combined detection of these two indicators can more comprehensively reflect liver function and bile metabolism abnormalities, especially showing significant value in the differential diagnosis of obstructive jaundice and the monitoring of liver damage.

[0035] 5. Data Statistics and Analysis SPSS 18.0 statistical software was used. Quantitative data are expressed as "mean ± standard deviation (x ± s)"; P < 0.05 indicates that the difference is statistically significant. 6. Test Results (1) Serum total bile acids (TBA) The results of total bile acid (TBA) in the serum of rats in the above 8 groups are shown in Table 1. Figure 1 and Figure 2 .

[0036] Table 1 Serum Indicators

[0037] Note: a, b, c, d, e, bc, and cd are significance markers.

[0038] As shown in the graphs, there was a significant difference between the model group and the control group (P<0.05), indicating that the model was successfully established. The results also showed that all probiotic groups had a significant decrease compared to the model group (p<0.05).

[0039] Among the experimental groups, the CECT5716 group showed the best performance in total bile acid (TBA), significantly lower than the GG, BB-12, M-63, and BB536 groups (P<0.05); the CECT5716 group was lower than the M-16V group, but the difference was not statistically significant (P>0.05). The M-16V, BB536, and M-63 groups showed significantly lower levels compared to the GG and BB-12 groups (P<0.05). The overall order of total bile acid (TBA) content in the experimental groups was: BB-12>GG>M-63>BB536>M-16V>CECT5716.

[0040] In terms of total bilirubin (TBIL), the M-16V group performed best, with the lowest value among all probiotic groups. There was no significant difference compared to the BB536, CECT5716, and M-63 groups (P>0.05), and the TBIL was significantly lower than the GG and BB-12 groups. The CECT5716, GG, BB536, and M-63 groups were all significantly lower than the BB-12 group (P<0.05). The overall order of total bilirubin (TBIL) content in the experimental groups was: BB-12>GG>M-63>CECT5716>BB536>M-16V.

[0041] In summary, based on the rat test results of TBA and TBIL, BB536, M-16V and CECT5716 are selected as the combined strains for the next step of the experiment for jaundice-related diseases.

[0042] Example 2: Validation of the effectiveness of different strain combinations in the treatment of jaundice 1. The viable cell count ratios of BB536, M-16V, and CECT5716 in different strain combinations are shown in Table 2.

[0043] Table 2. Viable cell count ratios of the three strains BB536, M-16V, and CECT5716 in different strain combinations.

[0044] 2. Experimental Grouping From 81 newborns, those with moderate to severe illness who had poor responses to traditional treatment were selected and divided into three groups based on their condition: a jaundice group (n=27), a hyperbilirubinemia group (n=25), and a cholestasis group (n=29). Each of the jaundice, hyperbilirubinemia, and cholestasis groups was further divided into five subgroups (control group, probiotic group 1, probiotic group 2, probiotic group 3, and probiotic group 4), with 5-7 individuals randomly selected from each subgroup. The four probiotic groups received probiotic adjuvant therapy in addition to the traditional treatment regimen (jaundice: phototherapy + albumin + intravenous immunoglobulin; hyperbilirubinemia: phototherapy + UDCA + albumin + nutritional support; cholestasis: ursodeoxycholic acid + fat-soluble vitamins + MCT formula milk); while the control group received only the traditional treatment regimen. The jaundice group and the hyperbilirubin group took probiotics for 7 days (1 sachet / day), and the cholestasis group took probiotics for 7 days (1 sachet twice a day for the first 3 days, and 1 sachet once a day for the next 4 days). After 7 days, the levels of total bile acid and total bilirubin were measured.

[0045] Probiotics Group 1: Lactobacillus fermentum ( Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V, Bifidobacterium longum subsp. ( Bifidobacterium long subsp long BB536, the ratio of the three strains of probiotics in each bag is 4:3:3, the total number of live bacteria is 10 billion CFU / bag, and it is supplemented with fructooligosaccharides and resistant dextrin.

[0046] Probiotics Group 2: Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp long BB536, the ratio of live bacteria of the two strains mentioned above in each bag of probiotics is 1:1, the total number of bacteria is 10 billion CFU / bag, and it is supplemented with the same fructooligosaccharides and resistant dextrin.

[0047] Probiotics Group 3: Lactobacillus fermentum ( Limosilactobacillus fermentum CECT5716 and Bifidobacterium breve ( Bifidobacterium breve M-16V, wherein the ratio of live bacteria of the two strains mentioned above in each bag of probiotics is 1:1, the total number of bacteria is 10 billion CFU / bag, and it is supplemented with the same fructooligosaccharides and resistant dextrin.

[0048] Probiotics Group 4: Lactobacillus fermentum ( Limosilactobacillus fermentum CECT5716 and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp longBB536, the ratio of live bacteria of the two strains mentioned above in each bag of probiotics is 1:1, the total number of bacteria is 10 billion CFU / bag, and it is supplemented with the same fructooligosaccharides and resistant dextrin.

[0049] How to add: Dissolve the probiotic powder completely in water, milk powder or breast milk and drink.

[0050] 4. Indicator Testing The test measures serum total bilirubin (TBIL), direct bilirubin (DBIL), and total bile acids (TBA) in the blood.

[0051] 5. Test Results (1) Neonatal jaundice In this test, all newborns with jaundice had a gestational age greater than 37 weeks. In the control group of 6, 3 were breastfed and 3 were mixed-fed; in probiotic group 1 of 6, 3 were breastfed and 3 were mixed-fed; in probiotic group 2 of 5, 1 was breastfed and 4 were mixed-fed; in probiotic group 3 of 5, 3 were breastfed and 2 were mixed-fed; and in probiotic group 4 of 5, 3 were breastfed and 2 were mixed-fed.

[0052] Table 3 Neonatal Jaundice - Blood Indicators

[0053] Note: a and b are significance markers, and the p-value shows the significance of the change between 7 days and 0 days.

[0054] The test results are shown in Table 3. In newborns with jaundice, taking probiotics can significantly reduce the levels of serum total bilirubin (TBIL) and total bile acid (TBA) (P<0.05).

[0055] Comparison of serum total bilirubin (TBIL) levels after 7 days of treatment revealed significant reductions in all four probiotic groups compared to the control group (P<0.05). Specifically, probiotic group 1 showed a significant reduction compared to probiotic groups 2-4 (P<0.05). Probiotic group 2 had higher TBIL levels than probiotic groups 3 and 4, but the difference was not statistically significant (P>0.05). The ranking of TBIL reduction was: probiotic group 1 > probiotic group 4 > probiotic group 3 > probiotic group 2.

[0056] Comparison of total bile acid (TBA) levels in the blood after 7 days of treatment revealed that the levels in the four probiotic groups were significantly lower than those in the control group (P<0.05). Among them, probiotic group 1 had the lowest level, which was close to the normal value and was significantly different from the other three groups (P<0.05).

[0057] After 7 days of treatment, all groups showed a significant decrease compared to day 0, including the control group. Among them, probiotic groups 1 to 4 showed extremely significant decreases in serum total bilirubin (TBIL) and total bile acid (TBA) (P<0.01).

[0058] In summary, the combination of three probiotic strains (i.e., probiotic group 1) during treatment of newborns with jaundice can improve the treatment effect and accelerate the recovery of the infants compared to other groups.

[0059] (2) Hyperbilirubinemia In this test, all newborns with hyperbilirubinemia had a gestational age greater than 37 weeks. In the control group, 4 out of 5 were formula-fed and 1 was mixed-fed. In probiotic group 1, 2 out of 5 were formula-fed and 3 were mixed-fed. In probiotic group 2, 4 out of 5 were formula-fed and 1 was mixed-fed. In probiotic group 3, 1 out of 5 were formula-fed and 4 were mixed-fed. In probiotic group 4, 3 out of 5 were formula-fed and 2 were mixed-fed.

[0060] Table 4. Hyperbilirubinemia - Blood Indicators

[0061] Note: a, b, and bc are significance markers; the p-value shows the significance of the change between 7 days and 0 days. As shown in Table 4, probiotics significantly reduced serum total bilirubin (TBIL) and total bile acid (TBA) levels in newborns with hyperbilirubinemia within 7 days (P<0.05).

[0062] Comparison of serum total bilirubin (TBIL) after 7 days of treatment revealed that the four probiotic groups showed a significant decrease compared to the control group (P<0.05), but there was no significant difference between the probiotic groups (P>0.05), with the lowest serum total bilirubin (TBIL) in probiotic group 1.

[0063] Comparison of total bile acid (TBA) levels in the blood after 7 days of treatment revealed that all four probiotic groups showed a significant decrease compared to the control group (P<0.05). Probiotic group 1 had the lowest TBA levels, which was not significantly different from probiotic groups 3 and 4 (P>0.05), but was significantly lower than probiotic group 2 (P<0.05).

[0064] All groups, including the control group, showed a significant reduction after 7 days of treatment compared to day 0. All probiotic groups showed highly significant reductions in serum total bilirubin (TBIL) and total bile acids (TBA) (P<0.01). The probiotic group 1, which showed the best effect, approached normal values ​​after 7 days of treatment.

[0065] Therefore, it can be concluded that supplementing newborns with hyperbilirubinemia with a combination of three probiotic strains from probiotic group 1 during treatment can accelerate the improvement effect.

[0066] (3) Cholestasis In this test, among the newborns with cholestasis, one had a gestational age of 31+6 weeks, and the rest had a gestational age greater than 37 weeks. In the control group of 7, 3 were breastfed, 3 were mixed-fed, and 1 was formula-fed. In probiotic group 1 of 7 (including 1 premature infant), 5 were breastfed and 2 were mixed-fed. In probiotic group 2 of 5, 4 were formula-fed and 1 was mixed-fed. In probiotic group 3 of 5, 4 were formula-fed and 1 was mixed-fed. In probiotic group 4 of 5, 3 were formula-fed and 2 were mixed-fed.

[0067] Table 5. Cholestasis - Blood Indicators

[0068] Note: a, b, and c are significance markers, and the p-value shows the significance of the change between 7 days and 0 days.

[0069] As shown in Table 5, probiotics significantly reduced serum total bilirubin (TBIL) and total bile acid (TBA) levels in newborns with cholestasis (P<0.01).

[0070] Comparison of serum direct bilirubin (DBIL) after 7 days of treatment revealed that the four probiotic groups were significantly lower than the control group (P<0.05), and probiotic group 1 was significantly lower than probiotic groups 2-4 (P<0.05); there was no significant difference among probiotic groups 2-4 (P>0.05).

[0071] Comparison of serum total bilirubin (TBIL) after 7 days of treatment revealed that the four probiotic groups had significantly lower levels compared to the control group (P<0.05), while there were no significant differences among the probiotic groups (P>0.05).

[0072] Comparison of total bile acid (TBA) levels in the blood after 7 days of treatment revealed that the levels in the four probiotic groups were significantly lower than those in the control group (P<0.05). Among them, probiotic group 1 was close to the normal value and significantly lower than probiotic groups 2-4 (P<0.05), while there was no significant difference among probiotic groups 2-4 (P>0.05).

[0073] Overall, all groups, including the control group, showed significant reductions after 7 days of treatment compared to day 0. The probiotic group showed highly significant reductions in serum direct bilirubin (DBIL), serum total bilirubin (TBIL), and total bile acids (TBA) (P<0.01).

[0074] Therefore, it can be concluded that supplementing newborns with cholestasis with a combination of 3 probiotic strains from probiotic group 1 during treatment can accelerate the improvement of the condition.

[0075] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A probiotic preparation for improving abnormal bile disorders in newborns, characterized in that, The probiotic preparation contains Lactobacillus fermentum (… Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp . longum BB536.

2. The probiotic preparation according to claim 1, characterized in that, The neonatal abnormal bile disorders include jaundice, hyperbilirubinemia, and / or cholestasis.

3. The probiotic preparation according to claim 1, characterized in that, The probiotic preparation contains *Lactobacillus fermentum* (Fermented Lactobacillus mucinus). Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp . longum The live bacteria ratio of BB536 is 2-8:1-5:1-5.

4. The probiotic preparation according to claim 1, characterized in that, The probiotic preparation contains *Lactobacillus fermentum* (Fermented Lactobacillus mucinus). Limosilactobacillus fermentum CECT5716, Bifidobacterium breve ( Bifidobacterium breve M-16V and Bifidobacterium longum subsp. ( Bifidobacterium longum subsp . longum The total number of live bacteria in BB536 is not less than 10 billion CFU.

5. The probiotic preparation according to claim 1, characterized in that, The probiotic preparation also contains fructooligosaccharides and / or resistant dextrin.

6. The probiotic preparation according to claim 1, characterized in that, The probiotic preparation is administered orally.

7. The probiotic preparation according to claim 1, characterized in that, The dosage forms of the probiotic preparations include capsules, tablets, or granules.

8. The probiotic preparation according to claim 1, characterized in that, The probiotic preparation also contains food-acceptable adjuvants, additives, fortifiers and / or raw materials.

9. The use of the probiotic preparation according to any one of claims 1-8 in the preparation of a medicament for the prevention and / or improvement of abnormal bile disorders in newborns.

10. The application according to claim 9, characterized in that, The neonatal abnormal bile disorders include jaundice, hyperbilirubinemia, and / or cholestasis.

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