Lactobacillus rhamnosus JM039 as well as composition and application thereof

Abstract

The invention belongs to the technical field of probiotic products, and particularly relates to lactobacillus rhamnosus JM039 as well as a composition and application thereof. The preservation number of the lactobacillus rhamnosus JM039 is GDMCC No: 64409. The lactobacillus rhamnosus JM039 is a lactobacillus rhamnosus strain. The composition is prepared from lactobacillus reuteri J1, lactobacillus gasseri JM1, lactobacillus paracasei JY062, lactobacillus paracasei with the preservation number of GDMCC.No.62990, lactobacillus plantarum JY039 and lactobacillus rhamnosus JM039, and is prepared from lactobacillus reuteri J1, lactobacillus gasseri JM1, lactobacillus paracasei JY062, lactobacillus paracasei with the preservation number of GDMCC.No.62990, lactobacillus plantarum The combined bacteria of the six probiotics can improve the levels of anti-inflammatory factors and intestinal epithelium tight junction proteins and reduce the levels of proinflammatory factors and claudin-2 mRNA, so that the intestinal microenvironment homeostasis and intestinal barrier integrity are maintained through double regulation of inflammatory factors and tight junction proteins, and necrotizing enterocolitis can be relieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotic products, and specifically relates to a Lactobacillus rhamnosus JM039 and a composition and application thereof. Background Art

[0002] The intestinal microenvironment is composed of intestinal epithelial cells, the local immune system, and local intestinal microorganisms. The three interact and complement each other, and jointly determine the homeostasis of the intestinal microenvironment. Among them, intestinal epithelial cells constitute the physical barrier of the intestine. This physical barrier includes the tight junction structure between intestinal epithelial cells, which plays an important role in maintaining the polarity of epithelial cells and regulating the permeability of the intestinal barrier. The local intestinal immune system includes immune cells and immune factors, which can protect the body from invasion by foreign pathogens. Intestinal inflammation and infection can cause damage to the intestinal physical barrier, and pathogens can easily enter the blood circulation system. Intestinal microorganisms participate in food digestion and absorption, and also play a protective role on intestinal epithelial cells through the inhibition of harmful bacteria by probiotics. In the maintenance of intestinal health, although simple antibiotics can kill harmful bacteria, they can also cause local flora disorders and affect intestinal health; long-term use of anti-inflammatory drugs can also inhibit the function of the immune system and destroy the homeostasis of the intestinal microenvironment. Therefore, imbalance in the intestinal microenvironment is associated with a variety of major diseases, including intestinal inflammation, atherosclerosis, etc. (Liu Xiaowei, Sun Ruijuan, Dong Erdan, et al. Current status and trends in research on intestinal homeostasis and related diseases. Advances in Physiological Sciences, 2013, 44(3): 206-212.).

[0003] Probiotics are currently one of the means to regulate the homeostasis of the intestinal microenvironment, which can make up for the shortcomings of chemical drugs such as antibiotics and form a beneficial protective effect on the intestine. For example, CN106399196A (a strain of Lactobacillus plantarum hew-a490 and its application) and CN108712906A respectively disclose one or several microbial products for regulating the homeostasis of the intestinal microenvironment, aiming to reduce the uncontrolled intestinal uptake of antigens, pro-inflammatory agents, bacteria or bacterial products, provide anti-inflammatory activity on the intestinal surface, and increase the intestinal barrier function.

[0004] However, the complexity of the intestinal microecology and the complexity of the biological characteristics and behaviors of probiotics have caused uncertainty and unpredictability in the development of probiotic intestinal microenvironment regulation products. More ideal probiotic intestinal microenvironment regulation products still need to be continuously investigated and developed. Summary of the invention

[0005] In view of the above problems, the object of the present invention is to provide a Lactobacillus rhamnosus JM039 and a composition and application thereof.

[0006] In view of the above purpose, the present invention first provides a Lactobacillus rhamnosus JM039. The strain is selected from the intestinal flora of infants and young children, and the preservation unit is Guangdong Microbiological Culture Collection Center, the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the preservation number is GDMCC No.: 64409, and the preservation date is March 11, 2024. It is identified and classified as Lactobacillus rhamnosus.

[0007] The present invention further provides a rhamnosus lactobacillus composition for regulating the homeostasis of intestinal microenvironment. The rhamnosus lactobacillus composition contains Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, Lactobacillus rhamnosus JM039 and auxiliary materials.

[0008] The Lactobacillus reuteri J1 is screened from traditional fermented dairy products in Tibet, China and preserved by Northeast Agricultural University. Its information is recorded in the public literature: Zhang Ziwei. Preliminary evaluation and genomics study of the probiotic properties of Lactobacillus reuteri J1 [D]. Northeast Agricultural University, 2019; Zhang Ziwei, Li Xuesong, Sun Linlin, et al. Evaluation of gastrointestinal tolerance of Lactobacillus reuteri J1 and in vitro study of its regulation of immune-related genes [J]. Food Industry Science and Technology, 2019, 40(18):7.

[0009] The Lactobacillus gasseri JM1 is isolated from healthy infant feces and preserved by Northeast Agricultural University; its information is recorded in: Sun Linlin. Research on the immunoregulatory effect of a strain of Lactobacillus gasseri and its regulatory pathway [D]. Northeast Agricultural University, 2020; CN202310047780.4 and other public documents.

[0010] The Lactobacillus paracasei JY062, namely Lactobacillus paracasei TD062, is isolated from traditional Tibetan fermented milk and preserved by Northeast Agricultural University; its information is recorded in: Zhang Yu, Zhao Jiayuan, Zheng Jiapeng, et al. Evaluation of adhesion and tolerance of a strain of high-yield extracellular polysaccharide-lowering Lactobacillus paracasei JY062 (TD062) [J]. China Dairy Industry, 2022, 50(4): 4-8, 13; CN202211072208.5 and other public documents.

[0011] The plant lactobacillus JY039 is isolated from traditional Tibetan fermented milk and preserved by Northeast Agricultural University; its information is recorded in: Zhao J, Wang L, Cheng S, et al. A Potential Synbiotic Strategy for the Prevention of Type 2 Diabetes: Lactobacillus paracasei JY062 and Exopolysaccharide Isolated from Lactobacillus plantarum JY039 [J]. Nutrients 2022, 14, 377.

[0012] In a preferred embodiment, the probiotic composition is a liquid preparation, in which the viable cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-9): (1-9): (1-9): (1-9): (1-9). Further preferably, the viable cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-2): (1-2): (1-2): (1-2): (1-2): (1-2). Still further preferably, the ratio of the viable cell counts of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is 1:1:1:1:1:1:1.

[0013] Preferably, in the liquid preparation, the viable counts of Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 are all ≥10 8 CFU / mL.

[0014] Preferably, in the liquid preparation, the viable counts of Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 are all ≥10 10 CFU / mL.

[0015] In a preferred embodiment, the probiotic composition is a solid preparation, and the auxiliary material includes a lyophilization protective agent; in the solid preparation, the viable cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-9): (1-9): (1-9): (1-9): (1-9). Further preferably, the viable cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-2): (1-2): (1-2): (1-2): (1-2): (1-2). Still further preferably, the ratio of the viable cell counts of Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei JY091, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is 1:1:1:1:1:1:1.

[0016] Preferably, the lyoprotectant is selected from at least one of skim milk, sucrose, trehalose, dextran, cysteine ​​and xylan.

[0017] Preferably, the solid preparation is one of tablets, capsules and powders. Further preferably, the capsule is an enteric-coated capsule.

[0018] Preferably, the probiotic composition may further contain dietary fiber and / or flavoring agents, including sweeteners and fruit flavors.

[0019] Studies have shown that the combination of the six probiotics has a better regulatory effect on the intestinal microenvironment homeostasis, thereby protecting the intestinal barrier. Therefore, another aspect of the present invention provides the use of the probiotic composition for regulating the intestinal microenvironment homeostasis in the preparation of an oral regulator for the intestinal microenvironment homeostasis of infants and young children.

[0020] Preferably, the oral regulator of intestinal microenvironment homeostasis in infants is an oral regulator of intestinal microenvironment homeostasis for preventing or improving necrotizing enterocolitis in infants. Necrotizing enterocolitis in infants is an acquired disease that mainly occurs in premature infants or sick newborns, characterized by necrosis of the mucosa or even the deep intestinal layer, mainly occurring in premature infants or sick newborns; clinically, it is mainly characterized by sudden onset, abdominal pain, diarrhea, and bloody stools.

[0021] The aforementioned Lactobacillus paracasei with the accession number of GDMCC.No.62990 was isolated from traditional fermented dairy products in Tibet by Northeast Agricultural University and named Lactobacillus paracasei JY091 (Lacticaseibacillus paracaseiJY091). The depository is Guangdong Microbiological Culture Collection Center; the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the deposit date is November 21, 2022. It is identified and classified as Lactobacillus paracasei.

[0022] Studies have shown that the present invention has the following beneficial effects:

[0023] 1. The Lactobacillus rhamnosus JM039 and the probiotic composition of the present invention can inhibit the levels of local intestinal inflammatory factors IL-1β, IL-6, and TNF-α, and increase the level of local intestinal anti-inflammatory factor IL-10, having a dual regulatory effect;

[0024] 2. The Lactobacillus rhamnosus JM039 and the probiotic composition of the present invention can increase the expression of intestinal epithelial tight junction-related proteins and RNA, including Claudin-1, Occludin, MUC2, and ZO-1; and reduce the expression of Claudin-2, thereby having a dual regulatory effect on intestinal epithelial tight junction-related proteins;

[0025] 3. The Lactobacillus rhamnosus JM039 and the probiotic composition of the present invention can play a protective role on the intestinal barrier through dual regulation of anti-inflammatory / pro-inflammatory factors and regulation of intestinal epithelial tight junction-related proteins and RNA, thereby preventing and alleviating symptoms of necrotizing enterocolitis, such as diarrhea, loose stools, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the determination of inflammatory cytokine levels in the colon tissues of the offspring mice in each group in Example 2;

[0027] Figure 2 The results of the determination of tight junction protein and mucin content in the colon tissue of each group of mice in Example 2;

[0028] Figure 3 The results of measuring the mRNA expression levels of tight junction proteins and mucins in the colon tissues of the offspring mice in each group in Example 2;

[0029] Among them, J1 is Lactobacillus reuteri J1; JM1 is Lactobacillus gasseri JM1; JY062 is Lactobacillus paracasei JY062; JY091 is Lactobacillus paracasei JY091; JY039 is Lactobacillus paracasei with a preservation number of GDMCC.No.62990; JM039 is Lactobacillus rhamnosus JM039; and F is the composite probiotic of the present invention. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments are only used to explain the present invention and are not used to limit the scope of protection of the present invention.

[0031] Example 1: Strain activation and preparation of solid or liquid probiotic composition

[0032] 1.1 Source of strains:

[0033] Lactobacillus reuteri J1: Screened from traditional fermented dairy products in Tibet, China, and preserved by Northeast Agricultural University; its information is recorded in the literature: Zhang Ziwei. Preliminary evaluation of the probiotic properties of Lactobacillus reuteri J1 and genomics research [D]. Northeast Agricultural University, 2019; Zhang Ziwei, Li Xuesong, Sun Linlin, et al. Evaluation of gastrointestinal tolerance of Lactobacillus reuteri J1 and in vitro study of its regulation of immune-related genes [J]. Food Industry Science and Technology, 2019, 40(18):7.

[0034] Lactobacillus gasseri JM1: screened from healthy infant feces and preserved by Northeast Agricultural University; its information is recorded in: Sun Linlin. Study on the immunomodulatory effect of a strain of Lactobacillus gasseri and its regulatory pathways [D]. Northeast Agricultural University, 2020.

[0035] Lactobacillus paracasei JY062, also known as Lactobacillus paracasei TD062: screened from traditional Tibetan fermented milk and preserved by Northeast Agricultural University; its information is recorded in the literature: Zhang Yu, Zhao Jiayuan, Zheng Jiapeng, et al. Evaluation of the adhesion and tolerance of a high-yield extracellular polysaccharide-lowering Lactobacillus paracasei JY062 (TD062) [J]. China Dairy Industry, 2022, 50(4): 4-8, 13.

[0036] Lactobacillus paracasei with the accession number of GDMCC.No.62990 (represented by Lactobacillus paracasei JY091 in the following examples): isolated from traditional Tibetan fermented dairy products.

[0037] Lactobacillus plantarum JY039: screened from traditional Tibetan fermented milk and preserved by Northeast Agricultural University; its information is recorded in the literature: Zhao J, Wang L, Cheng S, et al. A Potential Synbiotic Strategy for the Prevention of Type 2 Diabetes: Lactobacillus paracasei JY062 and Exopolysaccharide Isolated from Lactobacillus plantarum JY039[J]. Nutrients 2022, 14, 377.

[0038] Lactobacillus rhamnosus JM039: screened from the intestinal flora of infants and young children, preserved in Guangdong Provincial Microbiological Culture Collection Center, with the preservation number GDMCC No: 64409 and the preservation date of March 11, 2024.

[0039] 1.2 Strain activation and preservation

[0040] Take out the frozen stock of the strain from the -80℃ refrigerator, inoculate the bacterial solution in MRS liquid medium at a 5% inoculation volume, and culture at 37℃ for 18h. Use a sterile inoculation loop to streak the bacterial solution on MRS solid medium, and culture at 37℃ for 48h. Pick a single colony and culture it in MRS liquid medium at 37℃ for 18h, and pass it for two generations; the bacterial solution can be prepared with 80% glycerol at a ratio of 3:1 and stored in a -80℃ refrigerator for use. Repeat the above steps for the activation of the remaining strains.

[0041] 1.3 Preparation of liquid probiotic composition

[0042] Take activated Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei JY091 (with a preservation number of GDMCC.No.62990), Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 (with a preservation number of GDMCCNo:64409) and mix them with physiological saline in a ratio of 1:1:1:1:1:1:1, 1:2:7:4:1:2, and 1:9:1:8:5:3 in terms of viable bacteria count, respectively, to prepare a liquid probiotic composition. Among the six liquid probiotic compositions, the minimum viable bacteria count is not less than 10 8For example, when Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei JY091 (with a deposit number of GDMCC.No.62990), Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 are compounded in a ratio of 1:9:1:8:5:3 to prepare a liquid probiotic composition, the viable counts of Lactobacillus reuteri J1 and Lactobacillus paracasei JY062 are not less than 10 8 CFU / mL.

[0043] 1.4 Preparation of solid probiotic composition

[0044] Take 1.3 parts of the liquid probiotic composition, add skim milk (15% by mass) and trehalose (10% by mass) as freeze-drying protectants according to part 1.3 of Example 1 of CN115011532A, and perform vacuum freeze-drying to prepare probiotic composition powder.

[0045] Example 2 Protective effect of liquid probiotic composition on intestinal microenvironment and intestinal mucosa in neonatal rats with necrotizing enterocolitis

[0046] Modeling method and observation indicators:

[0047] The animals used in this experiment were SD rats, 6 to 8 weeks old, 25 males and 25 females. After 7 days of adaptive feeding in the animal room, they were caged together and after 100 newborn rats were born, they were randomly divided into 10 groups, 10 rats in each group.

[0048] Except for the normal group, which was not given modeling at 48 hours after birth, the other 9 groups were treated with hypoxia and cold stimulation to construct a necrotizing enterocolitis model, that is, hypoxia treatment for 1 min at an oxygen concentration of less than 1% combined with 4°C stimulation for 10 min, twice a day for 4 consecutive days. During the modeling period, mothers and offspring were separated from each other from 9 to 18 o'clock every day, and the same milk powder was gavaged at 9, 14 and 18 o'clock. During the modeling period, the fecal characteristics, activity status and feeding conditions of the offspring were observed to determine the modeling situation (see references: Nolan LS, Gong Q, Hofmeister HN, et al. A protocol for the induction of experimental necrotizing enterocolitis in neonatal mice [J]. STAR protocols, 2021, 2 (4): 100951; Sharma S, Kanwar SS. Effect of prebiotics on growth behavior of Lactobacillus plantarum and their impact on adherence of strict anaerobic pathogens to intestinal cell lines [J]. Journal of Food Safety, 2018, 38 (1): e12384). The offspring in the model group showed symptoms such as decreased activity and slow response, intolerance during feeding, and changes in stool shape.

[0049] At the same time as modeling, drug treatment was started, once a day, with each gavage volume of 0.2 mL / mouse. The normal control group and the model group were given 0.2 mL of normal saline by gavage as a control; the positive control group was given penicillin combined with aminoglycoside drugs [Azathioprine (Aza), injected at a dose of 3 ml / kg]; the remaining 7 experimental groups were given corresponding drug treatments according to the grouping in Table 1. After the modeling was completed for 4 days, drug treatment was continued for 3 days until death.

[0050] During the experiment, the stool shape of each group of mice was observed and the stool was scored. Stool looseness level: As = normal stool 0 points, Bs = semi-solid stool 1 point, Cs = watery stool 2 points. The stool looseness score of each group is the average of the stool looseness score of each animal. The stool looseness score of each animal = As x N1 + Bs x N2 + Cs x N3, where As, Bs, and Cs are the stool looseness level scores, and N1, N2, and N3 are the days with the corresponding stool looseness level (the highest stool looseness level is recorded daily). The loose stool scores of the normal group, model group, positive control group, single bacteria group 1 (J1 group), single bacteria group 2 (JM1 group), single bacteria group 3 (JY062 group), single bacteria group 4 (JY091 group), single bacteria group 5 (JY039 group), single bacteria group 6 (JM039 group), and compound probiotics group (F group) were 0.2±0.42, 4.5±1.08, 0.7±0.82, 2.1±0.99, 5.1±2.33, 5.4±0.69, 2.3±0.94, 1.6±0.69, 2.0±0.94, and 0.9±0.73, respectively. The diarrhea scores of group F and positive control group were significantly lower than those of the model group and the single bacteria groups (p<0.05), the single bacteria group 3 was higher than the model group (p<0.05), and the diarrhea scores of single bacteria groups 1, 4, 5, and 6 were significantly lower than those of the model group.

[0051] Before killing, blood was routinely collected to separate serum and frozen for future use. After killing, intestinal tissue was routinely collected and frozen for future use.

[0052] Table 1 Grouping and gavage conditions of animal experiments

[0053]

[0054] The serum of the offspring mice was taken out from the -80℃ refrigerator and thawed. After adding the lysis solution, it was allowed to stand for 10 minutes. It was centrifuged at 2500rpm for 20 minutes and the supernatant was taken. The levels of inflammatory factors such as TNF-α, IL-1β, IL-10, and IL-6 in the serum of the offspring mice were determined according to the instructions of the ELISA kit.

[0055] The colon tissue of the offspring was taken out from the -80℃ freezer, ground into powder with liquid nitrogen, added with the lysis solution of the kit and allowed to stand for 10 minutes, centrifuged at 2500rpm for 20 minutes, and the supernatant was collected. The content of tight junction proteins such as MUC2, Occludin, ZO-1, and Claudin-1 of the offspring was determined according to the instructions of the ELISA kit.

[0056] Gene-specific primers were designed using Primer 5.0 software (Table 2) and TB PremixExTaq TM II kit to prepare cDNA Real-time RT-PCR reaction system. 3 system for real-time RT-PCR reaction. The RT-PCR conditions are shown in Table 3. The reaction system and reaction procedure are shown in Table 3. GAPDH was used as the internal reference gene and 2 -ΔΔCt Methods To characterize the relative expression of the target gene, three parallel experiments were set up.

[0057] Table 2 RT-PCR primer information

[0058]

[0059] Table 3 RT-PCR conditions

[0060]

[0061] Observation results of each group:

[0062] In the following experimental results, serum indexes and protein and mRNA detection results are expressed as means and standard deviations. ANOVA analysis was used to compare data among multiple groups, and p<0.05 was considered to be statistically significant.

[0063] Results of serum inflammatory factor content determination in neonatal rats:

[0064] The effects of the six-strain probiotic composition on necrotizing enterocolitis were reflected by detecting the levels of inflammatory factors in the serum of offspring mice. Figure 1 As shown, compared with the normal group, the model group TNF-α, IL-1β, IL-6 significantly increased, and IL-10 significantly decreased (p<0.05). The positive control and each strain treatment group TNF-α, IL-1β, IL-6 significantly decreased, and IL-10 significantly increased (p<0.05). The effect of group F was more significant. Figure 1 D shows that the 6-strain probiotic composition can significantly increase the content of the anti-inflammatory factor IL-10, which is significantly higher than the positive control and single-bacteria treatment groups (p<0.05), but has no significant difference with the normal group. Figure 1 A to Figure 1 C It can be seen that the 6-strain probiotic composition can significantly reduce the content of pro-inflammatory factors TNF-α, IL-1β, and IL-6. Among them, the IL-6 content was significantly lower than that of the positive control and the single bacteria treatment group (p<0.05); IL-1β was significantly lower than that of the Lactobacillus reuteri J1, Lactobacillus paracasei JY062, Lactobacillus paracasei JY091, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 treatment groups (p<0.05); TNF-α was significantly lower than that of the Lactobacillus paracasei JY091 and Lactobacillus plantarum JY039 treatment groups (p<0.05), and there was no significant difference with the normal group.

[0065] The results showed that the 6-strain probiotic composition played a significant dual regulatory role in downregulating pro-inflammatory factors and upregulating the content of anti-inflammatory factors. The results showed that the 6-strain probiotic composition was ingested in the intestines of the offspring mice, mediating the differentiation of inflammatory factors, alleviating inflammation in the intestines of the offspring mice, and exerting the effect of alleviating necrotizing enterocolitis in the offspring mice through the dual regulatory effects of anti-inflammatory / pro-inflammatory effects. Lactobacillus rhamnosus JM039 also had a significant dual regulatory effect of downregulating pro-inflammatory factors and upregulating the content of anti-inflammatory factors.

[0066] Results of tight junction protein and mucin content determination in colon tissue of neonatal rats:

[0067] Several studies have shown that tight junction proteins between intestinal epithelial cells in mice with necrotizing enterocolitis are damaged, leading to increased intestinal permeability, which in turn allows pathogenic factors to enter the blood circulation through the intestine. This study measured the content of tight junction proteins and mucins (Claudin-1, Occludin, ZO-1, MUC2) in colon tissue of newborn rats with a necrotizing enterocolitis model. Figure 2 It can be seen that Claudin-1, Occludin, ZO-1, and MUC2 in the model group were significantly reduced, which is the reason for the increased intestinal permeability. Except for Lactobacillus plantarum JY039, Claudin-1 in the positive control and other strain treatment groups was significantly increased compared with the model group (p<0.05), among which the F group was higher than the positive control and other single bacteria treatment groups (p<0.05), and there was no significant difference with the normal group. MUC2 in the positive control and strain treatment groups was significantly increased compared with the model group, among which the F group was higher than other single bacteria treatment groups (p<0.05), and there was no significant difference with the positive control. Occludin in the positive control and strain treatment groups was significantly increased compared with the model group, among which the F group was higher than other single bacteria treatment groups (p<0.05), and there was no significant difference with the positive control. ZO-1 in the positive control and F group was higher than that in the model group and each single strain treatment group (p<0.05), and there was no significant difference between the F group and the positive control.

[0068] The increased expression of the above tight junction proteins is beneficial to improving the integrity of the intestinal barrier and reducing intestinal permeability, thereby inhibiting the entry of pathogenic factors into the blood circulation through the intestine.

[0069] Tight junction protein mRNA expression in colon tissue of neonatal rats:

[0070] Claudin-2 mediates the loss of intestinal barrier function, and overexpression of claudin-2 can aggravate colitis. The results of mRNA expression are shown in Figure 3. RT-PCR showed that compared with the normal group, the expression of Claudin-1, Occludin, ZO-1, and MUC2 mRNA in the model group was significantly decreased, while the expression of claudin-2 mRNA was significantly increased (p<0.05). Compared with the model group, the mRNA levels of Claudin-1, Occludin, ZO-1, and MUC2 in the positive control and strain treatment groups were significantly increased, while the expression of claudin-2 mRNA was significantly decreased (p<0.05). Among them, only the mRNA levels of Claudin-1, ZO-1, and MUC2 in group F were not significantly different from those in the positive control; the mRNA levels of Occludin in group F and Lactobacillus rhamnosus JM039 were not significantly different from those in the positive control; the mRNA levels of Claudin-2 in group F and Lactobacillus plantarum JY039 were not significantly different from those in the positive control.

[0071] The results showed that the intake of the 6-strain probiotic composition could not only upregulate Claudin-1, Occludin, ZO-1, and MUC2 mRNA, but also downregulate Claudin-2 mRNA. Through dual regulatory effects, it could improve the intestinal barrier function and alleviate necrotizing enterocolitis in mice. Lactobacillus rhamnosus JM039 also had dual regulatory effects of upregulating Claudin-1, Occludin, ZO-1, and MUC2 mRNA and downregulating Claudin-2 mRNA.

[0072] The examples of the present invention demonstrate that the 6-strain probiotic composition has a better effect on alleviating necrotizing enterocolitis in offspring mice than the single strains at the same concentration, and some indicators have no significant differences from the normal group and / or the positive control group. This indicates that the effect of the 6-strain probiotic composition on necrotizing enterocolitis in offspring mice is unexpectedly better than the single strains at the same concentration, and there is a complex synergistic effect between the strains in regulating necrotizing enterocolitis in offspring mice.

[0073] For those skilled in the art, under the technical principles of the present invention, the technical solutions of the present invention, such as the adjustment of the strain ratio, the preparation type or the adjustment of the auxiliary materials, do not deviate from the basic principles and inspirations of the present invention and should fall within the protection scope of the present invention.

Claims

1. A Lactobacillus rhamnosus JM039, characterized in that The preservation unit of the rhamnosus Lactobacillus JM039 is the Guangdong Provincial Microbiological Culture Collection Center, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; the preservation number is GDMCC No.: 64409, and the preservation date is March 11, 2024.

2. A composition containing Lactobacillus rhamnosus for regulating the homeostasis of intestinal microenvironment, characterized in that: The rhamnosus lactobacillus composition contains Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, Lactobacillus rhamnosus JM039 and auxiliary materials.

3. A Lactobacillus rhamnosus composition for regulating intestinal microenvironment homeostasis according to claim 2, characterized in that: The rhamnosus Lactobacillus composition is a liquid preparation. In the liquid preparation, the live cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-9):(1-9):(1-9):(1-9):(1-9):(1-9).

4. A Lactobacillus rhamnosus composition for regulating intestinal microenvironment homeostasis according to claim 3, characterized in that: In the liquid preparation, the live cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1~2):(1~2):(1~2):(1~2):(1~2):(1~2).

5. A Lactobacillus rhamnosus composition for regulating intestinal microenvironment homeostasis according to claim 4, characterized in that: In the liquid preparation, the ratio of the number of live bacteria of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is 1:1:1:1:1:1:

1.

6. The composition containing Lactobacillus rhamnosus for regulating the homeostasis of intestinal microenvironment according to claim 3, characterized in that: In the liquid preparation, the viable counts of Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 are all ≥10 8 CFU / mL.

7. The composition containing Lactobacillus rhamnosus for regulating the homeostasis of intestinal microenvironment according to claim 3, characterized in that: In the liquid preparation, the viable counts of Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 are all ≥10 10 CFU / mL.

8. The composition containing Lactobacillus rhamnosus for regulating the homeostasis of intestinal microenvironment according to claim 2, characterized in that: The rhamnosus Lactobacillus composition is a solid preparation, and the auxiliary material includes a lyophilization protective agent; in the solid preparation, the live cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1-9):(1-9):(1-9):(1-9):(1-9):(1-9).

9. The composition containing Lactobacillus rhamnosus for regulating the homeostasis of intestinal microenvironment according to claim 8, characterized in that: In the solid preparation, the live cell count ratio of the Lactobacillus reuteri J1, Lactobacillus gasseri JM1, Lactobacillus paracasei JY062, Lactobacillus paracasei with a preservation number of GDMCC.No.62990, Lactobacillus plantarum JY039, and Lactobacillus rhamnosus JM039 is: (1~2):(1~2):(1~2):(1~2):(1~2):(1~2).

10. Use of the Lactobacillus rhamnosus JM039 according to claim 1 or the Lactobacillus rhamnosus composition for regulating intestinal microenvironment homeostasis according to any one of claims 2 to 9 in the preparation of an oral regulator of intestinal microenvironment homeostasis in infants.

11. The use according to claim 10, characterized in that: The oral regulator of intestinal microenvironment homeostasis in infants and young children is an oral regulator of intestinal microenvironment homeostasis for preventing or improving necrotizing enterocolitis in infants and young children.

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