Strain for promoting development of infant intestine and application thereof

Abstract

The application discloses a strain for promoting the development of the intestinal tract of infants, and the strain is Lactobacillus paracasei ML-446, which is preserved in the China Center for Type Culture Collection on June 24, 2021, has a preservation number of CCTCC NO: M2021771, and is preserved in Wuhan, China, Wuhan University, and the taxonomic name of the strain is Lactobacillus paracasei ML-446. The strain is applied to functional food, and the intestinal tract homeostasis can be maintained and the development of the intestinal tract is promoted.

Description

Technical Field

[0001] This invention relates to the field of food health technology, and more specifically to a bacterial strain that promotes infant intestinal development and its application. Background Technology

[0002] Breast milk, as an infant's first food, has a significant impact on their health. Infants' gut microbiota is not fully established, and breast milk provides the microorganisms necessary for this development. Studies have shown that breast milk contains up to 100 microorganisms. 3 -10 4 CFU / mL. An infant ingests approximately 800 mL of milk per day, digesting about 1 × 10⁻⁶ CFU / mL. 5 ~1×10 7 CFU contains microorganisms. Breast milk helps infants gradually establish a stable gut microbiota. Clinical studies have shown that premature and low-birth-weight infants, whose gastrointestinal tracts are separated from the intrauterine environment too early, lack the nutrients, hormones, and growth factors necessary for growth and development, resulting in incomplete gastrointestinal function development, which poses a significant threat to infant health. Even in normal newborns, intestinal problems such as flatulence, diarrhea, and constipation are also present, which are closely related to incomplete intestinal development.

[0003] Currently, clinical practice often employs small, frequent meals, warm compresses, and the addition of probiotics to alleviate symptoms. For premature and low-birth-weight infants, antibiotics are used. However, for newborns, these medications have serious side effects and are not tolerated by everyone.

[0004] Recent studies have revealed a strong link between gut microbiota and intestinal development. Firstly, probiotics can inhibit apoptosis, promote cell proliferation, and maintain gut homeostasis. Experiments have shown that two proteins, p40 and p75, secreted by *Lactobacillus rhamnosus*, can effectively alleviate cytokine-induced apoptosis of intestinal epithelial cells by activating the epidermal growth factor receptor and its downstream Akt. Akt activation leads to the inactivation of apoptosis-promoting proteins, including caspase-3 and caspase-9, thereby improving cell viability. Other studies have shown that probiotics can stimulate intestinal epithelial cell proliferation by increasing R-spondins expression, thereby activating the Wnt / β-catenin pathway and repairing damaged intestinal mucosa. Secondly, probiotics also have a positive effect on regulating the function of tight junctions in the intestinal epithelium. Furthermore, probiotics can enhance the intestinal barrier function and maintain homeostasis by regulating the phosphorylation levels of the cytoskeleton and tight junction proteins.

[0005] Therefore, how to provide a strain that can maintain intestinal homeostasis and promote intestinal development and apply it to functional foods is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a strain of Lactobacillus paracasei ML-446, which can promote infant intestinal development and regulate intestinal homeostasis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A strain that promotes infant intestinal development, namely *Lactobacillus paracasei* ML-446, is deposited at the China Center for Type Culture Collection (CCTCC) on June 24, 2021, with accession number CCTCC NO: M 2021771, located at Wuhan University, Wuhan, China. The taxonomic name of the strain is... Lactobacillus paracasei ML-446.

[0009] The above-mentioned strains of bacteria promoting infant intestinal development are used in the preparation of functional foods that promote infant intestinal development and maintain intestinal homeostasis.

[0010] As a preferred embodiment of the above technical solution, the strain can promote the growth of intestinal villi and the proliferation of intestinal epithelial cells.

[0011] A functional food comprising the aforementioned Lactobacillus paracasei ML-446.

[0012] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a strain of Lactobacillus paracasei ML-446, which can promote infant intestinal development, regulate intestinal homeostasis, significantly promote the growth of intestinal villi, and promote the proliferation of intestinal epithelial cells. Its promotion may be related to upregulating the Wnt signaling pathway, inhibiting the Notch pathway, and promoting the proliferation of goblet cells and Paneth cells. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0014] Figure 1 The attached figure shows an HE staining image of rat pup intestinal tissue provided in Example 4;

[0015] Figure 2 The attached figure shows the immunoassay results of Ki67 intestinal tissue from rat pups provided in Example 4;

[0016] Figure 3 The attached figure is an immunohistochemical analysis diagram of the intestinal tissue of rat pups provided in Example 4;

[0017] Figure 4 The attached figure is an immunohistochemical analysis diagram of the intestinal tissue of rat pups provided in Example 4;

[0018] Figure 5 The attached figure is a PAS analysis diagram of the rat pup intestinal tissue provided in Example 4;

[0019] Figure 6 The attached figure is a PAS analysis diagram of the rat pup intestinal tissue provided in Example 4;

[0020] Figure 7 The attached figure shows the results of Western Blot analysis of WNT pathway expression in rat pups provided in Example 4;

[0021] Figure 8 The attached figure shows the results of Western Blot analysis of WNT pathway expression in rat pups provided in Example 4;

[0022] Figure 9 The attached figure shows the results of Western blotting notch pathway expression in rat pups provided in Example 4;

[0023] Figure 10 The attached figure shows the results of Western blotting notch pathway expression in rat pups provided in Example 4. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0025] Example 1: Screening of Lactobacillus paracasei ML-446 strain

[0026] This strain was isolated from breast milk. The specific method is as follows: Bacterial isolation was performed using a plating method. Breast milk samples were diluted 10-fold, and 100 μL was plated onto semi-selective culture plates using MRS-cys medium supplemented with X-gal. The plates were incubated at 37°C for 48 h, then allowed to stand in air for 4 h. Blue colonies were picked, purified, and identified for 16S rRNA. Genomic DNA was extracted from the bacteria using a kit. Then, using the genomic DNA as a template and universal primers as amplification primers, PCR was performed to amplify the 16S rDNA. After the reaction, the target fragment was confirmed by agarose gel electrophoresis, and the PCR amplification products were sent to Shanghai Sangon Biotech for sequencing. The sequence was submitted to the GenBank database, and a BLAST homology search was performed to find the 16S rDNA of a known taxonomic species with the highest homology to the target sequence. The bacteria were identified as *Lactobacillus paracasei* ML-446, which was deposited at the China Center for Type Culture Collection (CCTCC) on June 24, 2021, with accession number CCTCC NO: M 2021771, located at Wuhan University, Wuhan, China. The taxonomic name of the strain is... Lactobacillus paracasei ML-446.

[0027] Example 2: Preparation of Probiotic Suspension

[0028] The *Lactobacillus paracasei* ML-446 strain selected in Example 1 was inoculated into 10 ml of MRS liquid medium at a 2% (v / v) inoculum and cultured at 37°C for 24 h. The fermentation broth was centrifuged at 8000 r / min for 5 min at 4°C, the supernatant was discarded, and the bacterial resuspended in sterile PBS and washed twice. Finally, the bacterial concentration was adjusted to approximately 10⁻⁶ using the plate count method. 8 CFU / ml.

[0029] Example 3: Establishment of an intrauterine growth restriction model in rat pups

[0030] Intrauterine Growth Restriction (IUGR) Model in Pups: An IUGR model was established using a restricted diet during the mother's pregnancy. Sprague-Dawley pregnant rats were provided by Charles River Laboratory Animal Technology Co., Ltd., Beijing, China. Pregnant rats were housed individually. They were randomly divided into two groups: a normal diet group and a restricted diet group. Rats in the normal diet group were allowed unlimited food and water. From the first day of pregnancy to the day of parturition, rats in the restricted diet group received 50% of the food provided to the normal diet group. Both groups received adequate food and water on the day of parturition. IUGR was successfully established in newborn rats in the restricted diet group when their body weight was less than two standard deviations above the normal diet level.

[0031] Example 4: Verification of the promoting effect of ML-446 strain on intestinal development

[0032] The successfully modeled rat pups were divided into a negative control group (administered by gavage with 100 μL / 10 g PBS) and an experimental group (administered by gavage with 10 g / 10 g ML-446 strain suspension from Example 2). 8 (cfu / mL). During this period, the rats were allowed free access to breast milk. The rat pups were sacrificed by gavage after 10 days. Intestinal tissue was collected and stored at -80°C with tissue preservation solution for later use.

[0033] HE staining analysis of rat pup intestinal tissue

[0034] The jejunum, ileum, and colon of the pups were dehydrated with a gradient of alcohol and routinely embedded in paraffin. A 4 μm thick section of intestinal tissue was cut from each paraffin block, stained with hematoxylin and eosin (HE), and observed and photographed under an inverted microscope. Results are shown below. Figure 1 ;Depend on Figure 1 It is known that the addition of ML-446 has a certain promoting effect on the development of intestinal villi in young mice.

[0035] Immunohistochemical analysis of rat pup intestinal tissue

[0036] The jejunum, ileum, and colon of the pups were dehydrated with a gradient of alcohol and routinely embedded in paraffin. A 4 μm thick section of intestinal tissue was cut from each paraffin block and incubated with primary antibody (Ki67, lysozyme). The tissue was then evenly covered with proportionally diluted primary antibody and incubated overnight at 4°C. Secondary antibody incubation (HRP-goat anti-rabbit IgG) was performed. The sections were removed from the refrigerator and warmed to room temperature for 30 min, then washed three times with PBS for 5 min each. Proportionally diluted secondary antibody was added and incubated at room temperature for 50 min. The sections were washed three times with PBS for 5 min each. DAB staining and hematoxylin counterstaining were performed, and the results were observed and photographed under an inverted microscope. Results are shown below. Figure 2 , Figure 3 , Figure 4 ;Depend on Figure 2 It can be seen that in the ML-446 intervention group, the average optical density of Ki67 was significantly higher than that in the blank control group; from Figure 3 and Figure 4 It can be seen that the average optical density of Lysozyme in the jejunum and ileum of the ML-446 intervention group was higher than that in the blank control group.

[0037] PAS analysis of rat pup intestinal tissue

[0038] The jejunum, ileum, and colon of the pups were dehydrated with a gradient of alcohol and routinely embedded in paraffin. A 4 μm thick section of intestinal tissue was cut from each paraffin block, stained, and then stained with periodic acid solution for 15 min, followed by Schiff reagent (pre-warmed to room temperature) with a cap and protected from light for 30 min. The sections were rinsed with running water for 5 min, then stained with hematoxylin for 1 min, and washed with tap water. The sections were then differentiated in hematoxylin differentiation solution (1% hydrochloric acid alcohol) for 3 s, washed with tap water, and rinsed again with running water to achieve a blue reversal. The sections were observed and photographed under an inverted microscope.

[0039] See results Figure 5 and Figure 6 ;Depend on Figure 5 and Figure 6 It can be seen that the number of goblet cells in the jejunum, ileum and colon of the ML-446 intervention group was higher than that in the blank control group.

[0040] Western blot analysis of protein expression levels

[0041] The corresponding volume of lysed sample was then incubated on ice for 5 minutes. A low-temperature refrigerated centrifuge was then turned on and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was separated to obtain the protein extract. After protein extraction, protein quantification was performed by membrane transfer, blocking, incubation with primary and secondary antibodies, and finally analysis of the target bands using a gel imaging system. The results are shown in [Figure number missing]. Figure 7 , Figure 8 , Figure 9 and Figure 10 It can be seen that in the ML-446 intervention group, the expression of β-catenin protein in the jejunum, ileum and colon was higher than that in the control group; in the ML-446 intervention group, the expression of activated-notch protein in the jejunum and ileum was lower than that in the control group, while the expression of activated-notch protein in the colon was higher than that in the control group.

[0042] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0043] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A bacterial strain that promotes infant intestinal development, characterized in that, The strain is Lactobacillus paracasei ML-446, deposited at the China Center for Type Culture Collection (CCTCC) on June 24, 2021, with accession number CCTCC NO: M2021771, and the deposit address is Wuhan University, Wuhan, China.

2. The application of the strain that promotes infant intestinal development according to claim 1 in the preparation of functional foods that promote infant intestinal development and maintain intestinal homeostasis.

3. The application of the strain for promoting infant intestinal development according to claim 2 in the preparation of functional foods that promote infant intestinal development and maintain intestinal homeostasis, characterized in that, The strain can promote the growth of intestinal villi and the proliferation of intestinal epithelial cells.

4. A functional food, characterized in that, Includes the strain described in claim 1.

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