Lactobacillus paracasei HNGD-AL1 for degrading sulfur-containing amino acid, microbial inoculum and application of lactobacillus paracasei HNGD-AL1 in improvement of ulcerative colitis

Abstract

The invention belongs to the field of microorganisms, and relates to lactobacillus paracasei. In particular discloses a lactobacillus paracasei strain HNGD-AL1, the taxonomic name is Lactobacillus paracasei, the lactobacillus paracasei strain HNGD-AL1 is preserved in China Center for Type Culture Collection on February 27, 2025, the preservation number is CCTCC M 2025303, and the preservation address is No. 299 on eight road in Wuchang District, Wuhan, Hubei. The method for degrading the sulfur-containing amino acid by adopting the lactobacillus paracasei is found for the first time, the effect of degrading 53.39% of the sulfur-containing amino acid can be achieved by culturing the probiotics in a fermentation culture medium for 24 hours, and the probiotics can be colonized in intestinal tracts and play a role in degrading the sulfur-containing amino acid in intestinal contents; the content of metabolite hydrogen sulfide of sulfur-containing amino acid in the intestinal tract is reduced by 60.92%, intestinal tract tissue damage, permeability and inflammatory response are reduced, and the purpose of improving ulcerative colitis is achieved. The preparation method has a wide application prospect in the aspects of preparation of low-sulfur-content amino acid foods and probiotic preparations for improving colitis.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and relates to a strain of Lactobacillus paracasei. Background Art

[0002] Sulfur-containing amino acids, including methionine, cysteine, and cystine, are abundant in food and participate in important metabolic pathways such as protein synthesis, redox homeostasis, transmethylation, transsulfurization pathways, energy metabolism, and immune regulation. Sulfur-containing amino acids in food, as organic sulfur compounds, are partially metabolized by intestinal microorganisms upon entering the intestine to produce hydrogen sulfide (H2S), a major source of H2S in the intestine. Studies have found that high concentrations of H2S in the intestine can induce intestinal epithelial cell toxicity, cause intestinal inflammation and decreased immune function, and lead to damage to the intestinal mucosal barrier function. Reducing sulfur-containing amino acid intake reduces sulfur levels in the intestine, which can reduce the abundance of H2S-producing bacteria, reduce H2S levels in the intestine, and improve intestinal mucosal barrier function. Sulfur-containing amino acids in natural foods are almost all present in a bound form within proteins, and currently there is no safe, healthy, and sustainable method to reduce their levels. Sulfur-containing amino acids in food proteins are digested and absorbed in the intestines. During this process, microorganisms that do not produce H2S can also degrade sulfur-containing amino acids and reduce their content in the intestinal environment, thereby reducing H2S levels and improving colitis. Lactic acid bacteria, a recognized safe type of intestinal probiotic, have benefits such as enhancing immunity, inhibiting bacteria, regulating intestinal flora, and improving inflammation and metabolism. Some lactic acid bacteria have been reported to be able to utilize sulfur-containing amino acids and produce sulfur-degrading enzymes. Although the application with publication number CN 116515676 A discloses Lactobacillus paracasei E10 that regulates intestinal inflammatory response, the strain relieves intestinal inflammatory response by reducing the content of intestinal sulfate-reducing bacteria; the application with publication number CN110892940A discloses a strain of Lactobacillus paracasei that reduces the level of proinflammatory factors IL-6 and / or TNF-α, increases the level of anti-inflammatory factor IL-10, and reduces colitis tissue damage; Patent CN2020113697086 discloses a strain of Lactobacillus paracasei that inhibits the adhesion of intestinal pathogens and the production of intestinal inflammatory factors, thereby alleviating the inflammatory response caused by ETECH10407. It can be seen that even probiotics of the same genus, when used for the same disease, have different therapeutic effects due to different effects.

[0003] Therefore, using lactic acid bacteria to degrade sulfur-containing amino acids in the intestines, thereby reducing the amount of sulfur-containing amino acids in the intestinal environment, may become a highly promising and effective approach to achieve numerous health benefits, including improving colitis and intestinal health. To further explore new, safe, efficient, and highly active lactic acid bacteria capable of colonizing the intestine and degrading sulfur-containing amino acids, our research team conducted in-depth research. Summary of the Invention

[0004] To solve the above technical problems, the present invention proposes a strain of Lactobacillus paracasei HNGD-AL1 that degrades sulfur-containing amino acids, a bacterial agent, and use thereof in improving ulcerative colitis.

[0005] The technical solution of the present invention is achieved as follows: A strain of Lactobacillus paracasei HNGD-AL1, taxonomic name: Lactobacillus paracasei , deposited in the China Center for Type Culture Collection on February 27, 2025, with the deposit number CCTCC M 2025303, and the deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0006] The Lactobacillus paracasei HNGD-AL1 of the present invention is isolated and screened from sausages in Hefei, Anhui Province. Its colony characteristics are as follows: the colony diameter is about 2-4 mm, the colony is convex, the colony is milky white, round, opaque, and has a smooth surface and irregular colony edges. It is a Gram-positive bacterium, and the bacterial body is observed to be short rod-shaped and single through a scanning electron microscope.

[0007] The present invention studies the application of Lactobacillus paracasei HNGD-AL1 in improving ulcerative colitis and requests protection: A strain of Lactobacillus paracasei HNGD-AL1 and a bacterial agent containing the Lactobacillus paracasei HNGD-AL1.

[0008] Application of the above-mentioned Lactobacillus paracasei HNGD-AL1 or the above-mentioned bacterial agent.

[0009] Furthermore, the above application is to improve ulcerative colitis in an organism. Specifically, the above Lactobacillus paracasei HNGD-AL1 improves colitis by reducing the content of H2S in the intestinal tract of the organism.

[0010] The Lactobacillus paracasei HNGD-AL1 or the bacterial agent can also reduce the content of sulfur-containing amino acids in colon contents. The above application is achieved through the intestinal colonization ability of Lactobacillus paracasei HNGD-AL1.

[0011] The above-mentioned organisms are mammals, such as humans, rodents and other mammals.

[0012] The Lactobacillus paracasei HNGD-AL1 or the bacterial agent can also be used to reduce sulfur-containing amino acids in food.

[0013] The present invention has the following beneficial effects: 1. The present invention screened a strain of Lactobacillus paracasei HNGD-AL1 (HNGD-AL1) with high degradation of sulfur-containing amino acids from sausages in Hefei, Anhui Province. Lactobacillus paracaseiThis bacterium not only exhibits excellent acid and bile tolerance, but also has antibacterial and gastrointestinal colonization potential. This strain, Lactobacillus paracasei HNGD-AL1, is highly efficient at degrading sulfur-containing amino acids. Further research has revealed that this strain also exhibits excellent acid and bile tolerance, as well as the potential to colonize the gastrointestinal tract. Furthermore, this is the first time that a method has been discovered and employed to degrade sulfur-containing amino acids using Lactobacillus paracasei. This strain, in turn, reduces sulfur amino acid levels in the intestine, potentially improving ulcerative colitis.

[0014] 2. The present invention provides a method for improving ulcerative colitis by reducing the levels of sulfur-containing amino acids in the intestinal environment using lactic acid bacteria. This probiotic, cultured in a fermentation medium for 24 hours, can achieve a 53.39% degradation rate of sulfur-containing amino acids. This reduces abnormally elevated sulfur-containing amino acid levels in the intestine, restoring them to normal levels. It also reduces the content of H2S, a metabolite of sulfur-containing amino acids, in the intestine by 60.92%, reducing intestinal tissue damage, permeability, and inflammatory responses, thereby improving ulcerative colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 The phylogenetic tree and morphological diagram of strain HNGD-AL1.

[0017] Figure 2 Shown are the growth curve and acid production curve of strain HNGD-AL1 in MRS liquid medium as well as the optimal pH and temperature.

[0018] Figure 3 The effect of strain HNGD-AL1 on mouse body weight, disease activity index and colon tissue.

[0019] Figure 4 This is the effect of strain HNGD-AL1 on intestinal permeability in mice.

[0020] Figure 5 This is the effect of strain HNGD-AL1 on the levels of inflammatory factors in mouse colon tissue.

[0021] Figure 6 The effect of strain HNGD-AL1 on the content of sulfur-containing amino acids and H2S in the colon contents of mice. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0024] Example 1: Strain screening and identification (1) Isolation and purification of strains Ten grams of sausage from Hefei, Anhui Province, was added to 100 mL of sterile PBS buffer and shaken at 37°C, 150 rpm, for 2 hours. Then, 10 mL of the solution was diluted serially with PBS buffer. The diluted solution was evenly spread onto MRS solid selective medium containing 0.4 g / L bromocresol purple and incubated at 37°C for 72 hours. Plates with dispersed and diverse colonies were selected, and strains with distinct morphologies that produced yellow transparent circles were selected and streaked until free of contaminants. Strains that were Gram-positive and catalase-negative as determined by Gram staining and catalase assays were then inoculated into MRS liquid medium. After incubation for 24 hours, the supernatant was centrifuged at 8000 rpm for 15 minutes, and the sulfur-containing amino acid content was determined. Results Compared with the uninoculated MRS solution, the methionine content, cysteine content, and total sulfur-containing amino acid content of the solution inoculated with HNGD-AL1 strain decreased by 50.31%, 55.35%, and 54.51%, respectively.

[0025] (2) Morphological identification After activating strain HNGD-AL1 for three generations stored at -80°C, streak the cells onto MRS solid medium and incubate at 37°C for 48 hours before observing the colony morphology. Then, select the colonies for Gram staining and observe the cell morphology under a microscope.

[0026] Colony morphology: The colony diameter is about 2-4 mm, the colony is raised, the colony is milky white, round, opaque, with a smooth surface and irregular colony edges. It is a Gram-positive bacterium. Scanning electron microscopy showed that the bacteria were short rod-shaped, single ( Figure 1 ).

[0027] (3) 16S rRNA identification After activation for three generations, the cells were inoculated into MRS liquid medium and cultured at 37°C for 18 h. The bacteria were collected and the genomic DNA of strain HNGD-AL1 was extracted using the Ezup column-type bacterial genomic DNA extraction kit (Sanggong). PCR amplification was performed using the genomic DNA of strain HNGD-AL1 as a template with 16S rRNA universal primers 27F and 1492R.

[0028] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0029] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 60 s; and extension at 72°C for 10 min.

[0030] The purity and size of the amplified products were detected by electrophoresis, and the PCR products with the correct amplified length were sent to Shanghai Bioengineering for sequencing.

[0031] The 16S rRNA gene sequence of strain HNGD-AL1 obtained by sequencing has 1531 bases, such as Figure 1 As shown, the strain HNGD-AL1 has the closest homology to Lactobacillus paracasei, and the strain HNGD-AL1 was identified as Lactobacillus paracasei ( Lactobacillus paracasei ), and was deposited on February 27, 2025 in the China Center for Type Culture Collection, China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Postal Code 430072; the deposit number is CCTCC M 2025303.

[0032] Example 2: Study on strain characteristics (1) Drawing of strain growth curve The strain after activation for 3 generations was inoculated into MRS liquid medium with an inoculation loop, and the MRS liquid medium without strain was used as blank. The OD was measured every 2 h. 600 , three times in parallel. The results are as follows Figure 2 As shown in the figure, under static culture conditions at 37°C, the strain was in the lag phase from 0 to 10 h, entered the exponential growth phase after 10 h, and entered the stationary growth phase after about 25 h.

[0033] (2) Drawing of the optimal pH and temperature curves of the strain After the strain was activated, it was added to MRS broth at an inoculum volume of 0.01% (v / v) with an initial pH of 6.5. The temperatures were set at 27°C, 32°C, 37°C, 42°C, and 47°C for 12 hours. Finally, the OD was measured.600 To determine the growth of the strain under different temperature conditions. 600 Similarly, to determine the optimal pH value, 0.01% (v / v) inoculum of the strain was added to MRS broth and grown at pH 5, 6, 7, 8, and 9 for 12 h, and the OD was measured. 600 To determine the growth of the strain under different pH conditions. 600 The pH value of the value was determined as the optimal pH. Figure 2 As shown, the optimum temperature is 32°C and the optimum pH is 7.

[0034] (3) Acid and bile salt resistance test Preparation of artificial gastric juice: A pepsin solution (1:2500) with a concentration of 9 mg / mL was prepared in 0.01 M PBS buffer. The pH was then adjusted to 3.0 with 1 M HCl solution and sterilized by filtration through a 0.22 μm microporous membrane.

[0035] Preparation of artificial intestinal fluid: A trypsin enzyme solution with a concentration of 1 mg / mL (1:300) was prepared in 0.01 M PBS buffer. 0.3% (m / v) ox bile salt was then added. The pH was adjusted to 8.0 with 1 M HCl solution and sterilized by filtration through a 0.22 μm microporous membrane.

[0036] After activation for three passages, strain HNGD-AL1 was inoculated into MRS liquid medium and cultured at 37°C for 18 hours. Five mL of the bacterial culture was collected by centrifugation (6000 rpm for 10 minutes) and washed twice with equal volumes of 0.01 M PBS buffer. The washed bacteria were incubated with equal volumes of artificial gastric and intestinal fluid for 0 and 4 hours. The treated bacteria were serially diluted with PBS buffer, and the number of viable cells was determined by plate count. Plates containing 30 to 300 colonies were selected for count.

[0037] Survival rate = (number of viable bacteria after 4 h of treatment) / (number of viable bacteria after 0 h of treatment) × 100%.

[0038] Result analysis: The survival rates of strain HNGD-AL1 after 4 hours of treatment in artificial gastric fluid and artificial intestinal fluid are as follows: This indicates that strain HNGD-AL1 has good resistance to acid and bile salt solutions and has the potential to survive in the gastrointestinal tract.

[0039] (4) Cell hydrophobicity and self-aggregation ability test After activating the strain HNGD-AL1 for three generations, it was inoculated into MRS liquid medium and cultured at 37°C for 18 h. 5 mL of bacterial solution was collected by centrifugation (8000 r / min, 10 min), washed twice with an equal volume of 0.01 M PBS buffer, and then resuspended in PBS buffer to a concentration of OD 0. 600 The value is around 0.8, recorded as A0, and prepared into a lactic acid bacteria suspension.

[0040] Add 1 mL of chloroform to 3 mL of the lactic acid bacteria suspension and mix thoroughly. Then, let the mixture stand at room temperature for 20 minutes to allow the organic and aqueous phases to separate. Remove the organic phase and measure the OD value of the aqueous phase at 600 nm using PBS buffer as a blank. This value is recorded as A1. Repeat the experiment three times.

[0041] Cell hydrophobicity = [(A0-A1) / A0]×100%.

[0042] Take 4 mL of the lactic acid bacteria suspension and incubate at room temperature for 5 h. Then carefully aspirate 1 mL of the top layer of the suspension. Measure the OD value at 600 nm using PBS buffer as a blank and record it as A2. Repeat the experiment three times.

[0043] Cell self-aggregation ability = (1-A2 / A0) × 100%.

[0044] Result analysis: The cell hydrophobicity and self-aggregation ability of strain HNGD-AL1 are as follows: Cell hydrophobicity is a prerequisite for probiotics to adhere to intestinal epithelial cells and colonize the gastrointestinal tract to exert their beneficial effects. Cell self-aggregation can be used to assess the ability to adhere to intestinal cells and avoid pathogen colonization.

[0045] The results showed that strain HNGD-AL1 had good hydrophobicity and self-aggregation ability, and had the potential to colonize the intestine and inhibit pathogenic bacteria.

[0046] Example 3: Effect of Lactobacillus paracasei HNGD-AL1 on improving ulcerative colitis (1) Animal husbandry Forty 6-week-old male C57BL / 6 mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. and housed under illumination conditions of 25°C, 55% humidity, and a 12-h light / dark cycle.

[0047] (2) Experimental process The mice were randomly divided into 4 groups (CON-S group, CON-AL1 group, DSS-S group and DSS-AL1 group), with 10 mice in each group, and all were continuously fed with normal feed. The mice in CON-AL1 group and DSS-AL1 group were gavaged with Lactobacillus paracasei HNGD-AL1 (dose of 10 8 CFU / mL), 200 μL / mouse / day; mice in the CON-S and DSS-S groups were gavaged with an equal volume of normal saline. They were housed for 36 consecutive days. During the experiment, the CON-S and CON-AL1 groups drank distilled water. The DSS-S and DSS-AL1 groups were initially given distilled water for 7 days, followed by 5 days of distilled water containing 2% DSS (dextran sulfate sodium) for three consecutive days (to induce chronic colitis). Mouse body weights were recorded, and the weight change from the start to the end of the experiment was calculated. At the end of the experiment, mice were sacrificed by dislocation after eye extraction and blood collection. The colon was dissected and its length was measured, followed by hematoxylin and eosin staining.

[0048] (3) Weight record The results are as follows Figure 3 As shown in the data, at the end of the experiment, there was no significant difference in body weight and weight change between the CON-S group and the CON-AL1 group; compared with the DSS-S group, the body weight and weight change in the DSS-AL1 group were significantly increased, indicating that Lactobacillus paracasei HNGD-AL1 significantly improved the weight loss of mice with ulcerative colitis.

[0049] (4) Disease activity index score The disease activity index (DAI) of mice was calculated according to the scoring criteria set below.

[0050] The results are as follows Figure 3 As shown, at the end of the experiment, after three interventions, the disease activity index of mice in the CON-S and CON-AL1 groups remained unchanged. However, compared with the DSS-S group, the disease activity index of the DSS-AL1 group was significantly reduced, indicating that Lactobacillus paracasei HNGD-AL1 has an ameliorative effect on ulcerative colitis.

[0051] (5) Colon histopathological analysis The results are as follows Figure 3As shown, at the end of the experiment, there was no significant difference in colon length between the CON-S and CON-AL1 groups; however, the colon length of the DSS-AL1 group was significantly increased compared to the DSS-S group. The colonic tissue morphology of the CON-S and CON-AL1 groups was good, with intact crypt structure, numerous well-arranged goblet cells, and no inflammatory cell infiltration. In the DSS-S group, crypt structure was disrupted, the number of goblet cells was significantly reduced, and inflammatory cell infiltration was observed. Compared with the DSS-S group, the DSS-AL1 group showed visible crypt structure, restored goblet cell numbers, and reduced inflammatory cell infiltration. These results suggest that Lactobacillus paracasei HNGD-AL1 can alleviate colonic tissue damage in mice with ulcerative colitis.

[0052] (6) Intestinal permeability measurement Serum lipopolysaccharide (LPS), D-lactic acid (D-LA), and diamine oxidase (DAO) were determined according to the kit instructions. Figure 4 As shown, there was no significant difference in the levels of LPS, D-LA, and DAO in the serum of mice between the CON-S and CON-AL1 groups. However, the levels of LPS, D-LA, and DAO in the serum of the DSS-AL1 group were significantly lower than those in the DSS-S group. These results suggest that Lactobacillus paracasei HNGD-AL1 can alleviate ulcerative colitis by improving intestinal permeability.

[0053] (7) Determination of inflammatory factors The mouse colon tissue was thoroughly ground, and the inflammatory factors interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) in the tissue homogenate were determined according to the kit instructions. Figure 5 As shown, there was no significant difference in the levels of IL-6, IL-1β, TNF-α, and IL-10 in the colon of mice between the CON-S and CON-AL1 groups. Compared with the DSS-S group, the levels of IL-6, IL-1β, and TNF-α in the colon of the DSS-AL1 group were significantly reduced, while the level of IL-10 in the colon was significantly increased. These results suggest that Lactobacillus paracasei HNGD-AL1 can alleviate ulcerative colitis by reducing the levels of pro-inflammatory factors and increasing the levels of anti-inflammatory factors.

[0054] (8) Determination of methionine, cystine and H2S content in colon contents The contents of methionine and cystine in the colon contents of the CON-S, CON-AL1, DSS-S and DSS-AL1 groups were determined by amino acid analyzer after pretreatment according to GB5009.124-2016. Figure 6 As shown in the results, compared with the CON-S group, the contents of methionine and cystine in the colon contents of the CON-AL1 group were significantly decreased; compared with the DSS-S group, the contents of methionine and cystine in the colon contents of the DSS-AL1 group were significantly decreased.

[0055] Equal amounts of colon contents were taken from the CON-S, CON-AL1, DSS-S, and DSS-AL1 groups, respectively, and added to PBS buffer. 400 μM cysteine and 1 mM pyridoxal phosphate were then added and mixed evenly. 150 μL was added to a 96-well plate. Whatman quantitative filter paper was soaked in 20 mM lead acetate solution and dried, then placed on top of the plate. The plate was covered and incubated at 37°C for 18 h. The dark circles formed by lead sulfide on the filter paper were recorded using a camera, and then quantitative analysis was performed using ImageJ software. The results are shown in Figure 2. Figure 6 As shown in the results, compared with the CON-S group, the H2S content in the colon contents of the CON-AL1 group was reduced by 47.13%; compared with the DSS-S group, the H2S content in the colon contents of the DSS-AL1 group was reduced by 60.92%.

[0056] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus paracasei HNGD-AL1, whose taxonomic name is Lactobacillus paracasei , the deposit number is CCTCC M 2025303, and it was deposited in the China Center for Type Culture Collection on February 27, 2025. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A bacterial agent, characterized in that: The bacterial agent contains the Lactobacillus paracasei HNGD-AL1 according to claim 1.

3. The microbial agent according to claim 2, wherein: The concentration of Lactobacillus paracasei HNGD-AL1 in the bacterial agent is 10 7 ~10 9 CFU / mL.

4. Use of the Lactobacillus paracasei HNGD-AL1 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in reducing the sulfur-containing amino acids of a target substance.

5. The use according to claim 4, characterized in that: The target object is food or an organism.

6. The use according to claim 4, characterized in that: When the target object is an organism, the purpose of degrading sulfur-containing amino acids in the intestine is achieved by colonizing the intestine.

7. The use according to claim 5 or 6, characterized in that: The organism is a mammal.

8. The use according to claim 5 or 6, characterized in that: The sulfur-containing amino acids include methionine, cystine and cysteine.

9. Use of the Lactobacillus paracasei HNGD-AL1 according to claim 1 or the bacterial agent according to any one of claims 2 to 3 in preparing a medicine for improving ulcerative colitis.

10. The use according to claim 9, characterized in that: The drug improves colitis by reducing the content of H2S in the intestine.

Images