Lactobacillus casei as well as application and preparation thereof

Abstract

The invention provides a Lactobacillus casei MPEB0012108, which is preserved in the Guangdong Microbial Culture Collection Center (GDMCC), the preservation address is the 5th floor of the building 59, No.100 Courtyard, Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC No: 65113. The Lactobacillus casei MPEB0012108 is named as Lactobacillus casei MPEB0012108. The invention further provides a preparation. The preparation comprises the cheese cheese bacillus. The invention further provides an application of the cheese lactobacillus in preparation of probiotics capable of being administrated by intestines and stomach.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and more specifically to Lactobacillus casei and applications and preparations thereof. Background Art

[0002] Lactic acid bacteria are Gram-positive bacteria without spores and with low G+C content. They are widely found in food, environment, and the intestines of humans and animals. They are recognized as safe microorganisms and can be used as an alternative microecological preparation for livestock and poultry production. Lactic acid bacteria have important probiotic functions such as inhibiting the growth of pathogens, maintaining intestinal homeostasis, enhancing intestinal physical barriers, regulating the body's immunity and anti-tumor. The ability of lactic acid bacteria to colonize the intestine and tolerate the digestive tract environment is a necessary prerequisite for its probiotic properties.

[0003] Lactic acid bacteria grow anaerobically or facultatively anaerobically and can survive in acidic conditions of pH 3.0-4.5. In the animal intestine, it converts monosaccharides, especially lactose, into lactic acid, thereby lowering the intestinal pH, preventing foreign bacteria from colonizing the intestine, inhibiting the growth of pathogens such as Escherichia coli and Salmonella, and ultimately inhibiting the growth of lactic acid bacteria themselves. Lactic acid bacteria can also activate pepsin by regulating intestinal pH, promote gastrointestinal motility, help digestion and absorption of food, reduce flatulence and promote liver function. Summary of the invention

[0004] The invention provides a cheese Lactobacillus casei MPEB0012108, which is preserved in Guangdong Microbial Culture Collection Center (GDMCC), with a preservation address of 5th floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and a preservation number of GDMCC No: 65113.

[0005] The invention also provides a preparation comprising the above-mentioned Lactobacillus cheese.

[0006] The present invention also provides the use of the above Lactobacillus casei or the preparation in preparing probiotics that can be administered enterally.

[0007] Compared with the prior art, this application has the following beneficial effects:

[0008] (1) The Lacticaseibacillus casei MPEB0012108 of the present application was isolated from silage samples in Xinxiang City, Henan Province, and the strain has strong antioxidant capacity.

[0009] (2) The Lactobacillus casei MPEB0012108 of the present application has good bile salt tolerance, good gastrointestinal digestive juice tolerance, and strong survival ability in the human body.

[0010] (3) The Lacticaseibacillus casei MPEB0012108 of the present application has surface hydrophobicity, self-aggregation, and co-aggregation with pathogenic bacteria, and has intestinal adhesiveness.

[0011] (4) The Lacticaseibacillus casei MPEB0012108 of the present application has a strong antibacterial effect. Description of the Drawings

[0012] Figure 1 is the colony morphology of the Lacticaseibacillus casei MPEB0012108 of the present application on a solid medium.

[0013] Figure 2 is the Gram-stained cell morphology of the Lacticaseibacillus casei MPEB0012108 of the present application.

[0014] Figure 3 is the antibacterial result of the fermentation broth of the Lacticaseibacillus casei MPEB0012108 of the present application against Acinetobacter baumannii MPEB0012758.

[0015] Figure 4 is the antibacterial result of the fermentation broth of the Lacticaseibacillus casei MPEB0012108 of the present application against Salmonella enterica subsp. enterica MPEB0001613.

[0016] Figure 5 is the antibacterial result of the fermentation broth of the Lacticaseibacillus casei MPEB0012108 of the present application against Klebsiella pneumoniae MPEB0011218.

[0017] Figure 6 is the antibacterial result of the fermentation broth of the Lacticaseibacillus casei MPEB0012108 of the present application against Pseudomonas aeruginosa MPEB0011228.

[0018] Figure 7 is the antibacterial result of the fermentation broth of the Lacticaseibacillus casei MPEB0012108 of the present application against Staphylococcus gallinarum MPEB0009941.

[0019] Figure 8This is the antibacterial result of the fermentation liquid of Lactobacillus casei MPEB0012108 of the present application against Staphylococcus epidermidis MPEB0013698 (Staphylococcus epidermidis).

[0020] Figure 9 This is the antibacterial result of the fermentation liquid of Lactobacillus casei MPEB0012108 of the present application against Enterococcus faecium MPEB0011612 (Enterococcus faecium).

[0021] Figure 10 This is the antibacterial result of the fermentation liquid of Lactobacillus casei MPEB0012108 of the present application against Aeromonas hydrophila MPEB0012467 (Aeromonas hydrophila).

[0022] The cheese Lactobacillus casei (Lacticaseibacillus casei) MPEB0012108 provided by the present invention has a taxonomic name of Lacticaseibacillus casei, was deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on September 9, 2024, and the deposit number is: GDMCC No: 65113; the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0023] The following examples may enable those skilled in the art to more fully understand the present invention, but are not intended to limit the present invention in any way.

[0024] Lactobacillus casei is an important probiotic, which is widely used in dairy products, functional noodles, functional beverages, and improving feed digestibility and growth rate of livestock and poultry. Studies have shown that Lactobacillus casei can improve the intestinal microecological environment of animals, inhibit the reproduction of intestinal pathogens and improve the body's disease resistance. For example, adding Lactobacillus casei to feed can promote the increase of goblet cells, villus length and crypt depth in duck intestines; oral Lactobacillus casei has a good therapeutic effect on animals with colitis.

[0025] Reagents and culture medium involved in the embodiment:

[0026] MRS liquid culture medium (1L): 10.00g of peptone, 8.00g of beef extract powder, 4.00g of yeast extract powder, 20.00g of glucose, 2.00g of dimethyl hydrogen phosphate, 2.00g of diammonium hydrogen citrate, 5.00g of sodium acetate, 0.20g of magnesium sulfate, 0.04g of manganese sulfate, 1.00g of Tween, add 1000mL of ddH2O, pH 5.7±0.2, 121℃, high pressure sterilization for 20min for use.

[0027] MRS Solid Medium (1 L): Peptone 10.00 g, Beef Extract Powder 8.00 g, Yeast Extract Powder 4.00 g, Glucose 20.00 g, Dipotassium Hydrogen Phosphate 2.00 g, Diammonium Hydrogen Citrate 2.00 g, Sodium Acetate 5.00 g, Magnesium Sulfate 0.20 g, Manganese Sulfate 0.04 g, Tween 1.00 g, Agar 15.00 g. Add 1000 mL of ddH2O, adjust the pH to 5.7 ± 0.2, sterilize at 121 °C for 20 min, and reserve for later use.

[0028] Fermentation Medium (1 L): Peptone 10.00 g, Beef Extract Powder 8.00 g, Yeast Extract Powder 4.00 g, Glucose 20.00 g, Dipotassium Hydrogen Phosphate 2.00 g, Diammonium Hydrogen Citrate 2.00 g, Sodium Acetate 5.00 g, Magnesium Sulfate 0.20 g, Manganese Sulfate 0.04 g, Tween 1.00 g, Calcium Carbonate 0.005%, add 1000 mL of ddH2O, sterilize at 121 °C for 20 min, and reserve for later use.

[0029] YPD Liquid Medium (1 L): Peptone 10.00 g, Yeast Extract 5.00 g, Glucose 20.00 g. Add 1000 mL of ddH2O, sterilize at 105 °C for 45 min, and reserve for later use.

[0030] YPD Solid Medium (1 L): Peptone 10.00 g, Yeast Extract 5.00 g, Glucose 20.00 g, Agar 15.00 g. Add 1000 mL of ddH2O, sterilize at 105 °C for 45 min, and reserve for later use. (Add 0.05% CaCO3 as the medium for screening lactic acid strains)

[0031] MH Broth (1 L): Weigh 21.00 g of MH Broth, dissolve it in 1000 mL of ddH2O, sterilize at 121 °C for 20 min, and reserve for later use.

[0032] MH (A) Medium (1 L): Weigh 36.50 g of Mueller-Hinton Agar, add 1000 mL of ddH2O, sterilize at 121 °C for 20 min, then cool to 50 °C, pour the medium into plates, and after cooling and solidifying, invert and reserve for later use.

[0033] Simulated Gastric Juice: NaCl 0.2 g / 100 mL, Pepsin 0.35 g / 100 mL, adjust the pH to 3.0 with 1 mol / L HCl, filter and sterilize with a 0.22 μm filter membrane, and reserve for later use.

[0034] Simulated intestinal fluid: 1.1 g / 100 mL of NaHCO3, 0.2 g / 100 mL of NaCl, 0.1 g / 100 mL of Trypsin, 0.02 g / 100 mL of bovine bile salt, adjust the pH value to 8.0, filter and sterilize with a 0.22 μm filter membrane for standby.

[0035] Example 1

[0036] Isolation, purification and identification of Lacticaseibacillus casei MPEB0012108

[0037] The Lacticaseibacillus casei MPEB0012108 of the present application was isolated from a silage sample in Xinxiang City, Henan Province. The specific steps are as follows:

[0038] Collect silage samples from Xinxiang City, Henan Province, put them into a 1.5 mL centrifuge tube containing 900 μL of sterile distilled water, mix them with an oscillator, pipette 10 μL of the mixed liquid onto a YPD medium, streak plate, and culture in an incubator at 35 °C for 48 - 72 h, and observe the growth status of the colonies. Pick single colonies with different morphologies, sizes and colors and inoculate them into YPD liquid medium, place them in an incubator for constant temperature culture for 24 h, mix a part of the cultured strain with glycerol and store it in a cryotube, part of it is sub-packed and stored in a centrifuge tube, and part of it is subjected to 16s rRNA sequencing.

[0039] The Lacticaseibacillus casei MPEB0012108 of the present application is Gram-positive, showing white circular, smooth convex, neat edges and dull surface colonies on the plate. The colony morphology is shown in Figure 1 . The scanning electron microscope morphology of strain MPEB0012108 is shown in Figure 2 , and the bacterium is slender rod-shaped, but without branches and spores.

[0040] Example 2

[0041] Antioxidant analysis of strain MPEB0012108

[0042] 2.1 Hydroxyl radical scavenging activity of the fermentation broth of strain MPEB0012108

[0043] 5 mmol / L H2O2: Accurately weigh 0.0283 g of 30% H2O2, add distilled water to dissolve and make up the volume to 50 mL.

[0044] 5 mmol / L salicylic acid-ethanol solution: Accurately weigh 0.0345 g of salicylic acid, add absolute ethanol to dissolve and make up the volume to 50 mL.

[0045] 5 mmol / L FeSO4 solution: Accurately weigh 0.0695 g of FeSO4·7H2O, dissolve it in distilled water, and make up the volume to 50 mL.

[0046] Fermentation broth of strain MPEB0012108: Inoculate the seed liquid into the fermentation medium at an inoculation amount of 1% by volume, and carry out constant-temperature fermentation at 35 °C; centrifuge and filter the fermentation broth, and take the supernatant.

[0047] The reaction mixture contains 1 mL of FeSO4 (5 mmol / L), 1 mL of salicylic acid-ethanol solution (5 mmol / L), 1 mL of H2O2 (5 mmol / L), and 1 mL of the fermentation broth sample of strain MPEB0012108. After incubating at 37 °C for 30 min, measure the absorbance (A1) of the reaction mixture at 562 nm. The blank control measures the absorbance value (A0) by replacing the fermentation broth sample of strain MPEB0012108 with distilled water; the sample control measures the absorbance value (A2) by replacing the H2O2 solution (5 mmol / L) with distilled water. Each group of experiments is repeated three times. The hydroxyl radical scavenging rate is calculated as follows:

[0048] Hydroxyl radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0049] Where A0 is the absorbance of the control; A1 is the absorbance of the sample; A2 is the absorbance of the mixed solution without H2O2.

[0050] 2.2 DPPH radical scavenging activity of the fermentation broth of strain MPEB0012108

[0051] 0.2 mmol / L DPPH solution: Accurately weigh 0.0079 g of 2,2-diphenyl-1-picrylhydrazyl (DPPH), dissolve it in absolute ethanol, and make up the volume to 100 mL. Add 2 mL of the fermentation broth sample of strain MPEB0012108 and 2 mL of DPPH solution (0.2 mmol / L) to the measurement tube, add 2 mL of the fermentation broth sample of strain MPEB0012108 and 2 mL of absolute ethanol to the control tube, add 2 mL of distilled water and 2 mL of DPPH solution (0.2 mmol / L) to the blank tube, mix well, incubate in the dark at room temperature for 30 min, and measure the absorbance of the solution at 517 nm. Each group of experiments is repeated three times. The DPPH radical scavenging rate is calculated as follows:

[0052] DPPH radical scavenging rate (%) = [1 - (A1 - A2) / A0] × 100%

[0053] Where A0 is the absorbance of the blank tube; A1 is the absorbance of the measurement tube; A2 is the absorbance of the control tube.

[0054] Referring to Table 1, the results show that the scavenging rate of the fermentation broth of Lactobacillus casei MPEB0012108 against hydroxyl radicals is 92.95%, and the scavenging rate against DPPH radicals is 94.45%.

[0055] Table 1

[0056]

[0057] Example 3

[0058] Analysis of the bile salt tolerance of strain MPEB0012108

[0059] The lactic acid bacteria strain MPEB0012108 (2%, v / v) was inoculated into MRS-THIO broth (MRS broth supplemented with 0.20 wt% sodium thioglycolate) containing or not containing 0.30% (w / v) bile salt, and cultured anaerobically in an incubator at 37°C for 4 h. 100 μL was taken and spread on plates at 0 h and 4 h respectively, and the viable cell count was calculated at 37°C, with 3 parallel determinations. The tolerance rate (%) of the strain was determined by the MRS agar plate counting method, and the results are shown in Table 2.

[0060] Tolerance rate (%) = N 4h / N 0h × 100%

[0061] Where: N 4h , the viable cell count after strain treatment, CFU / mL; N 0h , the initial viable cell count, CFU / mL.

[0062] Table 2

[0063]

[0064] Example 4

[0065] Analysis of the gastrointestinal fluid tolerance of strain MPEB0012108

[0066] 4.1 Gastric juice tolerance of strain MPEB0012108

[0067] After the strain MPEB0012108 was activated and cultured, it was centrifuged at 12000 r / min for 6 min, the supernatant was removed, and the precipitate was washed twice with an equal volume of PBS solution to obtain a bacterial suspension of strain MPEB0012108 (1×10 8 CFU / ml).

[0068] Absorb 0.5 mL of the bacterial suspension of strain MPEB0012108, add it to 4.5 mL of simulated gastric juice, mix well, place it in an anaerobic condition at 37 °C for 3 h, take samples and dilute to an appropriate gradient, take 100 μL and spread it on a plate, culture it at 37 °C, calculate the viable count of lactic acid bacteria N1, and perform parallel determination 3 times. Calculate the survival rate of lactic acid bacteria in gastric juice according to the following formula, and the results are shown in Table 3.

[0069] Survival rate in simulated gastric juice (%) = N1 / N0 × 100%

[0070] Among them, N0 represents the viable bacterial colony count before treatment; N1 represents the viable bacterial colony count treated with simulated gastric juice.

[0071] Table 3

[0072]

[0073] 4.2 Intestinal juice tolerance of strain MPEB0012108

[0074] Centrifuge the reaction solution after reacting with simulated gastric juice for 3 h at 12,000 r / min for 6 min, remove the supernatant, wash it twice with PBS buffer solution and resuspend it with the same volume. Take the above bacterial suspension and add it to 4.5 mL of simulated intestinal juice, mix well, place it in an anaerobic condition at 37 °C for 4 h, take samples and dilute to an appropriate gradient, take 100 μL and spread it on a plate, culture it at 37 °C, calculate the viable count of lactic acid bacteria N2, and perform parallel determination 3 times,. Calculate the survival rate of lactic acid bacteria in intestinal juice according to the following formula, and the results are shown in Table 4.

[0075] Survival rate in simulated intestinal juice (%) = N2 / N1 × 100%

[0076] Among them, N1 / N2 represents the viable bacterial colony count treated with simulated gastric juice and intestinal juice.

[0077] Table 4

[0078]

[0079]

[0080] Example 5

[0081] Analysis of intestinal adhesion of strain MPEB0012108

[0082] 9.1 Surface hydrophobicity of strain MPEB0012108

[0083] The affinity of lactic acid bacteria for hydrocarbons such as xylene can reflect the surface hydrophobicity of the strain. The Bacteria Adhesion To Hydrocarbons (BATH) method was used to measure the affinity of lactic acid bacteria for xylene to evaluate the surface hydrophobic ability of lactic acid bacteria cells. The specific steps are as follows:

[0084] Absorb 2 mL of MPEB0012108 bacterial liquid into a 10 mL centrifuge tube, add 2 mL of xylene solution. Use the bacterial suspension without xylene solution as control group A0. Thoroughly mix the water and xylene phases by vortexing for 2 min, then place it at room temperature for incubation for 1 h to separate the two-phase system. Carefully aspirate the aqueous phase, use PBS as the blank control, and measure the OD 600 as A1. Each group has 3 parallel tubes, record the data. Calculate the hydrophobicity on the surface of MPEB0012108. The results are shown in Table 5.

[0085] Hydrophobicity rate of the strain surface (%) = (A0 - A1) / A0 × 100%

[0086] Where A0 represents the absorbance value of the control group; A1 represents the absorbance value of the experimental group.

[0087] 5.2 Auto-aggregation of strain MPEB0012108

[0088] Absorb 2 mL of MPEB0012108 bacterial liquid into a 10 mL centrifuge tube, vortex for 10 s, and place it at 37 °C for static incubation for 2 h (during this period, the centrifuge tube cannot be shaken). Then carefully aspirate 1 mL of the supernatant after static incubation, use PBS as the blank control, and measure the OD 600 , each group has 3 parallel tubes, record the data. Calculate the auto-aggregation rate of strain MPEB0012108 according to the following formula. The results are shown in Table 5.

[0089] Auto-aggregation rate (%) = (1 - (OD t / OD0)) × 100%

[0090] Where OD t represents the absorbance at t = x h; OD0 represents the absorbance at t = 0 h.

[0091] Table 5

[0092]

[0093] 5.3 Co-aggregation of strain MPEB0012108

[0094] Mix 2 mL of lactic acid bacteria and 2 mL of pathogenic bacteria suspension, place it at 37°C and let it stand for 4 h without shaking. Take the supernatant, use PBS as the blank control, measure OD600, with 3 parallel tubes in each group, and record the data. Calculate the cross-agglutination rate of strain MPEB0012108 according to the following formula, and the results are shown in Table 6. All of these pathogenic bacteria were purchased from Youyibang Biotechnology (Shanghai) Co., Ltd.

[0095] Cross-agglutination rate (%) = [(ODpat + ODlab) - 2 × ODmix] × 100 / (ODpat + ODlab)

[0096] Where ODpat represents the OD value of pathogenic bacteria at 0 h; ODlab represents the OD value of lactic acid bacteria at 0 h; ODmix represents the OD value of the mixed solution at 4 h.

[0097] Table 6

[0098] Pathogenic bacteria MPEB0012108 reciprocal agglutination rate MPEB0012758 10.85％ MPEB0001613 10.96％ MPEB0011218 10.25％ MPEB0011228 30.27％ MPEB0009941 12.82％ MPEB0013698 11.95％ MPEB0011612 11.95％ MPEB0012467 11.56％ MPEB0011884 9.51％ MPEB0010580 9.52％

[0099] Example 6

[0100] Analysis of the antibacterial ability of strain MPEB0012108

[0101] Pathogenic bacteria strains: Acinetobacter baumannii MPEB0012758; Salmonella enteritidis MPEB0001613; Klebsiella pneumoniae MPEB0011218; Pseudomonas aeruginosa MPEB0011228; Staphylococcus gallinarum MPEB0009941; Staphylococcus epidermidis MPEB0013698; Enterococcus faecium MPEB0011612, Aeromonas hydrophila MPEB0012467.

[0102] The antibacterial ability of the strain was determined by the plate coating method. Respectively pipette 100 μL of the pathogenic bacteria and coat them on the MHA solid medium; after activating strain MPEB0012108, centrifuge it at 8000 r / min for 10 min, take the fermentation supernatant, and filter it through a 0.22 μm microporous filter membrane to remove the residual lactic acid bacteria in the supernatant; divide the MH(A) petri dish into three areas, respectively pipette 10 μL of the sterile supernatant and drop it on the plate, use 10 μL of sterilized MRS liquid medium with the same pH value and sterilized hydrochloric acid solution with the same pH value as the control, and place it in an inverted constant temperature culture at 37°C for 24 - 48 h, and observe whether there is a clear zone formed in the area where the sterile fermentation broth is dropped. If a clear zone is formed, it proves that the fermentation broth has antibacterial activity, and take a photo and record it, see Figures 3 to 10 , indicating that strain MPEB0012108 has antibacterial activity against these pathogenic bacteria. Therefore, the strain MPEB0012108 of the present application is suitable for preparing probiotics that can be administered orally, such as probiotic tablets.

[0103] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and alterations can be made without departing from the spirit and scope of the present invention.

Claims

1. A cheese Lactobacillus casei MPEB0012108, deposited in Guangdong Microbial Culture Collection Center (GDMCC), deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, deposit number: GDMCC No: 65113.

2. A preparation comprising the Lactobacillus cheese as claimed in claim 1.

3. Application of Lactobacillus cheese as claimed in claim 1 or the preparation as claimed in claim 2 in preparing a probiotic for enteral administration.

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