Lactobacillus rhamnosus MFHZ40 and application thereof

Abstract

The invention relates to the technical field of functional microorganisms, in particular to lactobacillus rhamnosus MFHZ40 and application thereof. The strain is classified and named as Lactiplantibacillus rhamnosus, and is preserved in China General Microbiological Culture Collection Center on November 27, 2023, the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.29144. The invention further discloses a preparation method of the Lactiplantibacillus rhamnosus. Compared with commercialized strains in the market, the lactobacillus rhamnosus MFHZ40 provided by the invention has stronger surface hydrophobicity, auto-polymerization capability, adhesion characteristic and gastrointestinal fluid tolerance, and is a lactobacillus with high adhesion, good gastrointestinal fluid tolerance and blood sugar reduction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of functional microorganisms, in particular to a lactobacillus rhamnosus MFHZ40 and application thereof. BACKGROUND

[0002] At present, diabetes has become the most common metabolic disease. High blood sugar content in serum is considered to be the main factor of metabolic diseases such as diabetes. Therefore, reducing the serum blood sugar level is directly related to the health of the human body. So far, a large number of experiments have confirmed that taking some lactobacillus and its related products has the effect of reducing the serum blood sugar content of the human body and reducing the complications caused by diabetes. Lactobacillus is the main probiotic in the human intestinal tract, and as a safe and effective blood sugar-lowering microbial preparation, it has shown potential efficacy in relieving high blood sugar in diabetes. However, at present, there are still few reports on the relief of high blood sugar by lactobacillus, and the screening method lacks integrity and systematicness.

[0003] Probiotics are a class of active microorganisms that are beneficial to the host, and are a general term for active beneficial microorganisms that colonize the human intestinal tract and reproductive system, produce definite health effects, and thus improve the microecological balance of the host and play a beneficial role in the intestinal tract. The most widely used probiotics at present are lactobacillus and bifidobacterium, etc. Lactobacillus rhamnosus, as a kind of lactobacillus, can be added to food to improve the taste, texture and flavor of food. However, lactobacillus rhamnosus has poor resistance to gastric acid and gastric colonization ability, and it is difficult to colonize the stomach tissue to play a role in reducing blood sugar. Therefore, there is an urgent need in the market to screen a lactobacillus rhamnosus with good gastric mucosa adhesion, resistance to gastric acid and blood sugar-lowering effect. SUMMARY

[0004] The purpose of the present application is to improve the integrity and systematicness of the method for screening blood sugar-lowering lactobacillus, so as to screen a lactobacillus with high adhesion, good gastrointestinal fluid tolerance and blood sugar-lowering effect.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0006] The present application provides a lactobacillus rhamnosus MFHZ40, which is classified and named as Lactiplantibacillus rhamnosus, and has been preserved in the China General Microbiological Culture Collection Center on November 27, 2023, at the address of No. 1, Beichen West Road, Haidian District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, with the preservation number of CGMCC No. 29144.

[0007] Preferably, the type of the functional food is a solid beverage.

[0008] Preferably, the solid beverage is compounded by protein powder, malt dextrin and lactobacillus rhamnosus MFHZ40.

[0009] Preferably, the mass ratio of the protein powder, malt dextrin and lactobacillus rhamnosus MFHZ40 is 90-110:5-7:4-6.

[0010] Preferably, the mass ratio of mung bean protein powder, black bean protein powder and millet protein powder in the protein powder is 10-30:20-40:30-70.

[0011] Preferably, the viable count of the lactobacillus rhamnosus MFHZ40 is 6-7x10 10 CFU / g.

[0012] The application also provides application of the lactobacillus rhamnosus MFHZ40 in preparation of a medicine for reducing blood sugar.

[0013] Compared with the prior art, the application has the following beneficial effects:

[0014] The application evaluates the in-vitro adhesion characteristics of the lactobacillus rhamnosus MFHZ40 through surface hydrophobicity, self-polymerization ability and Caco-2 cell adhesion test, and combines the evaluation results of inhibition of a-glucosidase activity in in-vitro culture medium and gastrointestinal fluid tolerance of the strain, to confirm that the application screens a lactic acid bacteria with high adhesion, good gastrointestinal fluid tolerance and blood sugar reduction.

[0015] The lactobacillus rhamnosus MFHZ40 involved in the application has stronger surface hydrophobicity, self-polymerization ability, adhesion characteristics and gastrointestinal fluid tolerance ability than market commercial strains, the surface hydrophobicity is 90.95%, the self-polymerization ability is 29.77%, the Caco-2 cell adhesion number is 19 / cell, and the artificial gastrointestinal fluid tolerance is 97.96% and 98.31%, respectively. BRIEF DESCRIPTION OF DRAWINGS

[0016] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only belong to the embodiments of the application, and those skilled in the art can also obtain other drawings according to the provided drawings without creative labor.

[0017] Figure 1 It is the morphological identification diagram of the lactobacillus rhamnosus MFHZ40 in Example 1, wherein the left side diagram is the colony morphology diagram, and the right side diagram is the electron microscope diagram.

[0018] Figure 2Figure for evaluation of high adhesion and hypoglycemic ability of Lactobacillus rhamnosus MFHZ40 in Example 1; wherein A is surface hydrophobicity; B is self-aggregation ability; B is Caco-2 cell adhesion; D is α-glucosidase inhibition rate.

[0019] Figure 3 Figure for evaluation of tolerance of Lactobacillus rhamnosus MFHZ40 in simulated gastrointestinal fluid in Example 1; wherein A is the growth of Lactobacillus rhamnosus MFHZ40 in simulated gastric juice; B is the viable count of Lactobacillus rhamnosus MFHZ40 in simulated gastric juice; C is the growth of Lactobacillus rhamnosus MFHZ40 in simulated intestinal juice; D is the viable count of Lactobacillus rhamnosus MFHZ40 in simulated intestinal juice.

[0020] Figure 4 Figure of scanning electron microscope of lactic acid bacteria grain protein solid beverage; wherein A is grain protein solid beverage without Lactobacillus rhamnosus MFHZ40, B is lactic acid bacteria grain protein solid beverage; the scale of both from left to right is 100 μm, 50 μm, 20 μm.

[0021] Figure 5 Figure of particle size distribution of lactic acid bacteria grain protein solid beverage; wherein A-B is grain protein solid beverage without Lactobacillus rhamnosus MFHZ40, C-D is lactic acid bacteria grain protein solid beverage.

[0022] Figure 6 Figure of zeta potential of lactic acid bacteria grain protein solid beverage; wherein A is grain protein solid beverage without Lactobacillus rhamnosus MFHZ40, B is lactic acid bacteria grain protein solid beverage.

[0023] Figure 7 Protein digestibility of lactic acid bacteria grain protein solid beverage; wherein A is grain protein solid beverage without Lactobacillus rhamnosus MFHZ40, B is lactic acid bacteria grain protein solid beverage.

[0024] Deposit Description

[0025] Lactobacillus rhamnosus MFHZ40, classified as Lactiplantibacillus rhamnosus, strain number MFHZ40, has been preserved in the China General Microbiological Culture Collection Center (CGMCC) on November 27, 2023, the address of the preservation is No. 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, the preservation number is CGMCC No. 29144. DETAILED DESCRIPTION

[0026] The technical solutions provided by the present application are described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application. All raw materials and reagents in the following examples are commercially available.

[0027] Example 1

[0028] Screening of a strain of Lactiplantibacillus rhamnosus MFHZ40

[0029] (1) Screening of Lactiplantibacillus rhamnosus MFHZ40

[0030] Samples were taken from the salted radish pool of Hangzhou Xiaoshan Dangshan Jiangcai Food Co., Ltd. using a 3-point sampling method, and 3 samples were collected at each point. After collection, mix well and place in a sterile cryotube of 15 ml, immediately put into liquid nitrogen for preservation, and then placed in a -70°C ultra-low temperature refrigerator.

[0031] Take 1 ml of the above sample in physiological saline for 10-fold gradient dilution, evenly spread on MRS medium, then directional culture in a 36°C incubator for 72h, observe the colony morphology, select the white, round, smooth, moist, short rod-shaped, single or paired distribution, non-motile, no spore, gram-positive colony, and its morphology in the medium and electron microscope is as shown in Figure 1 .

[0032] A strain MFHZ40 was screened through surface hydrophobicity, self-aggregation ability, and cell colonization. It was identified as Lactiplantibacillus rhamnosus.

[0033] (2) Evaluation of surface hydrophobicity of Lactiplantibacillus rhamnosus MFHZ40

[0034] The test strain was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and cultured at 37°C for 24h. The bacterial cells were collected by centrifugation. The bacterial cells were washed with sterile PBS solution and resuspended in sterilized 0.1M KNO3. The absorbance OD 600 of the bacterial cell suspension was adjusted. 3mL of the bacterial cell suspension was taken, 1mL of dimethylbenzene was added, and after vortexing, it was left to stand at room temperature for 30min. The water phase was carefully aspirated and its absorbance at 600nm was measured.

[0035] The above results show that the surface hydrophobicity of Lactiplantibacillus rhamnosus MFHZ40 is 90.95% ± 0.23%. Figure 2 A, the surface hydrophobicity of Lactiplantibacillus rhamnosus MFHZ40 is 90.95% ± 0.23%.

[0036] (2) Evaluation of self-aggregation ability of Lactiplantibacillus rhamnosus MFHZ40

[0037] The test strain was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v) and incubated at 37°C for 24 h. The bacterial cell precipitate was collected by centrifugation. The bacterial cells were washed with sterile PBS solution and then resuspended in sterilized PBS. The absorbance OD of the bacterial cell suspension was adjusted to 0.1 at 600 nm. 600 2 mL of the bacterial cell suspension was taken, vortexed, and then incubated at 37°C for 2 h. The supernatant was carefully taken and the absorbance at 600 nm was measured.

[0038] The above results show that, as Figure 2 B, the self-aggregation ability of Lactobacillus rhamnosus MFHZ40 was 29.77% ± 3.91%

[0039] (3) Cell colonization test of lactic acid bacteria

[0040] The concentration of the digested Caco-2 cells was adjusted to 10 5 cell / mL, 1 mL was taken and inoculated into a 6-well plate, and incubated at 37°C in a 5% CO2 incubator. The culture medium was replaced once every other day (on the day before the experiment, DMEM medium without double antibodies was added to the well plate). After the cells were completely attached and grew into a monolayer, the culture medium was removed and the cells were washed twice with sterile PBS to remove excess cells that were not attached to the bottom of the plate. Then, 1 mL of the bacterial cell suspension was added to the washed Caco-2 cells (an equal amount of DMEM was used as a control), and incubated at 37°C in a 5% CO2 incubator for 2 h for cell adhesion. After adhesion, each well was washed with sterile PBS to remove unbound lactic acid bacteria and metabolic secretions. Methanol was used for fixation, and after natural air drying, Gram staining was performed. Under an oil immersion lens, a random field of view was selected, and the number of lactic acid bacteria adhered to each 10 Caco-2 cells was observed and counted.

[0041] Figure 2 C is a representative microscope image of lactic acid bacteria adhering to cells. It can be seen that lactic acid bacteria densely surround (adhere to) cells, and the number of cell adhesion is 19. Based on the above results of surface hydrophobicity and self-aggregation ability, as shown in Table 1, Lactobacillus rhamnosus MFHZ40 showed strong adhesion.

[0042] Table 1 Adhesion properties of Lactobacillus rhamnosus MFHZ40

[0043]

[0044] (4) In vitro blood glucose-lowering test of Lactobacillus rhamnosus

[0045] The test strain was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and incubated at 37°C for 24 h. After centrifugation, the bacterial pellet was washed with 0.85% physiological saline and resuspended. The bacterial suspension was mixed with an α-glucosidase solution, incubated at 37°C for 10 min, and then 1 mol / L Na2CO3 was added as a reaction stopping solution. After the reaction was completed, the absorbance of the reaction solution was measured at 405 nm. Phosphate buffer was used as a blank control for the α-glucosidase solution and the sample to be tested, and the α-glucosidase inhibition rate of the lactic acid bacteria was determined.

[0046] As Figure 2 D, the α-glucosidase inhibition rate of L. rhamnosus MFHZ40 was 12.16 ± 0.70%, indicating that the strain had a certain blood glucose-lowering ability in vitro.

[0047] (5) Evaluation of the gastric tolerance of L. rhamnosus MFHZ40

[0048] The test strain was inoculated into MRS liquid medium at an inoculation amount of 2% (v / v), and incubated at 37°C for 24 h. After centrifugation, the bacterial pellet was washed with 0.85% physiological saline and resuspended. The bacterial suspension was mixed with an α-glucosidase solution, incubated at 37°C for 10 min, and then 1 mol / L Na2CO3 was added as a reaction stopping solution. After the reaction was completed, the absorbance of the reaction solution was measured at 405 nm. Phosphate buffer was used as a blank control for the α-glucosidase solution and the sample to be tested, and the α-glucosidase inhibition rate of the lactic acid bacteria was determined. 8 CFU / mL. After thorough mixing, it was placed in a 37°C incubator. Samples were taken at 0 h, 1.5 h, and 3 h, and cultured with MRS solid medium at 37°C to determine the viable cell count.

[0049] The above results show that, as Figure 3 As shown in A-B, the viable cell count of L. rhamnosus MFHZ40 in simulated gastric juice decreased slowly, and the survival rate of L. rhamnosus MFHZ40 after 3 hours was 97.96% ± 0.13%.

[0050] (6) Evaluation of the intestinal tolerance of L. rhamnosus MFHZ40

[0051] After 3 h of culture in artificial gastric juice, the bacterial pellet was resuspended in an equal volume of artificial gastric juice and mixed, and incubated at 37°C. Samples were taken at 0 h, 4 h, and 8 h, and cultured with MRS solid medium at 37°C to determine the viable cell count.

[0052] The above results show that, as Figure 3 As shown in C-D, L. rhamnosus MFHZ40 still maintained a good survival rate in simulated intestinal juice, and the survival rate after 8 hours was 98.31% ± 0.21%.

[0053] Example 2

[0054] Application of lactobacillus rhamnosus MFHZ40 in preparation of lactic acid bacteria solid beverage of coarse cereal protein

[0055] (1) The ratio design of the lactic acid bacteria solid beverage of coarse cereal protein

[0056] Three samples (green bean, black bean, and millet three kinds of coarse cereal protein powder) were mixed in different ratios according to Table 2, and the optimal ratio of the three was determined by comprehensive evaluation of their brewing characteristics (solubility, stability, and caking rate).

[0057] Table 2 Different mixing ratios of coarse cereal protein powder

[0058]

[0059] Through comprehensive evaluation of their brewing characteristics (solubility, stability, and caking rate), the optimal mixing ratio of green bean, black bean, and millet three kinds of coarse cereal protein powder was determined to be 10:40:50, and then 5% lactobacillus rhamnosus powder and 6% malt dextrin were added. The evaluation results are shown in Table 3 and Table 4.

[0060] The specific process of the lactic acid bacteria solid beverage of coarse cereal protein is as follows: the three kinds of coarse cereal protein powder are mixed in a mass ratio of 10:40:50, 6% malt dextrin is added to the mixed powder, and then the mixture is subjected to high-pressure shearing, high-pressure homogenization, and spray drying. Finally, 5% lactobacillus rhamnosus powder (the viable count of lactobacillus rhamnosus MFHZ40 is 6.4 x 10 10 CFU / g) is added to obtain the lactic acid bacteria solid beverage of coarse cereal protein.

[0061] Table 3 Solubility and caking rate of the lactic acid bacteria solid beverage of coarse cereal protein with different ratios

[0062]

[0063] Table 4 Stability of the lactic acid bacteria solid beverage of coarse cereal protein with different ratios

[0064]

[0065]

[0066] The stability and solubility of the lactic acid bacteria solid beverage of coarse cereal protein were evaluated in terms of in vitro digestion characteristics, color, loose bulk density, particle size, turbidity, zeta potential, and rheological properties, and the results are shown in Table 5 and Table 6. Figures 4-7As shown, under the process, the gastrointestinal digestion characteristics of the lactic acid bacteria coarse cereal protein solid beverage are increased by 29.77% and 45.63%, respectively, the loose bulk density is as low as 0.21±0.01 g / ml, the solubility is increased by 27.8%, the hygroscopicity is reduced by 26.2%, the zeta potential is-14.49±0.55 mV, and the stability is strong; small particles are presented and the particle size is evenly distributed in the range of 2-30 μm, and the tan delta value presents a trend of first increasing and then decreasing, indicating that the product has strong gel dynamic rheological characteristics.

[0067] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. Lactobacillus rhamnosus MFHZ40, characterized in that, The strain classification name is Lactiplantibacillus rhamnosus, which has been preserved in the China General Microbiological Culture Collection Center on November 27, 2023, the address of the preservation center is No. 1, Xibei Road, Beichen, Beijing, China Institute of Microbiology, Chinese Academy of Sciences, and the preservation number is CGMCC No. 29144.

2. Use of the Lactobacillus rhamnosus MFHZ40 in claim 1 in the preparation of a functional food with the function of reducing blood sugar.

3. Use according to claim 2, characterized in that, The type of the functional food is a solid beverage.

4. Use according to claim 3, characterized in that, The solid beverage is compounded by protein powder, malt dextrin and Lactobacillus rhamnosus MFHZ40.

5. Use according to claim 4, characterized in that, The mass ratio of the protein powder, malt dextrin and Lactobacillus rhamnosus MFHZ40 is 90-110:5-7:4-6.

6. Use according to claim 4, characterized in that, The mass ratio of mung bean protein powder, black bean protein powder and millet protein powder in the protein powder is 10-30:20-40:30-70.

7. Use according to claim 4, characterized in that, The viable cell number of the Lactobacillus rhamnosus MFHZ40 is 6-7 x 10 10 CFU / g.

8. Use of the Lactobacillus rhamnosus MFHZ40 in claim 1 in the preparation of a drug for reducing blood sugar.

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