CN112322554A - Lactobacillus rhamnosus strain, microbial inoculum, preparation method and application - Google Patents
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Abstract
The invention discloses a Lactobacillus rhamnosus (Lactobacillus rhamnoides) strain with the preservation number of CGMCC No.13310 and application thereof in improving type 2 diabetes caused by intestinal barrier damage. The invention also discloses a microbial inoculum prepared by the strain and a preparation method of the microbial inoculum. The invention adopts lactobacillus rhamnosus CGMCC No.13310 and a microbial inoculum prepared by the same for the first time, and discloses a new application of the lactobacillus rhamnosus CGMCC No.13310 in improving type 2 diabetes caused by intestinal barrier damage, in particular effects of reducing fasting blood glucose and improving tolerance of glucose and insulin.
Description
Technical Field
The invention mainly relates to the field of microorganisms, in particular to a lactobacillus rhamnosus strain, a microbial inoculum, a preparation method and application thereof.
Background
In recent years, with the rapid development of Chinese economy, the living conditions of people are greatly improved, particularly in the aspect of diet, the people are more and more beautified, and high-calorie and high-fat foods such as fried chicken and cola are seen everywhere in our life. However, consumers are unaware that long-term, overeating of such foods, in addition to providing them with a palatable experience, may be accompanied by various potential hazards. High fat diets increase intestinal permeability by reducing expression of genes encoding intestinal tight junction-associated proteins (e.g., Claudin-1 and occludin), thereby disrupting the integrity of the intestinal barrier, and subsequently activating non-specific immunity of the body to cause chronic mild inflammation, ultimately leading to insulin resistance and promoting the development of type 2 diabetes.
For type 2 diabetes caused by the damage of the intestinal barrier, the focus cannot be eradicated by simply relying on the conventional type 2 diabetes drugs (such as sulfonylurea drugs, biguanide drugs, glucosidase inhibitors and the like), the recurrence probability is extremely high, in addition, the drugs have side effects of different degrees on patients, slight discomfort of the gastrointestinal tract is caused, and serious injuries are caused to organs such as liver, kidney and the like.
Lactic acid bacteria are a special group of microorganisms existing in the human body, and generally have various probiotic functions, such as promoting the absorption of nutrients by the human body, synthesizing various vitamins essential to the human body, inhibiting bacteria, reducing blood ammonia and cholesterol, regulating immunity and the like. In the present day that the research of microbial flora is advanced, although the human beings have rather deeply understood the connection between the microbial flora and specific diseases of human bodies, especially chronic diseases, the lactic acid bacteria resource clinically used for preventing and treating type 2 diabetes caused by the impaired intestinal barrier is almost zero so far.
Therefore, the invention is especially provided.
Disclosure of Invention
The invention aims to provide a lactobacillus strain which has the functions of reducing fasting blood glucose and improving the tolerance of glucose and insulin and can improve type 2 diabetes caused by the damage of intestinal barrier, and application, a microbial inoculum and a preparation method thereof.
The invention provides a Lactobacillus rhamnosus (Lactobacillus rhamnosus) strain, the preservation number of the strain is CGMCC No.13310, and application of the strain in improving type 2 diabetes caused by intestinal barrier damage, wherein the strain improves type 2 diabetes caused by intestinal barrier damage in a mode of reducing fasting blood glucose and improving tolerance of glucose and insulin.
The invention provides a microbial inoculum which comprises lactobacillus rhamnosus belonging to the lactobacillus rhamnosus strain.
The invention also provides a preparation method of the microbial inoculum, which is characterized by comprising the following steps in sequence:
(1) inoculating lactobacillus rhamnosus CGMCC No.13310 into MRS culture medium for fermentation to obtain fermentation liquid;
(2) collecting thalli from the fermentation liquor obtained in the step (1) and cleaning;
(3) and (3) re-suspending the thalli obtained in the step (2) in skim milk, and freeze-drying to obtain a microbial inoculum product.
Preferably, the inoculation amount of the lactobacillus rhamnosus CGMCC No.13310 in the step (1) is 1.25x107-1x108CFU/mL; preferably 2.5x107-7.5x107CFU/mL; more preferably 5x107CFU/mL. When the inoculation amount is too small, the strain propagation speed is slow, so that the finally obtained thallus multiplication effect is lower than the optimal inoculation amount range; when the inoculation amount is too large, the strain individuals die or stop growing due to the limitation of living space and nutrition after a certain time, and the number of the finally obtained viable bacteria of the microbial inoculum is lower than the optimal inoculation amount range.
Preferably, the fermentation process in the step (1) is static culture at the temperature of 25-45 ℃ for 12-36 h; preferably 30-40 ℃, and preferably 18-30 h; more preferably 37 ℃ for 24 hours. The lactobacillus rhamnosus is an anaerobic bacterium, and can quickly grow and propagate under the condition of static culture. When the fermentation temperature is lower than the preferable range value, the metabolism of the strain is slow, and the thallus turnover rate is reduced.
Preferably, the method for collecting the thalli in the step (2) is centrifugal collection, the centrifugal speed is 4000-12000g, and the time is 8-12 min; preferably 8,000-12,000g, 9-11 min; more preferably 10,000g for 10 min.
Preferably, the skim milk used in step (3) is prepared from skim milk powder and water, and the use amount of the skim milk powder is 6-12% of the mass of the skim milk. The preparation method can comprise the following steps: adding skimmed milk powder into distilled water, mixing, dissolving, sterilizing at 95-125 deg.C for 5-20 min, and cooling.
Preferably, the viable count of the lactobacillus rhamnosus CGMCC No.13310 in the microbial inoculum product in the step (3) is more than or equal to 109CFU/g。
Compared with the prior art, the invention adopts the lactobacillus rhamnosus CGMCC No.13310 and the microbial inoculum prepared by the lactobacillus rhamnosus CGMCC No.13310 for the first time, and discloses a new application of the lactobacillus rhamnosus CGMCC No.13310 in improving type 2 diabetes caused by intestinal barrier damage.
Drawings
FIG. 1 is a graph showing the result of fasting blood glucose test in an embodiment of the present invention.
Fig. 2 is a graph showing the results of an experiment on glucose tolerance in an example of the effect of the present invention.
FIG. 3 is AUC of results of glucose tolerance test of an example of the effect of the present inventionglucoseArea comparison graph.
FIG. 4 is a graph showing the results of an experiment on insulin resistance in an example of the effect of the present invention.
Detailed Description
In order to more clearly illustrate the technical solution of the present invention, the technical solution of the present invention will be further described with reference to the specific embodiments
The invention discloses a Lactobacillus rhamnosus (Lactobacillus rhamnoides) strain, the preservation number of which is CGMCC No.13310, the strain is preserved in China general microbiological culture Collection center (CGMCC) at 11-15 th month in 2016, and the preservation address is as follows: west road No.1, north chen, chaoyang district, beijing, zip code: 100101.
example one
The embodiment of the invention provides a preparation method of a lactobacillus rhamnosus CGMCC No.13310 microbial inoculum.
Material preparation
Preparing a fermentation strain: dissolving lyophilized powder of Lactobacillus rhamnosus CGMCC No.13310 with small amount of sterile distilled water, taking one ring by using an inoculating ring, streaking on MRS solid culture medium (purchased from Merck Co., Germany), performing anaerobic culture at 37 ℃ for 48h, taking out, picking out a single bacterial colony by using the inoculating ring, putting into 10mL of MRS liquid culture medium (purchased from Merck Co., Germany), uniformly dispersing the bacterial colony in the liquid culture medium by using a vortex oscillator, performing anaerobic culture at 37 ℃ for 48h, taking out, inoculating into the MRS liquid culture medium by using an inoculation amount of 2% (v/v), performing anaerobic culture at 37 ℃ for 24h, centrifuging the culture at 15,000rpm for 10min, discarding the supernatant, washing the thallus with sterile distilled water for 2 times, suspending with sterile distilled water of the original culture volume to obtain a fermentation strain seed liquid for fermentation, wherein the bacterial concentration of the Lactobacillus rhamnosus CGMCC No.13310 of the seed liquid is 2.5x109 CFU/mL。
Preparation of skim milk: mixing 10 wt% of skimmed milk powder with distilled water, dissolving completely, sterilizing at 125 deg.C for 5min, and cooling to room temperature.
The preparation of the microbial inoculum is carried out according to the following steps:
(1) inoculating seed liquid of fermentation strain of Lactobacillus rhamnosus CGMCC No.13310 in an inoculum size of 2% (v/v, the seed liquid accounts for the volume percent of the fermentation liquid, the same below) in MRS liquid culture medium, and standing and culturing at 37 deg.C for 24h to obtain fermentation liquid;
(2) centrifuging the fermentation liquor obtained in the step (2) for 10min under 10000g, discarding the supernatant, and washing the precipitate (thallus) with sterile distilled water twice;
(3) and (3) resuspending the bacterium obtained in the step (2) with skim milk (the mass percentage of the vegetation in the material preparation is 10%) in the original culture volume, and freeze-drying to obtain the lactobacillus rhamnosus CGMCC No.13310 bacterium agent.
Example two
The embodiment of the invention provides a preparation method of a lactobacillus rhamnosus CGMCC No.13310 microbial inoculum.
Material preparation
Preparing a fermentation strain: as in the examples.
Preparation of skim milk: uniformly mixing 12% of skimmed milk powder with distilled water, dissolving completely, sterilizing at 95 deg.C for 20min, and cooling to room temperature.
The preparation of the microbial inoculum is carried out according to the following steps:
(1) inoculating seed liquid of fermentation strain of Lactobacillus rhamnosus CGMCC No.13310 in MRS liquid culture medium with inoculum size of 0.5% (v/v, the seed liquid accounts for volume percent of the fermentation liquid, the same below), and standing at 25 deg.C for 36 hr to obtain fermentation liquid;
(2) centrifuging the fermentation liquor obtained in the step (2) for 8min at 12000g, removing supernatant, and washing a precipitate part (thallus) twice by using sterile distilled water;
(3) and (3) resuspending the bacterium obtained in the step (2) with skim milk (the mass percentage of the vegetation in the material preparation is 12%) in the original culture volume, and freeze-drying to obtain the lactobacillus rhamnosus CGMCC No.13310 bacterium agent.
EXAMPLE III
The embodiment of the invention provides a preparation method of a lactobacillus rhamnosus CGMCC No.13310 microbial inoculum.
Material preparation
Preparing a fermentation strain: as in the examples.
Preparation of skim milk: mixing 6 wt% of skimmed milk powder with distilled water, dissolving completely, sterilizing at 100 deg.C for 15min, and cooling to room temperature.
The preparation of the microbial inoculum is carried out according to the following steps:
(1) inoculating a seed solution of a fermentation strain of lactobacillus rhamnosus CGMCC No.13310 in an inoculum size of 4% (v/v, the seed solution accounts for the volume percent of the fermentation broth, the same below) in an MRS liquid culture medium, and performing standing culture at 45 ℃ for 12h to obtain the fermentation broth;
(2) centrifuging the fermentation liquor obtained in the step (2) for 12min at 8000g, discarding the supernatant, and washing the precipitate (thallus) with sterile distilled water twice;
(3) and (3) resuspending the bacterium obtained in the step (2) with skim milk (the mass percentage of the vegetation in the material preparation is 6%) in the original culture volume, and freeze-drying to obtain the lactobacillus rhamnosus CGMCC No.13310 bacterium agent.
Example four
The embodiment of the invention provides a preparation method of a lactobacillus rhamnosus CGMCC No.13310 microbial inoculum.
Material preparation
Preparing a fermentation strain: as in the examples.
Preparation of skim milk: uniformly mixing 8% of skimmed milk powder with distilled water, dissolving completely, sterilizing at 120 deg.C for 10min, and cooling to room temperature.
The preparation of the microbial inoculum is carried out according to the following steps:
(1) inoculating a fermentation strain seed liquid of lactobacillus rhamnosus CGMCC No.13310 into an MRS liquid culture medium in an inoculum size of 3% (v/v, the seed liquid accounts for the volume percent of the fermentation liquid, the same applies below) aseptically, and performing standing culture at 30 ℃ for 18h to obtain the fermentation liquid;
(2) centrifuging the fermentation liquor obtained in the step (2) for 9min at 11000g, discarding the supernatant, and washing the precipitate (thallus) twice with sterile distilled water;
(3) and (3) resuspending the bacterium obtained in the step (2) with skim milk (the mass percentage of the vegetation in the material preparation is 8%) in the original culture volume, and freeze-drying to obtain the lactobacillus rhamnosus CGMCC No.13310 bacterium agent.
EXAMPLE five
The embodiment of the invention provides a preparation method of a lactobacillus rhamnosus CGMCC No.13310 microbial inoculum.
Material preparation
Preparing a fermentation strain: as in the examples.
Preparation of skim milk: mixing 9 wt% of skimmed milk powder with distilled water, dissolving completely, sterilizing at 115 deg.C for 12min, and cooling to room temperature.
The preparation of the microbial inoculum is carried out according to the following steps:
(1) inoculating a seed solution of a fermentation strain of lactobacillus rhamnosus CGMCC No.13310 into an MRS liquid culture medium in an inoculum size of 1% (v/v, the seed solution accounts for the volume percent of the fermentation liquid, the same applies below) aseptically, and performing standing culture at 40 ℃ for 30h to obtain the fermentation liquid;
(2) centrifuging the fermentation liquor obtained in the step (2) for 11min under 9000g, discarding the supernatant, and washing the precipitate (thallus) with sterile distilled water twice;
(3) and (3) resuspending the bacterium obtained in the step (2) with skim milk (the mass percentage of the vegetation in the material preparation is 9%) in the original culture volume, and freeze-drying to obtain the lactobacillus rhamnosus CGMCC No.13310 bacterium agent.
The viable count of lactobacillus rhamnosus CGMCC No.13310 in the microbial inoculum prepared by each embodiment of the invention is shown in the following table.
| Example number | Viable count (CFU/g) |
| Example one | 3x1010 |
| Example two | 1.2x109 |
| EXAMPLE III | 8x109 |
| Example four | 2.4x109 |
| EXAMPLE five | 1.8x1010 |
Effect example measurement of improving Effect of Lactobacillus rhamnosus CGMCC No.13310 microbial inoculum on type 2 diabetes caused by impaired intestinal Barrier
Establishing an animal model: 40 SPF-grade adult C57BL/6 mice (Shanghai Slek laboratory animals, Inc.) are purchased, adaptive feeding is performed for 1 week, 30 mice are selected from the mice at week 2 and fed with high-fat feed (with the fat content of 60%), the rest 10 mice are used as control groups and fed with normal feed, and the blood sugar of the mice in the high-fat diet group is remarkably increased until week 12 later, which indicates that the type 2 diabetes caused by the damage of the intestinal barrier of the mice occurs, and the establishment of an animal experimental model is successful.
Animal experiments: selecting 15 mice successfully modeled, randomly dividing 5 mice in each group into 3 groups (a model control group, a high-dose intervention group and a low-dose intervention group), and taking 5 mice fed with normal feed as a normal control group. According to the method shown in Table 1, feeding corresponding feed to each group, and using bacterial suspension (prepared from the microbial inoculum prepared by the method in example one, wherein the viable count of the bacterial suspension I: Lactobacillus rhamnosus CGMCC No.13310 is 10)9CFU/mL and II: the viable count of the lactobacillus rhamnosus CGMCC No.13310 is 108CFU/mL) or blank sample (10% skim milk prepared in example one) was gavaged (10mL/Kg · d · BW) for 4 weeks.
TABLE 1 Experimental animals grouping and gavage method
During the experiment, the test animals were tested for each test according to the method described in table 2.
Table 2 detection indexes, processing time and sampling time
The results of the fasting plasma glucose test are shown in FIG. 1. The fasting blood glucose value of the model control group mouse is always maintained at a higher level (12mmol/L) and is obviously higher than that of the normal control group (9.5mmol/L), which indicates that the establishment of the model of type 2 diabetes caused by the damage of the intestinal barrier is successful. After each intervention group is subjected to lactobacillus rhamnosus CGMCC No.13310 intervention (gastric perfusion is carried out by using bacterial suspension of lactobacillus rhamnosus CGMCC No. 13310), the fasting blood glucose level of the mouse is obviously reduced and is obviously lower than that of a model control group, and the blood glucose reducing effect is enhanced along with the prolonging of intervention time. And the intervention effect of the high-dose intervention group is due to the low-dose intervention group. The experimental results show that the lactobacillus rhamnosus CGMCC No.13310 has positive regulation and control and relieving effects on the fasting blood glucose of the type 2 diabetic mice caused by the damaged intestinal barrier.
The results of the glucose tolerance test are shown in fig. 2. The normal control group mice had the strongest glucose tolerance, and their blood glucose values were most rapidly decreased after glucose intake. The glucose tolerance of the mouse in the model control group is the worst, and the blood glucose value of the mouse in the model control group is reduced the slowest after reaching the peak value after glucose intake, which indicates that the glucose metabolism capability of the mouse in the model control group is negatively affected by intestinal barrier damage caused by high fat diet, and the model is successfully established. After the mice in the high and low dose intervention groups take glucose, the blood sugar value decline rate is obviously superior to that of the model control group, and the mice have the trend of improving the normal control group. AUC was obtained by integrating the area under each set of curves in FIG. 2glucoseArea, results are shown in FIG. 3. The area of the normal control group AUC is the smallest, and the area of the model control group is the largest, which shows that the high fat diet reduces the glucose metabolism ability of the mice and increases the glucose content in body fluid. The AUC value of the low-dose intervention group is slightly lower than that of the model control group, while the AUC value of the high-dose intervention group is remarkably lower than that of the model control group and is close to that of the normal control group, which shows that the Lactobacillus rhamnosus CGMCC No.13310 can improve the glucose tolerance of mice with type 2 diabetes caused by the damage of the intestinal barrier, and has a certain dose-effect relationship on the regulation effect of the type 2 diabetes caused by the damage of the intestinal barrier, and the larger the dose is, the more obvious the blood glucose reducing effect is.
The results of the insulin resistance experiments are shown in figure 4. After 30min of insulin injection, the blood sugar value of each group of mice is reduced to a certain degree, but compared with other groups, the blood sugar value of the model control group is always kept at a higher level. The blood sugar values of the mice in the high and low dose intervention groups are inversely observed, the blood sugar values are almost kept in the same trend as those of the normal control group in the whole experimental process and are obviously lower than those of the model control group, and the experimental phenomenon shows that the intervention of lactobacillus rhamnosus CGMCC No.13310 can enhance the sensitivity of the type 2 diabetes model mice caused by the damaged intestinal barrier to insulin.
The results show that the microbial inoculum of lactobacillus rhamnosus CGMCC No.13310 has positive regulation and control and relieving effects on type 2 diabetes caused by the damage of intestinal barrier, has certain hypoglycemic effect, can enhance the sensitivity to insulin, and the effect is enhanced along with the increase of the dosage of the microbial inoculum.
Comparative example
The inoculation amount, the culture temperature and the culture time in the first embodiment are adjusted one by one, lactobacillus rhamnosus CGMCC No.13310 microbial inoculum prepared by the following group of different methods is obtained, and the viable count of lactobacillus rhamnosus CGMCC No.13310 in each group of microbial inoculum is shown in Table 3.
TABLE 3 comparative example shows the viable count of Lactobacillus rhamnosus CGMCC No.13310 in the prepared microbial inoculum
From the results shown in table 3, the inoculum size, the culture temperature and the culture time in the preparation method of the microbial inoculum are adjusted, when the inoculum size, the culture temperature and the culture time are out of the optimal ranges, lactobacillus rhamnosus CGMCC No.13310 can still grow and be prepared into the corresponding microbial inoculum, however, the proliferation effect is obviously reduced, and the number of viable bacteria of the obtained microbial inoculum is low. Because the blood sugar regulation effect of lactobacillus rhamnosus CGMCC No.13310 depends on the number of bacterial cells, the microbial inoculum with low viable count can not reach the biological target of regulating various indexes of blood sugar in vivo.
The invention provides lactobacillus rhamnosus CGMCC No.13310, a microbial inoculum thereof and a preparation method of the microbial inoculum. Through related experiments, the microbial inoculum of lactobacillus rhamnosus CGMCC No.13310 has positive regulation and control and relieving effects on type 2 diabetes caused by damaged intestinal barrier, has a certain hypoglycemic effect and can enhance the sensitivity to insulin. Furthermore, the preparation process of the microbial inoculum is optimized, so that the prepared microbial inoculum product has better relevant effect.
The lactobacillus rhamnosus CGMCC No.13310 provided by the invention and the application, the microbial inoculum and the preparation method thereof are described in detail above. The principles and embodiments of the present invention are explained herein using specific examples, which are presented only to assist in understanding the method and its core concepts. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
Claims (10)
1. A Lactobacillus rhamnosus (Lactobacillus rhamnoides) strain is characterized in that the preservation number of the strain is CGMCC No. 13310.
2. Use of a lactobacillus rhamnosus strain as claimed in claim 1 for improving type 2 diabetes caused by an impaired intestinal barrier.
3. The use of claim 2, wherein the improvement is a reduction in fasting glucose, an improvement in glucose and insulin tolerance.
4. An agent comprising lactobacillus rhamnosus belonging to the lactobacillus rhamnosus strain described in claim 1.
5. A method for preparing the microbial inoculum as described in claim 4, which comprises the following steps in sequence:
(1) inoculating lactobacillus rhamnosus CGMCC No.13310 into MRS culture medium for fermentation to obtain fermentation liquid;
(2) collecting thalli from the fermentation liquor obtained in the step (1) and cleaning;
(3) and (3) re-suspending the thalli obtained in the step (2) in skim milk, and freeze-drying to obtain a microbial inoculum product.
6. The method for preparing the microbial inoculum according to claim 5, wherein the inoculation amount of Lactobacillus rhamnosus CGMCC No.13310 in the step (1) is 1.25x107-1x108 CFU/mL。
7. The method for preparing the microbial inoculum according to claim 5, wherein the fermentation process in the step (1) is static culture at 25-45 ℃ for 12-36 h.
8. The method for preparing microbial inoculum according to claim 5, wherein the step (2) of collecting the microbial inoculum is centrifugal collection, the centrifugal speed is 4000-12000g, and the time is 8-12 min.
9. The method for preparing microbial inoculum according to claim 5, wherein the skim milk used in the step (3) is prepared from skim milk powder and water, and the use amount of the skim milk powder is 6-12% of the mass of the skim milk.
10. The method for preparing the microbial inoculum according to claim 5, wherein the viable count of Lactobacillus rhamnosus CGMCC No.13310 in the microbial inoculum product in the step (3) is more than or equal to 109CFU/g。
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Cited By (4)
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| CN112322528A (en) * | 2020-11-03 | 2021-02-05 | 江南大学 | Lactobacillus rhamnosus capable of intervening metabolic syndrome and application thereof |
| CN113396974A (en) * | 2021-07-01 | 2021-09-17 | 光明乳业股份有限公司 | Application of lactobacillus paracasei in preparation of fermented dairy product in type 2 diabetes |
| CN115386518A (en) * | 2022-08-18 | 2022-11-25 | 齐鲁工业大学 | Lactobacillus rhamnosus strain taking D-psicose as carbon source and derivative product and application thereof |
| CN116694537A (en) * | 2023-07-28 | 2023-09-05 | 善恩康生物科技（苏州）有限公司 | Lactobacillus rhamnosus and application thereof in preparation of products for treating type 2 diabetes |
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| CN116694537A (en) * | 2023-07-28 | 2023-09-05 | 善恩康生物科技（苏州）有限公司 | Lactobacillus rhamnosus and application thereof in preparation of products for treating type 2 diabetes |
| CN116694537B (en) * | 2023-07-28 | 2023-10-31 | 善恩康生物科技（苏州）有限公司 | Lactobacillus rhamnosus and application thereof in preparation of products for treating type 2 diabetes |
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