CN115011532B

CN115011532B - Lactobacillus paracasei JY062 preparation, and preparation method and application thereof

Info

Publication number: CN115011532B
Application number: CN202210944148.5A
Authority: CN
Inventors: 满朝新; 姜毓君; 张宇; 苏悦; 崔政盈
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2022-08-08
Filing date: 2022-08-08
Publication date: 2022-11-15
Anticipated expiration: 2042-08-08
Also published as: CN115011532A

Abstract

The invention discloses a preparation method of a lactobacillus paracasei JY062 preparation, which comprises the following steps: step one, lactobacillus paracasei JY062 (Lactobacillus paracasei JY 062) inoculating to an MRS culture medium by 5 percent of inoculation amount, after culturing for 16-18 h at 32-37 ℃, streaking three regions on the MRS solid culture medium, selecting a single bacterial colony in the MRS culture medium after culturing, centrifuging at 6000-8000 r/min for at least 10 minutes after subculturing two generations, collecting fermentation supernatant, washing bacterial sludge with PBS for at least two times, then re-suspending with PBS, and enabling the viable count of bacterial suspension to reach at least 10 ⁹ CFU/mL. The invention also discloses a preparation obtained by the preparation method and application thereof. The preparation obtained by the invention has higher viable count and good application effect.

Description

Lactobacillus paracasei JY062 preparation, and preparation method and application thereof

Technical Field

The invention relates to a lactobacillus paracasei JY062 preparation, a preparation method and an application thereof, in particular to a lactobacillus paracasei JY062 preparation for improving hepatic injury caused by glycolipid metabolic disorder, a preparation method and an application thereof, and belongs to the technical field of microorganisms.

Background

Glycolipid metabolic disorders (GLMD) are a series of diseases which cause sugar and lipid metabolism disorders under the influence of genetic, psychological and environmental factors. The main pathological manifestations of GLMD are neuroendocrine dysfunction, insulin Resistance (IR), metabolic inflammation and gut flora disorders. The main clinical manifestations of the disease include Type 2 Diabetes mellitis, T2DM), hyperlipidemia, obesity, non-alcoholic fatty liver disease (NAFLD), atherosclerosis (AS), and the like, which occur singly or in combination. The cause of glycolipid metabolic disorder diseases is complex, the symptoms of the glycolipid metabolic disorder diseases continuously change along with the change of the disease course, especially, the occurrence of complications brings more obstacles to the treatment of the diseases, the clinical diagnosis and treatment mainly comprise single disease species and branch diagnosis and treatment, the focus is mainly on a specific disease, and the prevention and control effect of the diseases is not ideal if comprehensive integral treatment is needed when the existence of a plurality of disease complications is ignored. The combined use of hypoglycemic and hypolipidemic drugs such as sulfonylureas, biguanides, alpha-glucosidase inhibitors and the like is generally adopted clinically, but some side effects of the drugs limit the long-term clinical application of the drugs, and the hypoglycemic and hypolipidemic drugs cannot improve liver injury. Therefore, the development of a novel natural and safe hypoglycemic agent or adjuvant therapy agent becomes important.

A great deal of research shows that the probiotics can regulate and control the internal environment homeostasis of the intestinal tract of an organism and relieve the glycolipid metabolic disturbance of an animal model or a clinical patient to different degrees by reducing inflammatory reaction, relieving oxidative stress reaction, improving the intestinal tract barrier, enhancing immune regulation and the like. In recent years, with the intensive study on the interaction between a host and an intestinal flora, the probiotics are considered to be feasible and have important research value for relieving GLMD by mediating the intestinal flora of patients. The invention develops a lactobacillus paracasei strain capable of improving glycolipid metabolic disturbance.

Disclosure of Invention

The invention aims to provide a preparation method of a lactobacillus paracasei JY062 preparation, and the preparation obtained by the method has higher viable count and good application effect.

Meanwhile, the invention provides a preparation obtained by the preparation method of the lactobacillus paracasei JY062 preparation.

Meanwhile, the invention provides application of the lactobacillus paracasei JY062 preparation in preparing a medicine for improving hepatic injury caused by glycolipid metabolic disorder.

Meanwhile, the invention provides application of the lactobacillus paracasei JY062 preparation in preparation of a medicine for improving glycolipid metabolic disorder.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the invention discloses a preparation method of a lactobacillus paracasei JY062 preparation, which comprises the following steps:

step one, lactobacillus paracasei JY062 (Lactobacillus paracasei JY 062) inoculating to an MRS culture medium by 5 percent of inoculation amount, after culturing for 16-18 h at 32-37 ℃, streaking three regions on the MRS solid culture medium, selecting a single bacterial colony in the MRS culture medium after culturing, centrifuging at least 10 min at 6000-8000 r/min after subculturing two generations, collecting fermentation supernatant, washing bacterial sludge at least twice by PBS, then re-suspending by PBS, and enabling the viable count of bacterial suspension to at least reach 10 ⁹ CFU/mL；

Step two, centrifuging the bacterial suspension, collecting bacterial sludge, adding a component I of the preparation solution into the bacterial sludge, wherein the volume ratio of the bacterial sludge to the component I is 1;

step three, adding the component II of the preparation solution into the bacterial suspension II, and uniformly mixing to obtain a bacterial suspension III;

step four, placing the bacterial suspension III in a constant-temperature water bath kettle, standing for at least 20 min at 35-37 ℃, and performing vacuum freeze drying to obtain bacterial powder;

the first component of the preparation solution comprises the following components in percentage by mass: 10-20% of skim milk, 8-12% of trehalose, 4-6% of maltodextrin, 2-4% of sodium L-glutamate and the balance of water;

the second component of the preparation solution comprises: 2 '-fucosyllactose (2' -FL), wherein the adding volume of the 2 '-fucosyllactose (2' -FL) is 1 to 3 percent of the first component, and the second component is 5 to 10wt percent of 2 '-fucosyllactose (2' -FL) aqueous solution;

the viable count of the bacterial powder is at least 1.23 × 10 ¹¹ CFU/mL。

The MRS culture medium has the following formula: 5.0 g of peptone, 10.0 g of tryptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 2.6 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate and 1 mL of Tween-80; the preparation method of the MRS culture medium comprises the following steps: accurately weighing the reagents according to the formula, fixing the volume in 1000 mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 min, and storing at 4 ℃ for later use.

The MRS solid culture medium has the following formula: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 14.0 g of agar and 1 mL of Tween-80; the preparation method of the MRS solid culture medium comprises the following steps: accurately weighing the above reagents according to the above formula, fixing the volume in 1000 mL of distilled water, mixing well, adjusting pH to 5.8, sterilizing at 121 deg.C for 15 min,

putting into a constant temperature water bath kettle at 60 ℃ for standby.

The vacuum freeze drying process comprises the following steps: placing the treated bacterial suspension III into an ultra-low temperature refrigerator for pre-freezing at the temperature of minus 80 ℃ for 2 hours; and (3) starting a vacuum freeze dryer in advance, putting the pre-frozen bacterial suspension III on a partition plate of the vacuum freeze dryer, vacuumizing and freeze-drying, wherein the temperature of a cold trap of the freeze dryer is-53.2 ℃, the vacuum degree is 0.162 mbar, and the time is 24 hours.

The PBS is 0.1mol/L, and the pH value is 6.8.

Meanwhile, the invention discloses a preparation obtained according to the preparation method.

Meanwhile, the invention discloses application of the preparation in preparing a medicament for improving hepatic injury caused by glycolipid metabolic disturbance.

Meanwhile, the invention discloses application of the preparation in preparing a medicine for improving glycolipid metabolic disorder.

The invention has the following beneficial effects:

the invention discloses lactobacillus paracasei JY062, and belongs to the technical field of food microorganisms. The lactobacillus paracasei JY062 preparation used by the invention comprises the following components in a preparation formula according to the total mass fraction of 100 percent: comprises 10 to 20 percent of skim milk, 8 to 12 percent of trehalose, 4 to 6 percent of maltodextrin, 2 to 4 percent of sodium L-glutamate and the balance of water; the second component of the preparation formula is 2 '-fucosyllactose (2' -FL), and the adding volume of the 2 '-fucosyllactose (2' -FL) is 1 to 3 percent of that of the first component; the 2 '-fucosyllactose (2' -FL) has good probiotic function and synergistic probiotic effect, and can improve the activity of Lactobacillus paracasei JY062 bacteria in the preparation. The lactobacillus used in the invention is JY062 which is separated from Tibetan dairy products and is used for improving the hepatic injury of mice with glycolipid metabolic disturbance induced by high-sugar and high-fat diet; obtaining liver and serum samples, and detecting the gene expression related to the fat factor and insulin level and the fat-insulin axis path so as to evaluate the influence of lactobacillus paracasei JY062 on glycolipid metabolism disorder; the result shows that Lactobacillus paracasei JY062 activates the expression of the APN-AMPK signal path, reduces the expression of SREBP-1c, ACC and FAS in lipid metabolism, and up-regulates the expression of GLUT-4 and PGC-1 alpha in sugar metabolism, thereby improving the glycolipid index and further relieving liver injury.

Drawings

FIG. 1 is a graph showing the results of measurement of viable count of a preparation;

FIG. 2 is a graph of the change in body weight and blood glucose of a mouse, wherein a) is the body weight of the mouse and b) is the blood glucose of the mouse;

FIG. 3 is a graph showing the glucose tolerance change in mice, wherein a) is the blood glucose level in mice, and b) is the glucose tolerance in mice;

FIG. 4 is a graph of the change in mouse adipokine and insulin, wherein a) is the leptin value (LEP), b) is the adiponectin value (ADPN), c) is the insulin value (INS), d) is the glucagon-like peptide-1 value (GLP-1), e) is the free fatty acid value (FFA);

FIG. 5 is a graph showing changes in gene expression of adiponectin-related genes (adipoQ and Adipor-2), AMPK, lipid metabolism-related genes (SREBP-1 c, FAS, ACC), and carbohydrate metabolism-related genes (GLUT-4, PGC-1. Alpha.) in mouse liver, wherein a) is the adipoQ gene, b) is the Adipor-2 gene, c) is the AMPK gene, d) is the SREBP-1c gene, e) is the FAS gene, f) is the ACC gene, g) is the GLUT-4 gene, and h) is the PGC-1. Alpha. Gene;

FIG. 6 is a graph of HE staining of mouse liver, a) is liver of N groups of mice; b) Liver of HFD group mice; c) The liver of LPL group mice; d) Liver of LPM group mice; e) The liver of mice in LPH group; thin arrows represent hepatocyte edema; thick arrows represent steatosis;

FIG. 7 is a graph of HE staining of mouse pancreas, a) is N groups of mouse pancreas; b) Pancreas of mice in HFD group; c) The pancreas of mice in LPL group; d) The pancreas of mice in LPM group; e) The pancreas of mice in LPH group.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

In the invention, the Latin name of lactobacillus paracasei JY062 isLactobacillus paracasei JY062, deposited in the northeast agriculture university dairy products focus laboratory, the Lactobacillus paracasei JY062 of the invention has prior art sources: adhesion and tolerance evaluation of high-yield exopolysaccharide hypoglycemic lactobacillus paracasei JY062 (TD 062) [ J]The chinese dairy industry, 2022, 50 (4).

Example 1

1.1 Experimental reagents

(1) MRS culture medium

For culturing lactobacillus paracasei JY062, the formula of the culture medium is as follows: 5.0 g of peptone, 10.0 g of tryptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 2.6 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate and 1 mL of Tween-80; the preparation method of the MRS culture medium comprises the following steps: accurately weighing the reagents according to the formula, fixing the volume in 1000 mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 min, and storing at 4 ℃ for later use.

(2) High-fat high-sugar feed

The high-fat high-sugar feed comprises 65% of basic feed, 20% of cane sugar, 10% of lard oil, 2.5% of cholesterol, 1% of sodium cholate, 1.5% of egg, sesame oil, peanut and the like which are mixed and blended.

(3) Adipokine, insulin Elisa kit (shanghai xinle); primeScript RT reagent Kit, SYBR Premix Ex TaqTM II (Dalianbao Biotechnology engineering Co., ltd.).

(4) Gene primer:

TABLE 1 Gene primer sequences

1.2 culture of Lactobacillus paracasei JY062

Inoculating lactobacillus paracasei JY062 to an MRS culture medium by 5% of inoculation amount, culturing at 37 ℃ for 18 hours, streaking three regions on the MRS solid culture medium, selecting a single bacterial colony in the MRS culture medium after culturing, centrifuging at 6000 r/min after subculturing for two generations for 10 min, collecting fermentation supernatant, washing bacterial sludge twice by PBS (0.1 mol/L, pH value 6.8), then re-suspending by PBS, and enabling the number of viable bacteria of bacterial suspension to reach about 10 ⁹ CFU/mL。

1.3 preparation of Lactobacillus paracasei JY062 preparation

(1) Preparation of a bacterial suspension of the formulation

Optimization group 1: the Lactobacillus paracasei JY062 is cultured according to the conditions, so that the viable count of the bacterial suspension reaches about 10 ⁹ CFU/mL, centrifuging, concentrating, collecting, adding the prepared preparation solution (2 '-fucosyllactose (2' -FL) mixed therein), and mixing to obtain bacterial suspension. Placing the bacterial suspension in a constant-temperature water bath kettle, standing at 37 ℃ for 20 min, and aseptically transferring into a glass plate.

Optimization group 2: the Lactobacillus paracasei JY062 is cultured according to the conditions, so that the viable count of the bacterial suspension reaches about 10 ⁹ CFU/mL, after centrifugation, concentration and collection, the prepared preparation solution (2 '-fucosyllactose (2' -FL) was added to the bacterial sludge and mixed well to prepare a bacterial suspension, and 5wt% of the solubilized 2 '-fucosyllactose (2' -FL) was added and further mixed well. Placing the bacterial suspension in a constant-temperature water bath kettle, standing at 37 ℃ for 20 min, and aseptically transferring into a glass plate.

Specifically, the first component of the preparation solution comprises the following components in percentage by mass: 15% of skim milk, 10% of trehalose, 5% of maltodextrin, 3% of sodium L-glutamate and the balance of water;

the second component of the preparation solution comprises: 2 '-fucosyllactose (2' -FL), said 2 '-fucosyllactose (2' -FL) being added in a volume corresponding to 2% of component one.

Control group: the conditions were the same as those in the optimized group for the ordinary lyoprotectant without the addition of 2 '-fucosyllactose (2' -FL).

(2) Vacuum freeze drying method

And pre-freezing the prepared bacterial suspension in an ultra-low temperature refrigerator at-80 ℃ for 2 h. And (3) starting a vacuum freeze dryer in advance, quickly putting the pre-frozen sample on a partition plate, and vacuumizing for freeze drying. The temperature of the cold trap of the freeze dryer is-53.2 ℃, the vacuum degree is 0.162 mbar, and the time is 24 hours. And (4) collecting the bacterial powder after freeze-drying is finished, and determining the bacterial activity in the bacterial powder.

(3) Carrying out 10-time serial gradient dilution on the freeze-dried bacterial powder by using sterile PBS (phosphate buffer solution), adding 1 mL of diluent into a culture dish after reaching a proper gradient, and determining the number of viable bacteria by adopting a plate counting method, wherein the culture medium is MRS agar. The culture dish is placed in an incubator at 37 ℃ for 48 hours for data statistics.

(4) Viable count of lactobacillus paracasei JY062 preparation: as can be seen from FIG. 1, the viable count obtained by optimizing the formulation of group 2 was 1.23X 10, which is higher ¹¹ CFU/mL, and has good application effect.

1.4 mouse protocol

60 male C57BL/6J mice are selected for the experiment, the age of the mice is about three weeks, and the mice are randomly divided into 5 groups: n: (normal group), HFD: model group, LPH: high dose lactobacillus paracasei JY062 formulation group, LPM: medium dose lactobacillus paracasei JY062 formulation group and LPL: low dose lactobacillus paracasei JY062 formulation group. The temperature of the rat room is 22 +/-2 ℃, the humidity is 55 +/-5%, the rat room is alternately used for 12 hours day and night, and the rat room is fed with the basal feed adaptively for one week. Starting from the second week, the HFD, LPL, LPM and LPH groups were fed with high-fat high-sugar diet. After three weeks of modeling, the random blood sugar of the mice is measured, and the mice with the blood sugar value higher than 11.1 mmol/L are successful models. Then, 9 am, LPH, LPM and LPL groups were gavaged 10 mL/kg at a dose of 10 mL/kg, respectively, every day ⁹ 、10 ⁸ And 10 ⁷ The bacterial suspension of the Lactobacillus paracasei JY062 of CFU/mL and the normal saline with the same dosage for intragastric administration of the N and HFD groups.

In this example, the number of viable bacteria in the bacterial powder was 1.23X 10 ¹¹ CFU/mL, adding physiological saline to the bacterial powder for gradient dilution to 10 ⁹ 、10 ⁸ And 10 ⁷ CFU/mL; and (4) intragastrically filling the prepared bacterial suspension.

1.5 mouse weight determination

Mouse body weights were measured and recorded weekly from the beginning of the first week to the end of the experiment.

As a result: the change in body weight of the mice in each group during the feeding period can be seen from fig. 2 a), the mice in the N groups are always on a normal diet during the feeding period, and the body weight of the mice in the group tends to be gradually flat after increasing. The body weight of the mice in the HFD group is increased by 25.5 g compared with that in the N group and is higher than that in the normal diet mice in the second to fourth weeks of the test, the body weight of the mice in the HFD group is increased by 45.50 percent compared with that in the N group, the body weight of the mice in the HFD group is continuously increased in the fifth to tenth weeks of the test, while the body weight of the mice in the LPL group, the LPM group and the LPH group of the Lactobacillus paracasei JY062 is increased slowly and is obviously lower than that in the HFD group, and the body weight of the mice in the LPL group, the LPM group and the LPH group is increased by 4.025 g, 3.57 g and 3.07 g and is lower than that in the HFD group (P < 0.05) compared with that in the fourth week of the test. In conclusion, the lactobacillus paracasei JY062 has the effect of relieving the weight gain of the glycolipid metabolic disorder mice, and the effect of the high-dose lactobacillus paracasei JY062 is more remarkable.

1.6 mouse blood glucose and oral glucose tolerance assays

1.6.1 mouse blood glucose assay: from the beginning of the first week to the end of the experiment, the body weight and Fasting Blood Glucose (FBG) before meals of the mice were measured at fixed time each week, before measurement, the mice were fasted overnight without water deprivation, and the FBG values were measured by taking tail blood. The results are shown in FIG. 2 b), the fasting plasma glucose of the mice in each group during the test period. It can be observed from the figure that the blood glucose levels of the N groups of mice were stable throughout the test period. From the second to the fourth week of the experiment, there was no difference in blood glucose in the other groups compared to the N group; in the fifth week of the experiment, the blood glucose of the mice in the HFD group was significantly increased to 8.83 mmol/L, which indicates that the sugar metabolism of the mice was changed after feeding high-fat high-sugar diet. The HFD group of mice was consistently at high blood glucose levels for five to eleven weeks of the study. The blood glucose levels in the LPL group, LPM group and LPH group were less different than those in the HFD group in five to seven weeks of the experiment, while the blood glucose levels in the LPL group, LPM group and LPH group were decreased than those in the HFD group in the eighth to eleventh week of the experiment. At the end of the eleventh week of the experiment, the blood glucose levels in LPL, LPM and LPH groups were 7.44 mmol/L, 7.00 mmol/L and 6.29 mmol/L, respectively, while the blood glucose level in HFD group was 9.17 mmol/L.

1.6.2 oral glucose tolerance test in mice: at the end of the experiment in the eleventh week, the mice were fasted overnight for 12 h without water deprivation, and 3 mice were selected to be given 40% glucose solution and were gavaged at a volume of 5 mL/kg, with a gavage standard of 2 g/kg glucose. Blood glucose levels were measured at fasting and glucose load 30 min, 60 min, 120 min, respectively, and the area under the glucose curve (AUC) was calculated.

As can be seen from FIG. 3 a), the blood glucose metabolism of the N group of mice was normal after gavage of glucose, the blood glucose level first gradually increased, reaching a peak at 30 min, with a maximum blood glucose value of 12.06 mmol/L, and then its value rapidly decreased and approached the normal blood glucose value at 2 h (6.83 mmol/L). The results show that the islet cells of the N groups of mice have normal functions and the body has good regulating capacity on blood sugar. However, in the HFD, LPL, LPM and LPH group mice with high-sugar and high-fat diet intervention, blood glucose level rapidly increased after gavage glucose and reached a peak at 30 min, which was 18.6 mmol/L,17.0 mmol/L,16.7 mmol/L and 14.7 mmol/L respectively, and the difference was significant compared with the group N (P < 0.05). The blood sugar value of the mice shows a slow descending trend after 30 min, the blood sugar value of each group of mice does not recover to the initial level at 2 h, the blood sugar concentration of the HFD group of mice is still at a high level and is 12.6 mmol/L, the blood sugar value of the LPL group of mice, the LPM group of mice and the LPH group of mice is obviously lower than that of the HFD group of mice (P is less than 0.05), the blood sugar value of the LPL group of mice reaches 11.07 mmol/L at 2 h, the blood sugar value of the LPM group of mice reaches 9.53 mmol/L, and the blood sugar value of the LPH group of mice reaches 7.77 mmol/L. The result shows that the lactobacillus paracasei JY062 can obviously inhibit abnormal rise of blood sugar after the glucose is perfused into the stomach of a glycolipid metabolic disorder mouse, can normalize the function of islet cells, has dose dependence, and has the best effect of the high-dose lactobacillus paracasei JY 062.

According to the oral glucose tolerance curve, the area under the peak curve (AUC) is made, and the AUC can be used to represent the degree of impaired glucose tolerance, and the larger the AUC value is, the more serious the impaired glucose tolerance is, and the result is shown in fig. 3 b). As can be seen from the figure, the AUC values for the HFD group are significantly higher than for the N group. Compared with the N group, the AUC value of the HFD group is increased by 62.41 percent (P < 0.05), which indicates that the blood sugar metabolism of the mice in the HFD group is abnormal and the glucose tolerance capability is impaired. Whereas AUC values of LPL, LPM and LPH groups were significantly reduced compared to HFD group (P < 0.05), and AUC value of LPH group was lowest, reduced by 27.08% compared to HFD group. The result shows that the lactobacillus paracasei JY062 can enhance the glucose tolerance of the glycolipid metabolic disorder mouse body to a certain extent, the effect is dose-dependent, and the effect of the high-dose lactobacillus paracasei JY062 is the best.

1.7 measurement of mouse fat factor and insulin

At the end of the experiment, five groups of mice were fasted overnight for 12 h, weighed, bled by the eyeballs, blood samples were centrifuged at 3000 Xg for 10 min at 4 ℃, and serum was collected and its content was determined according to the kit instructions.

As a result: as shown in fig. 4, after feeding with high-sugar and high-fat diet, the contents of Leptin (LEP), insulin (INS) and Free Fatty Acid (FFA) in HFD group mice were significantly increased (P < 0.05), 143%, 117% and 68.8%, respectively, while the contents of Adiponectin (ADPN), glucagon-like peptide-1 (GLP-1) were significantly decreased (P < 0.05), 64.25% and 71.02%, respectively, in LPL, LPM and LPH group mice, as compared to HFD group mice, the contents of Leptin (LEP), insulin (INS) and Free Fatty Acid (FFA), adiponectin (ADPN), glucagon-like peptide-1 (GLP-1) were significantly decreased (P < 0.05); among them, the LPH group showed the best effect, and showed 54.34%, 39.95% and 40.68% decrease in Leptin (LEP), insulin (INS) and Free Fatty Acid (FFA) and 153% and 135% increase in Adiponectin (ADPN) and glucagon-like peptide-1 (GLP-1), respectively (P < 0.05), compared with the HFD group.

1.8 quantitative PCR analysis of mouse liver tissue

mRNA in mouse liver tissue was extracted according to the kit instructions and reverse-transcribed into cDNA, and the resulting cDNA was reacted according to the reaction system and reaction conditions shown in Table 2 below.

TABLE 2 Real-time RT-PCR reaction System

TABLE 3 Real-time RT-PCR reaction conditions

As a result: as shown in FIG. 5, the relative expression levels of adiponectin related genes (AdipoQ and AdipoR-2), AMPK, glucose metabolism related genes (GLUT-4 and PGC-1. Alpha.) in the liver of the HFD group mice were significantly decreased (P < 0.05), and the relative expression levels of the lipid metabolism related genes (SREBP-1 c, ACC and FAS) were significantly increased (P < 0.05) compared to the N group mice after feeding the mice on the high-sugar high-fat diet. In the LPL, LPM and LPH groups, relative gene expression levels of adiponectin-related genes (AdipoQ and AdipoR-2), AMPK, sugar metabolism-related genes (GLUT-4 and PGC-1. Alpha.) were significantly up-regulated (P < 0.05), and relative gene expression levels of lipid metabolism-related genes (SREBP-1 c, ACC and FAS) were significantly down-regulated (P < 0.05) as compared to the HFD group, wherein the LPH group was most effective, and relative gene expression levels of adiponectin-related genes (AdipoQ and AdipoR-2), AMPK, sugar metabolism-related genes (GLUT-4 and PGC-1. Alpha.) were up-regulated by 182%, 202%, 384%, 138% and 185%, and relative gene expression levels of lipid metabolism-related genes (SREBP-1 c, ACC and FAS) were down-regulated by 47.2%, 44.6% and 50.9% (P < 0.05), respectively

1.9 mouse liver and pancreas HE staining

Mouse liver and pancreatic tissues were fixed in 4% paraformaldehyde solution for 24H, then dehydrated, cleared, paraffin embedded, sectioned and stained (H & E). The samples were then imaged under an optical microscope and examined histologically.

As a result: the liver tissue structure of N mice is slightly abnormal, part of liver cells are slightly edematous, the nucleus is clear, and cytoplasm vacuoles as shown by a thin arrow in figure 6; inflammatory cell infiltration and steatosis are not seen in the liver parenchyma; the liver tissue structure of the mice in the HFD group is severely abnormal, and large-area liver cell steatosis is observed, so that a large number of fat drops with different sizes can be seen, as shown by thin arrows in figure 6; some cells were heavily edematous, the cells were swollen and the cytoplasm vacuolated, as indicated by the thick arrows in FIG. 6. Compared with the HFD group, after different doses of the Lactobacillus paracasei JY062 preparation are used for dry prognosis, the degree of edema and steatosis of the liver cells is reduced, the arrangement of the liver cells is homogenized, the gap between the liver cells is reduced, and the effect of the LPH group is best similar to the state of the liver cells of N groups of mice.

As shown in fig. 7, the pancreatic tissue structure of N groups of mice was clear, with no vacuolization and inflammatory cell infiltration; the islets are spherical cell cluster-like structures, are distributed among acini, have clear boundaries with surrounding glands, are regularly arranged in the cells in the islets, have higher cell density and are rich in cytoplasm; the islets of the HFD mice were distributed between the acini, with unclear boundaries with the surrounding glands, and the number of islets was also significantly reduced, with a large number of inflammatory cell infiltrates visible, as indicated by the arrows in FIGS. 7 b) and 7 c). Compared with the HFD group, after different doses of the Lactobacillus paracasei JY062 preparation are dried, the islet cells are arranged regularly, the number of the islets is increased gradually, the boundaries of the islets and the surrounding glands are clear gradually, and partial telangiectasia phenomena occur in the M group, as shown by arrows in figure 7 d). The results show that the lactobacillus paracasei JY062 has a repairing effect on pancreas of mice with glycolipid metabolism disorder, is dose-dependent, and has the most obvious effect in an LPH group.

The Lactobacillus paracasei JY062 preparation selected by the invention has the capability of regulating and controlling the level of fat factors and insulin on a fat-insulin axis, and can reduce the expression of SREBP-1c, ACC and FAS in lipid metabolism and up-regulate the expression of GLUT-4 and PGC-1 alpha in glucose metabolism through an APN-AMPK pathway to improve glycolipid disturbance, thereby relieving liver injury. The Lactobacillus paracasei JY062 preparation can be applied to the alleviation and prevention of multi-organ injury caused by glycolipid metabolic disorder, and provides theoretical reference for developing safe and effective probiotic related products which are easy to produce and store and have the effect of alleviating liver injury caused by glycolipid metabolic disorder.

Example 2

The purpose of this example is to provide a preparation method of lactobacillus paracasei JY062 preparation, including the following steps:

step one, lactobacillus paracasei JY062 (Lactobacillus paracasei JY 062) inoculating to an MRS culture medium by 5 percent of inoculation amount, after culturing for 16 h at 32 ℃, streaking three regions on the MRS solid culture medium, selecting a single bacterial colony in the MRS culture medium after culturing, centrifuging for 20 min at 8000r/min after subculturing for two generations, collecting fermentation supernatant, washing bacterial sludge by PBS, wherein the PBS is 0.1mol/L, the pH value is 6.8, after washing for three times, re-suspending by PBS, and enabling the viable count of bacterial suspension to reach 10 ⁹ CFU/mL；

step four, placing the bacterial suspension III in a constant-temperature water bath, standing for 30 min at 35 ℃, and performing vacuum freeze drying to obtain bacterial powder;

the first component of the preparation solution comprises the following components in percentage by mass: 10% of skim milk, 8% of trehalose, 4% of maltodextrin, 2% of sodium L-glutamate and the balance of water;

the second component of the preparation solution comprises: 2 '-fucosyllactose (2' -FL), the added volume of the 2 '-fucosyllactose (2' -FL) is equivalent to 1% of the first component, and the second component is 10wt% of 2 '-fucosyllactose (2' -FL) water solution;

the viable count of the bacterial powder is 10 ¹¹ CFU/mL。

The formula of the MRS culture medium is as follows: 5.0 g of peptone, 10.0 g of tryptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 2.6 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate and 1 mL of Tween-80; the preparation method of the MRS culture medium comprises the following steps: accurately weighing the reagents according to the formula, fixing the volume in 1000 mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 min, and storing at 4 ℃ for later use.

The formula of the MRS solid culture medium is as follows: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 14.0 g of agar and 1 mL of Tween-80; the preparation method of the MRS solid culture medium comprises the following steps: accurately weighing the above reagents according to the above formula, fixing the volume in 1000 mL of distilled water, mixing well, adjusting pH to 5.8, sterilizing at 121 deg.C for 15 min,

putting into a constant temperature water bath kettle at 60 ℃ for standby.

The vacuum freeze drying process comprises the following steps: placing the treated bacterial suspension III into an ultra-low temperature refrigerator for pre-freezing at the temperature of minus 80 ℃ for 2 hours; and (3) starting the vacuum freeze dryer in advance, putting the pre-frozen bacterial suspension III on a partition plate of the vacuum freeze dryer, vacuumizing and freeze-drying, wherein the temperature of a cold trap of the freeze dryer is-53.2 ℃, the vacuum degree is 0.162 mbar, and the time is 24 hours.

The formulation obtained according to the preparation method described in this example.

Use of the formulation according to this example for the preparation of a medicament for ameliorating hepatic injury caused by a disturbance of glycolipid metabolism.

Use of a formulation according to this example in the manufacture of a medicament for ameliorating a disorder of glycolipid metabolism.

Example 3

This example differs from example 2 only in that: the first component comprises the following components in percentage by mass: 20% of skim milk, 12% of trehalose, 6% of maltodextrin, 4% of sodium L-glutamate and the balance of water;

the second component of the preparation solution comprises: 2 '-fucosyllactose (2' -FL), wherein the adding volume of the 2 '-fucosyllactose (2' -FL) is 3% of that of the first component, and the second component is 8wt% of 2 '-fucosyllactose (2' -FL) aqueous solution.

The above description is only of the preferred embodiments of the present invention, and it should be noted that: it will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the principles of the invention, and such modifications and adaptations are intended to be within the scope of the invention.

Claims

1. A preparation method of a Lactobacillus paracasei JY062 preparation is characterized by comprising the following steps:

step one, lactobacillus paracasei JY062 (Lactobacillus paracasei JY 062) inoculating 5% of inoculum size to an MRS culture medium, culturing for 16-18 h at 32-37 ℃, streaking three regions on the MRS solid culture medium, selecting a single bacterial colony in the MRS culture medium after culturing, centrifuging at 6000-8000 r/min for at least 10 min after subculturing two generations, collecting fermentation supernatant, washing bacterial sludge for at least two times by PBS, then re-suspending by PBS, and enabling the viable count of bacterial suspension to reach at least 10 ⁹ CFU/mL；

the second component of the preparation solution comprises: 2' -fucosyllactose, wherein the adding volume of the 2' -fucosyllactose is 1 to 3 percent of that of the first component, and the second component is 5 to 10 weight percent of 2' -fucosyllactose aqueous solution;

the viable count of the bacterial powder is at least 1.23 × 10 ¹¹ CFU/mL。

2. The preparation method according to claim 1, wherein the formulation of the MRS medium is as follows: 5.0 g of peptone, 10.0 g of tryptone, 5.0 g of beef extract, 5.0 g of yeast powder, 20.0 g of glucose, 2.6 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.58 g of magnesium sulfate, 0.25 g of manganese sulfate and 1 mL of Tween-80; the preparation method of the MRS culture medium comprises the following steps: accurately weighing the components according to the formula, fixing the volume in 1000 mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 min, and storing at 4 ℃ for later use.

3. The preparation method according to claim 1, wherein the formulation of the MRS solid medium is as follows: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0 g of glucose, 2.0 g of diammonium hydrogen citrate, 2.0 g of dipotassium hydrogen phosphate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, 14.0 g of agar and 1 mL of Tween-80; the preparation method of the MRS solid culture medium comprises the following steps: accurately weighing the components according to the formula, fixing the volume in 1000 mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 min,

putting into a constant temperature water bath kettle at 60 ℃ for standby.

4. The preparation method according to claim 1, wherein the vacuum freeze-drying process comprises: placing the treated bacterial suspension III into an ultra-low temperature refrigerator for pre-freezing at the temperature of minus 80 ℃ for 2 hours; and (3) starting a vacuum freeze dryer in advance, putting the pre-frozen bacterial suspension III on a partition plate of the vacuum freeze dryer, vacuumizing and freeze-drying, wherein the temperature of a cold trap of the freeze dryer is-53.2 ℃, the vacuum degree is 0.162 mbar, and the time is 24 hours.

5. The method of claim 1, wherein the PBS is 0.1mol/L and the pH value is 6.8.

6. A preparation obtained by the production method according to any one of claims 1 to 5.

7. Use of the formulation of claim 6 in the manufacture of a medicament for ameliorating a disorder of glycolipid metabolism.

8. Use of the formulation of claim 6 for the manufacture of a medicament for ameliorating hepatic injury caused by a disturbance of glycolipid metabolism.