CN117603828B - Lactobacillus rhamnosus LRa66 with blood glucose and blood lipid reducing functions and application thereof - Google Patents
Lactobacillus rhamnosus LRa66 with blood glucose and blood lipid reducing functions and application thereof Download PDFInfo
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- CN117603828B CN117603828B CN202310481728.XA CN202310481728A CN117603828B CN 117603828 B CN117603828 B CN 117603828B CN 202310481728 A CN202310481728 A CN 202310481728A CN 117603828 B CN117603828 B CN 117603828B
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Abstract
The invention relates to a lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat and application thereof, wherein the lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat is named as lactobacillus rhamnosus Lacticaseibacillus rhamnosus LRa66 strain, and the preservation number is CGMCC No.24282. The strain has good gastric acid resistance, intestinal juice resistance and bile salt resistance, can obviously improve metabolic syndrome reaction, especially hyperglycemia and hyperlipidemia, particularly shows that triglyceride, total cholesterol and low-density lipoprotein cholesterol in serum are reduced, improves serum IL-10 level, and inhibits the generation of TNF-alpha and IL-1 beta.
Description
Technical Field
The invention belongs to the technical field of probiotics, relates to lactobacillus rhamnosus LRa66 with blood sugar and blood fat reducing functions and application thereof, and in particular relates to lactobacillus rhamnosus LRa66 with blood sugar and blood fat reducing functions, a culture, a probiotic and application thereof.
Background
The pathological states of obesity, hypertension, dyslipidemia, diabetes, hyperuricemia, incidence of fatty liver and co-occurrence of hyperinsulinemia are called Metabolic Syndrome (MS). MS is the state in which multiple metabolic risk factors for cardiovascular disease build up in an individual. The main component of MS is obesity, especially visceral obesity, diabetes or impaired glucose regulation, dyslipidemia characterized by hypertriglyceridemia (TG) and low high density lipoprotein cholesterol (HDL-C) blood, and hypertension. In addition, MS is also involved in sustained low inflammatory response and abnormal blood coagulation and dissolution.
Obesity is a chronic, recurrent, progressive disease state, and in addition, obesity is associated with a variety of metabolic disorders including cardiovascular disease, hyperlipidemia, nonalcoholic fatty liver, type 2 diabetes, and the like. Studies have shown that obesity is essentially an excessive accumulation of lipids and inhibition of energy metabolism. Intestinal flora is a major contributor to the occurrence of diet-induced obesity and related metabolic dysfunction.
The incidence of obesity and related disorders such as type 2 diabetes mellitus, cardiovascular and cerebrovascular diseases, etc. has an increasing trend year by year. In addition to host genes, diet and other environmental factors can directly regulate body weight and insulin resistance, also have an effect on the composition of the intestinal flora. Dysbacteriosis occurs in obese and type 2 diabetics, and the most of the microorganisms reduced are butyrate-producing bacteria and bacteria with metabolic sulfate and antioxidant effects. Recent studies have shown that chronic inflammation caused by endotoxin bleeding due to dysregulation of intestinal flora is one of the important factors in the development of metabolic diseases such as obesity and diabetes. Dysbacteriosis and unbalance can cause systemic chronic reaction inflammation, thereby causing islet beta cell injury and insulin resistance reduction, and simultaneously affecting the absorption of other cells in the body to sugar so as to convert energy, so that the human body is difficult to normally operate, and finally a series of complications are caused.
Probiotics are active microorganisms and proper amount of administration is beneficial to host health. Both current clinical and experimental evidence suggests that intestinal flora is a potential causative agent of MS, and that all disease indications exhibited by it are the result of many factor interactions. Intestinal flora metabolites, such as represented by short chain fatty acids, are important causative factors of obesity, and are capable of regulating multiple metabolic functions, energy intake and even appetite in the host. Polysaccharides, short chain fatty acids, are both energy sources and can bind to G protein-coupled receptor 41 or GPR43 to regulate hormones or inflammatory responses. Meanwhile, in the complex interaction of intestinal flora and a host, the flora can generate pro-inflammatory factors, such as lipopolysaccharide, and induce the host to generate immune response related proteins so as to influence the stable state and composition of the flora, thereby causing imbalance of the intestinal flora and translocation of the flora, and further exacerbating the progress of obesity and insulin resistance. Choline, cholesterol and polysaccharides in foods can be directly metabolized by intestinal flora or further metabolized by a host and flora in combination to produce bioactive substances, e.g., choline can cause cardiovascular disease, and cholesterol can activate G protein-coupled cholic acid receptor 5 to increase energy consumption and GLP-1 secretion to protect the heart. Intestinal flora exacerbates the progression of fatty liver and NALFD by producing ethanol, altering choline and cholic acid metabolism, stimulating hepatocyte adipogenesis, and increasing intestinal permeability. Recent studies have demonstrated that acquired immunity is involved in deregulating the antibacterial flora, and thus there is increasing evidence that the intestinal flora can serve as a target for the treatment of MS.
Therefore, it is important to screen probiotics which can effectively reduce hyperglycemia and hyperlipidemia as main indexes for metabolic syndrome. At the same time, this has great significance for developing probiotics with higher health care value, and opens up new paths and solutions for alleviating metabolic syndrome by using dietary strategies.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat and application thereof, in particular to lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat, a culture, a probiotic and application thereof.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
In a first aspect, the invention provides lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat, the lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat is classified and named as lactobacillus rhamnosus Lactobacillus rhamnosus, the preservation number is CGMCC No.24282, and the preservation date is 2022, 1 month and 10 days.
The invention separates and stores a new lactobacillus rhamnosus strain capable of improving or reducing hyperglycemia and hyperlipidemia from human breast milk samples, which is named lactobacillus rhamnosus Lactobacillus rhamnosus LRa strain, has good gastric acid resistance, intestinal juice resistance and cholate resistance, can obviously improve metabolic syndrome reaction, especially hyperglycemia and hyperlipidemia, and particularly shows that Triglyceride (TG), total Cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) in serum are reduced, thereby effectively improving serum IL-10 level and inhibiting the production of TNF-alpha and IL-1 beta. LRa66 significantly reduced higher FINS levels in T2DM mice. Compared with the prior art, the lactobacillus rhamnosus LRa66 provided by the invention has better blood sugar and blood fat reducing effects and obvious advantages. When the strain is used for improvement or treatment, drug resistance is not generated, adverse reactions of patients are not caused in the improvement or treatment process, and the strain can be used for preparing products for improving or reducing hyperglycemia and blood fat, such as medicines and the like.
The screening steps of the LRa66 strain related to the invention are as follows:
(1) Selecting a human breast milk sample, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on an MRS solid culture medium (the culture medium contains an antibiotic mupirocin lithium salt which can inhibit most other strains except bifidobacteria, medicines are purchased from Haibo organisms), culturing at 37 ℃ for 48: 48 h, selecting colonies with different forms, performing streak purification on the surface of the MRS solid culture medium, selecting single colonies, performing expanded culture with the MRS liquid culture medium at 37 ℃, and preserving with glycerol with the mass concentration of 30%.
(2) And (3) carrying out in-vitro physiological characteristic test on the preserved 30 single bacteria derived from human milk, screening out a single strain with the best gastrointestinal fluid tolerance (artificial simulation), caco-2 cell adhesion capability, bacteriostasis capability and functional oligosaccharide utilization capability, and identifying the single strain.
In a second aspect, the present invention provides a culture of rhamnose cheese fungus LRa66 having hypoglycemic and hypolipidemic functions, the preparation method of the culture comprising: lactobacillus rhamnosus LRa66 according to the first aspect is inoculated in a medium and cultured at 18-24 h (e.g. 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24h, etc.) at 30-37 ℃ (e.g. 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 37 ℃, etc.).
Other specific point values within the above numerical ranges are all selectable, and will not be described in detail herein.
The invention preferably selects the culture conditions, the lactobacillus rhamnosus LRa66 strain can reach the growth stabilization period, and has more excellent carbon source utilization capacity.
The formula of the culture medium comprises the following components: peptone, beef extract, glucose, sodium acetate, yeast powder, diammonium hydrogen citrate, K 2PO4·3H2O、MgSO4·7H2O、MnSO4 and cysteine hydrochloride.
In a third aspect, the invention provides a probiotic with blood glucose and blood lipid reducing functions, which comprises lactobacillus rhamnosus LRa66 of the first aspect.
Preferably, in the probiotic agent, the viable count of lactobacillus rhamnosus LRa66 is not lower than 1×10 8 CFU/mL or 1×10 8 CFU/g, e.g. 1×108 CFU/mL、2×108 CFU/mL、5×108 CFU/mL、8×108 CFU/mL、1×109 CFU/mL、5×109 CFU/mL、1×1010 CFU/mL, etc. Other specific point values within the numerical range can be selected, and will not be described in detail herein.
The probiotic preparation comprises freeze-dried powder, capsules, tablets and granules.
Preferably, the probiotic agent with blood glucose and blood lipid reducing functions further comprises a protective agent and/or a prebiotic.
The protective agent comprises skimmed milk powder.
The prebiotic comprises any one or a combination of at least two of xylooligosaccharide, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, inulin, trehalose, soybean oligosaccharide, resistant dextrin, spirulina, polydextrose, alpha-lactalbumin or lactoferrin.
The lactobacillus rhamnosus LRa66 strain related to the invention can be applied to related products alone or in combination with other strains.
Preferably, the probiotic with the functions of reducing blood sugar and blood fat further comprises lactobacillus casei LC89 which is classified and named as lactobacillus casei Lactobacillus casei, and the preservation number is CGMCC No.15409, and the preservation date is 2018, 3, 5 days.
Preferably, the ratio of the viable count of lactobacillus casei LC89 to lactobacillus rhamnosus LRa66 is 3:1 to 1:3, for example, 3:1, 2:1, 1:1, 1:2, 1:3, etc., and other specific values within the numerical range may be selected, which will not be described in detail herein.
Preferably, the probiotic with the functions of reducing blood sugar and blood fat further comprises lactobacillus plantarum Lp90 which is classified and named as lactobacillus plantarum Lactobacillus plantarum, and the preservation number is CGMCC No.10453, and the preservation date is 2015, 1, 27.
Preferably, the ratio of the viable count of lactobacillus plantarum Lp90 to lactobacillus rhamnosus LRa66 is 3:1-1:3, for example, 3:1, 2:1, 1:1, 1:2, 1:3, etc., and other specific point values within the numerical range can be selected, which will not be described in detail herein.
The invention also creatively discovers that the lactobacillus rhamnosus LRa66 strain can be compounded with lactobacillus casei Lactobacillus casei LC strain and lactobacillus plantarum Lactobacillus plantarum Lp to obtain a compound probiotic used for reducing blood sugar and blood fat, has excellent effects, and compared with a single LRa66 strain, the compound probiotic has more remarkable effect of improving or treating hyperglycemia and hyperlipidemia, and shows that the LRa66 strain, the LC89 strain and the Lp90 strain have synergistic effect on the effects.
In the composite probiotic agent, the three strains have better synergistic effect when meeting the specific mass proportion relation.
In a fourth aspect, the invention provides the use of lactobacillus rhamnosus LRa66 according to the first aspect or the culture according to the second aspect or the probiotic according to the third aspect in the preparation of a medicament having hypoglycemic and hypolipidemic functions.
Compared with the prior art, the invention has the following beneficial effects:
The invention separates and stores a new lactobacillus rhamnosus strain capable of improving or reducing hyperglycemia and hyperlipidemia from human breast milk samples, and names the lactobacillus rhamnosus strain as lactobacillus rhamnosus LRa66 strain, the strain has good gastric acid resistance, intestinal juice resistance and bile salt resistance, caco-2 cell adhesion capability, bacteriostasis and strong capability of utilizing functional oligosaccharides, can obviously improve metabolic syndrome reaction, especially hyperglycemia and hyperlipidemia, and has the specific expression that compared with LRa66 group, HFD group serum TG, TC, LDL-C is obviously increased, and supplementing LRa66 can reduce HFD-induced dyslipidemia. Non-alcoholic fatty liver examples serum Triglyceride (TG), total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) levels were reduced, and T2DM examples were effective in improving serum IL-10 levels and inhibiting the production of TNF- α and IL-1β. LRa66 reduced higher FINS levels in T2DM mice. Provides an alternative for improving or reducing hyperglycemia and blood lipid. When the strain is used for improvement or treatment, drug resistance is not generated, adverse reactions of patients are not caused in the improvement or treatment process, and the strain can be used for preparing products for improving or reducing hyperglycemia and blood fat, such as medicines and the like.
Detailed Description
In order to further describe the technical means adopted by the present invention and the effects thereof, the following describes the technical scheme of the present invention in combination with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
Lactobacillus rhamnosus LRa66 used below is from a micro Kang Yisheng (su zhou) stock limited company strain library, and the strain is classified and named lactobacillus rhamnosus Lactobacillus rhamnosus, and is preserved in China general microbiological culture Collection center (CGMCC) for 1 month 10 of 2022, and has an address of North Star Xiyu 1, beijing, chaoyang area, 3, postal code 100101 and a preservation number of CGMCC No. 24282;
The lactobacillus casei LC89 used below is from a strain library of micro Kang Yisheng bacteria (Suzhou) stock, the strain is classified and named as lactobacillus casei Lactobacillus casei, and is preserved in China general microbiological culture Collection center (CGMCC) in 3 months and 5 days of 2018, the address is No. 3 in the West way 1 of the Chat area North Star of Beijing, the post code is 100101, and the preservation number is CGMCC No. 15409;
The lactobacillus plantarum Lp90 used below is from a strain library of a micro Kang Yisheng (Suzhou) stock, and is classified as lactobacillus plantarum Lactobacillus plantarum, and is preserved in China general microbiological culture Collection center (CGMCC) of China 1 month 27, with the address of Beijing Kogyo area North Star Xiya No. 1, accession number of 3, mail code 100101 and the preservation number of 10453;
RPMI 1640 medium was purchased from the Withanbozier Life technologies Co., ltd;
fetal bovine serum, PBS and trypsin were purchased from Thermo company;
MRS solid medium: weighing peptone 10 g, beef extract 10 g, glucose 20 g, sodium acetate 2 g, yeast powder 5 g, diammonium citrate 2 g, K 2PO4·3H2O 2.6 g、MgSO4·7H2O 0.1 g、MnSO4 0.05 g, agar 20 g and cysteine hydrochloride 0.5 g, dissolving with deionized water, adding 1mL Tween 80, fixing volume to 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use;
MRS liquid medium: weighing peptone 10 g, beef extract 10 g, glucose 20 g, sodium acetate 2 g, yeast powder 5g, diammonium citrate 2 g, K 2PO4·3H2O 2.6 g、MgSO4·7H2O 0.1 g、MnSO4 0.05 g and cysteine hydrochloride 0.5 g, dissolving with deionized water, adding 1 mL Tween 80, constant volume to 1L, sterilizing, and cooling.
Example 1
The method for screening lactobacillus rhamnosus capable of improving or reducing hyperglycemia and blood lipid comprises the following steps:
(1) Selecting a human breast milk sample, performing 10-time gradient dilution with physiological saline with the mass concentration of 0.9%, diluting for 3 times, coating on an MRS solid culture medium (the culture medium contains an antibiotic mupirocin lithium salt which can inhibit most other strains except bifidobacteria, medicines are purchased from Haibo organisms), culturing at 37 ℃ for 48: 48 h, selecting colonies with different forms, performing streak purification on the surface of the MRS solid culture medium, selecting single colonies, performing expanded culture with the MRS liquid culture medium at 37 ℃, and preserving with glycerol with the mass concentration of 30%.
(2) In vitro physiological characteristic test is carried out on the preserved single bacteria from human milk, and a single strain with the best gastrointestinal fluid tolerance (artificial simulation), caco-2 cell adhesion capability, bacteriostasis capability and functional oligosaccharide utilization capability is screened out, which is specifically as follows:
A. gastrointestinal fluid tolerance test:
Gastric juice is simulated manually: preparing 0.5% NaCl solution, adding 0.3% pepsin, adjusting pH to 2.5 with 1mol/L HCl, dissolving completely, and filtering with 0.22 μm microporous membrane for sterilization.
Manually simulating intestinal juice: preparing a 0.5% NaCl solution, adding 0.1% trypsin, adjusting the pH value to 8.0 by using 0.1mol/L NaOH, and filtering and sterilizing by using a 0.22 mu m microporous filter membrane after the solution is fully dissolved for later use.
Anaerobic culturing the strain to be selected in artificial gastric juice and intestinal juice for 3 hours, taking digestion liquid of 0 hour and 3 hours for detecting viable count, and calculating the survival rate. Strain survival rate (%) =b/a×100%, where a represents the number of viable bacteria (CFU/mL) of the bacteria for 0h, and B represents the number of viable bacteria (CFU/mL) of the bacteria for 3 h.
B. Caco-2 cell adhesion Capacity test:
Activating strains: the strain to be selected is inoculated into an anaerobic glass tube containing 0.1% of L-cysteine hydrochloride MRS liquid culture medium according to the inoculation amount of 2%, the culture is carried out for 14 hours at 37 ℃, fermentation liquor is taken, the bacteria are collected after centrifugation, PBS is used for washing 3 times, the bacteria are suspended in a DMEM culture solution without double antibodies, and the concentration of the bacterial suspension is regulated to be 10 8 CFU/mL.
Caco-2 cell culture: in a cell culture flask, 5mL of DMEM complete culture solution (containing 20% fetal calf serum, 1% penicillin and streptomycin solution) is added, the culture solution is incubated in a 5% CO 2 constant temperature incubator at 37 ℃ and changed 1 time a day, and after the cell state is good (70% -80% fusion), digestion and passage are carried out by using 0.2% digestive juice (pancreatin-EDTA).
Adhesion test: adjusting the concentration of digested Caco-2 cells to 1X 10 5 cells/mL, inoculating 1mL of the cells into a 12-well cell culture plate, incubating the cells to a monolayer in an incubator with a concentration of 5% CO 2, washing the cells twice with sterile PBS, digesting one well with pancreatin, and counting the cells with a blood cell counting plate; 1mL of suspension of the strain to be selected is respectively added into other holes, after the cells are incubated for 2 hours at 37 ℃ in a 5% CO 2 incubator, the sterile PBS is used for washing the cells for 5 times, the non-adhered bacterial suspension is removed, 0.2mL of pancreatin EDTA buffer solution is added into each hole to digest the cells for 5 minutes, after the digestion is finished, 0.8mL of PBS is added for blowing evenly, and bacterial liquid is taken for dilution and viable bacteria counting.
C. Antibacterial ability test:
The inhibition ability of pathogenic bacteria is tested by adopting an oxford cup method: the antagonistic strain was inoculated at 2% (V/V) into an anaerobic glass tube containing 0.1% L-cysteine hydrochloride MRS liquid medium, and cultured at 37℃for 12 hours. The pathogenic bacterial strains of escherichia coli, salmonella and staphylococcus aureus are respectively inoculated into a liquid beef extract peptone culture medium, cultured overnight at 37 ℃ and a rotating speed of 250rpm by a constant-temperature shaking table, and then pathogenic bacterial suspension is prepared. Cooling MRS solid culture medium to about 55deg.C, mixing with pathogenic bacteria suspension at a certain ratio to make the number of pathogenic bacteria in the system be in the order of 10 6 CFU/mL, then quickly pouring in a plate in which oxford cup is placed, taking out oxford cup after the culture medium is cooled and solidified, injecting 200 μl antagonistic strain bacteria liquid into each hole, placing the plate in a constant temperature incubator at 37deg.C after light cover, culturing for proper time, observing, and measuring the diameter of the bacteriostasis zone with vernier caliper.
D. Functional oligosaccharide availability test:
Sugar-free medium: 1% of peptone, 1% of beef extract, 0.5% of yeast extract, 0.2% of diammonium hydrogen citrate, 0.019% of K 2HPO40.2%、MgSO4 0.058%、MnSO4, 0.1% of tween-80 and 0.1% of L-cysteine hydrochloride.
Inulin, fructo-oligosaccharide, and galacto-oligosaccharide were purchased from Quantum Gao Ke (China) biological Co., ltd. And xylo-oligosaccharide (XOS) was purchased from Shandong Li Biotech Co., ltd.
Adding xylo-oligosaccharide (XOS), inulin, fructo-oligosaccharide and galacto-oligosaccharide into the sugarless culture medium according to 2% to obtain the culture medium containing oligosaccharide as carbon source. Inoculating the strain to be selected into a culture medium containing oligosaccharide, standing and culturing at a constant temperature of 37 ℃ for 12 hours, and measuring the OD600 absorbance to judge the utilization capacity of the strain to the functional strain.
LRa66 is the best strain combining the above gastrointestinal fluid tolerance (artificial simulation), caco-2 cell adhesion, bacteriostasis and experiments on the ability to utilize functional oligosaccharides.
Example 2
In this example, the strains obtained by screening in example 1 were subjected to morphological identification and 16S rRNA molecular biology identification, as follows:
(1) Morphological identification:
The strain was inoculated in MRS solid medium, cultured at 37℃for 48 h, and then observed under a microscope. The observation shows that the colony is milky white, is semicircular convex, has smooth and moist surface and neat edge.
(2) 16S rRNA molecular biology identification:
The strain preserved at-80℃was removed and inoculated into MRS liquid medium, and cultured at 37℃to 18 h. Sucking 1 mL bacterial liquid into a centrifuge tube, centrifuging 10 min under 8000 rpm, removing supernatant, and collecting bacterial body.
Extracting genome of the strain, adding bacterial universal primer for PCR amplification, and delivering amplified product to Shanghai biological engineering Co., ltd for sequencing identification. The strain is subjected to sequencing analysis, and the 16S rDNA sequence of the strain is shown as SEQ ID No. 1.
SEQ ID No: 1：
CGCCACCGGCTTCGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATCCATTGTAGCACGTGTAGCCCAGGTCATAAGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGCTTCAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCAACACCTAGCATTCATCGTTTACGTCAGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCATCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAACCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGATCACGCCGACAACAGTTACTCTGCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGCGGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTTTGGGCCGTGTCTCAGTCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGGATCATTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAAGCGATAGCTGACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGATTTATGCGGTATTAGCATCTGTTTCCAAATGTTATCCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCACTCGTTCAAAATTAAATCAAGATGCAAGCACCTTTCAATAATCAGAA.
And (3) comparing the sequences obtained by sequencing with nucleic acid sequences in GeneBank, wherein the results show that the strain is lactobacillus rhamnosus.
Example 3
This example demonstrates the gastric acid resistance of lactobacillus rhamnosus LRa66 as follows:
(1) Preparing artificial gastric juice:
The artificial gastric juice contains 0.20% of NaCl and 0.30% of pepsin by mass fraction, the pH is respectively regulated to 2.0, 2.5 and 3.0 by using HCl, and the artificial gastric juice is filtered and sterilized for standby.
(2) Gastric acid resistance test:
1.0 mL lactobacillus rhamnosus LRa66 bacterial suspension (the concentration is 1×10 9 CFU/mL, the bacterial liquid concentration is measured by a method in national standard food safety national standard microbiological detection of lactic acid bacteria detection, GB 4789.35-2016), is mixed with 9.0 mL pH artificial gastric juice of 2.0, 2.5 and 3.0 respectively, and is subjected to anaerobic stationary culture at 37 ℃, sampling is carried out after the beginning (0 h) and the treatment of 3 h respectively, the viable count is determined by a pour culture method, and the survival rate is calculated according to the following formula:
survival (%) =n1/n0×100%,
Wherein, N1: viable count after artificial gastric juice treatment of 3 h; n0: viable count of 0 h. The test results are shown in Table 1.
TABLE 1
As can be seen from table 1, lactobacillus rhamnosus LRa66 has good gastric acid resistance, and the survival rate can reach more than 80.0% when incubated in artificial gastric juice with the pH value of 2.0 for 3 h; incubating the mixture in artificial gastric juice with the pH of 2.5 for 3 h, wherein the survival rate can reach more than 90.4%; the survival rate can reach more than 95.6 percent after the artificial gastric juice with the pH value of 3.0 is incubated in the artificial gastric juice for 3 h. The good acid resistance creates conditions for preparing products for improving or reducing hyperglycemia and blood fat.
Example 4
This example demonstrates the intestinal fluid resistance of lactobacillus rhamnosus LRa66 as follows:
(1) Preparing artificial intestinal juice:
trypsin was dissolved in PBS at pH 8.0 to a final concentration of 0.8 g/L and filtered through a 0.22 μm filter to prepare simulated intestinal fluid.
(2) Intestinal juice resistance test:
1.0 mL of lactobacillus rhamnosus LRa66 bacterial suspension (the concentration is 1 multiplied by 10 9 CFU/mL, the bacterial liquid concentration is measured by a method in national standard of food safety national standard food microbiology detection lactobacillus detection, GB 4789.35-2016) is added into 9 mL simulated intestinal juice (pH 8.0), and after uniform mixing, the bacterial liquid is cultured at 37 ℃ for 8 h, and then the viable count is detected. The viability of the candidate probiotics was calculated by the following formula with MRS solid medium incubated at 37 ℃ for 48 h:
Survival (%) = [ log CFU N1/log CFU N0] ×100%;
wherein n1=the number of viable probiotic candidates after treatment with simulated intestinal fluid, n0=the number of viable probiotic candidates before treatment. The test results are shown in table 2:
TABLE 2
As can be seen from Table 2, the lactobacillus rhamnosus LRa66 has good intestinal juice resistance, and the survival rate can reach more than 74.3% after being incubated in artificial intestinal juice for 3 h. The good intestinal juice resistance creates conditions for preparing products for improving or reducing hyperglycemia and blood fat.
Example 5
This example demonstrates the bile salt tolerance of lactobacillus rhamnosus LRa66 as follows:
Lactobacillus rhamnosus LRa66, adjusted to a concentration of 1X 10 9 CFU/mL, was inoculated into 100mL of MRS-THIO (MRS contains 0.2% sodium thioglycolate) medium containing 0.3% bile salts, incubated at 37℃for 4h, and absorbance was measured at 600 nm. The experiment uses lactobacillus rhamnosus LGG as a control bacterium, and the culture method of the LGG bacterium is the same as that of LRa 66. The greater OD 600 value indicates a greater ability of the lactic acid bacteria to tolerate bile salts. The test results are shown in table 3:
TABLE 3 Table 3
As can be seen from table 3, after 4 hours of culture the OD600 of lactobacillus rhamnosus LRa66 was 0.38, higher than that of lactobacillus rhamnosus LGG, which suggests that lactobacillus rhamnosus LRa66 is higher in bile salt tolerance than that of lactobacillus rhamnosus LGG.
Example 6
The embodiment provides lactobacillus rhamnosus LRa66 bacterial powder, and the lactobacillus rhamnosus LRa66 bacterial powder is prepared by the following method:
Inoculating lactobacillus rhamnosus LRa66 strain into MRS liquid culture medium, culturing at 37deg.C for 24 h times for activation, and continuously activating for 2 times to obtain activating solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing at 37 ℃ for 24 h to obtain bacterial solution; centrifuging the bacterial liquid at 8000 g for 10 min to obtain lactobacillus rhamnosus thalli;
Re-suspending the thalli with 10% skimmed milk powder aqueous solution to 1×10 10 CFU/mL to obtain bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 1h, and then freeze-drying to obtain the lactobacillus rhamnosus LRa66 bacterial powder, wherein the number of viable bacteria of the detected bacterial powder is 300 hundred million CFU/g.
The lactobacillus casei LC89 bacterial powder is prepared by the same method, and the viable count of the bacterial powder is 300 hundred million CFU/g.
The lactobacillus plantarum Lp90 bacteria powder is prepared by the same method, and the viable count of the bacteria powder is 300 hundred million CFU/g.
Example 7
The present embodiment provides a method for reducing blood glucose and blood lipid in obese mice:
(1) The molding method comprises the following steps: female C57 BL/6J mice (6 weeks old) were purchased from Shanghai Laike laboratory animal center. 50 mice were kept under light and temperature controlled barrier maintenance conditions (light/dark cycle, 12 h/12 h; temperature, 22 ℃ + -1 ℃; relative humidity, 55% + -5%). After 2 weeks of acclimation, all mice were randomly divided into 5 groups, each group consisting of 10 mice, each: normal control group (CTL), model group (HFD) fed HFD (60% fat); the probiotic intervention group (LRa 66) was fed HFD (60% fat) at 3x 10 9 CFU/day; the probiotic intervention group (LC 89+ Lp 90) was fed HFD (60% fat) at 3x 10 9 CFU/day (viable count 1:1); the probiotic intervention group (lra66+lc89+lp 90) was fed HFD (60% fat) at 3×10 9 CFU/day (viable count 1:1:1). The tail vein Fasting Blood Glucose (FBG) level was determined by a glucometer (On Call Plus, hangzhou, china). Both CTL and HFD groups were given equal volumes of solvent by gavage-i.e. 200 μl of saline per mouse. All mice were given their respective feeds by gavage for 8 weeks. All mice were free to eat and drink throughout the study. Body weight was measured every 2 weeks.
(2) Serum collection: after the experiment is finished, all mice are fasted for 12 hours and are sacrificed by anesthesia with 10% chloral hydrate, the heart is punctured and fresh blood is collected, and after centrifugation for 10min at 3000 r/min, serum is preserved at-80 ℃.
(3) Serum biochemical analysis: triglyceride (TG), total Cholesterol (TC), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) levels in serum were measured using commercial kits (Nanjing's institute of biological engineering, nanjing, china) according to the instructions prescribed by the kits.
The body weight (g) changes of each group of mice are shown in Table 4.
TABLE 4 Table 4
From the results in Table 4, it can be seen that: the change in body weight of the mice showed the same trend at the 2 nd peripheral surface. Notably, the body weight gain trend was inhibited in the probiotic-intervention group after week 2 compared to the HFD group, with the lra66+lc89+lp 90 group being better inhibited. Supplementing LRa66 or a complex bacterial agent was suggested to reduce HFD-induced weight abnormalities.
The blood lipid results of each group are shown in Table 5.
TABLE 5
From the results in Table 5, it can be seen that: the probiotic intervention group mice had relatively reduced serum TG, TC and LDL-C compared to the HFD group; meanwhile, compared with a CTL group, the HDL-C of the mice serum of the probiotic intervention group is raised to a certain extent, which suggests that the supplementation of LRa66 or the compound microbial agent can reduce HFD-induced dyslipidemia.
Example 8
The present example explores the therapeutic effect of probiotics on non-alcoholic fatty liver mice:
(1) Animal model: 50 male Sprague Dawley rats, 6 weeks old and weighing 170-190 g, free of specific pathogen (Specific Pathogen Free, SPF), were fed 5 per cage for 12h light/dark cycles at tightly controlled temperature 22+ -2deg.C, with free feeding and drinking. After 2 weeks of acclimation, rats were randomly assigned to normal Control (CTL) groups (n=10) and HFD (high-fat set) groups (n=40). Animals in the CTL group were periodically fed regular normal rat diet, and animals in the HFD group were fed high fat diet. After 10 weeks of feeding, the HFD group 40 rats were further evenly randomized to HFD groups, probiotic LRa 66-interfered groups (LRa 66 at 3×10 9 CFU/day), probiotic LC89+ Lp 90-interfered groups (LC 89+ Lp90 at 3×10 9 CFU/day, viable count 1:1), probiotic LRa66+ LC89+ Lp 90-interfered groups (LRa 66+ LC89+ Lp90 at 3×10 9 CFU/day, viable count 1:1:1). To eliminate the biological rhythmic effects, the gastric lavage was continued for 4 weeks at 9:00 am each day.
(2) Serum biochemical analysis: the liver alanine Aminotransferase (ALT), liver aspartate transferase (AST) and Total Cholesterol (TC) and Triglyceride (TG) levels were measured using commercial kits (institute of biotechnology, built in south kyo).
(3) Inflammatory factor determination: the levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-10 (IL-10) were detected using a commercial ELISA kit (Nanjing, china) for the institute of biological engineering. As shown in table 6.
TABLE 6
From the results in Table 6, it can be seen that: the serum ALT, AST, TC, TG levels were higher in the HFD group compared to the CTL group. The probiotic-intervention group rats had reduced serum ALT, AST, TC, TG levels compared to the HFD group. Compared with HFD group, the content of TNF-alpha and IL-1 beta in rat serum is reduced after the intervention of probiotics, the content of IL-10 is improved, which shows that the intervention of probiotics can inhibit inflammatory reaction to a certain extent.
Example 9
This example explores the therapeutic effect of probiotics on type 2 diabetic (T2 DM) mice:
(1) Animal model: SPF male Kunming mice at 6 weeks of age were purchased from Shanghai Laek's test animal center and acclimatized in a climate controlled environment (22+ -1deg.C and 55+ -5% humidity, 12h light/dark cycles) for 1 week. Normal group eat standard feed (NC, n=8), diabetic group (n=32) eat high fat feed. After 4 weeks, all mice fasted 12 h. The diabetes group was intraperitoneally injected with streptozotocin (100 mg/kg. BW) and the CTL group was injected with an equal volume of buffer. Fasting glycemia (FBG) was measured by a glucometer (On Call Plus, hangzhou, china). After 1 week, mice with FBG.gtoreq.7.1 mmol/L were considered as T2DM. T2DM mice were randomly divided into 4 groups (8 per group): (1) T2DM group, oral administration of 0.2 mL sterile saline lavage; (2) LRa66 group, LRa66 was dissolved in sterile physiological saline, and 0.2 ml,3×10 9 CFU/day LRa66 was intragastric; (3) LC 89+Lp90 group, 3× 9 CFU/day gastric lavage, viable count 1:1; (4) Lra66+lc89+lp 90 group, 3× 9 CFU/day gastric lavage, viable count 1:1:1.CTL groups were given only 0.2 mL sterile saline. FBG was measured weekly. After 6 weeks of LRa66 treatment, blood was collected by retroorbital bleeding after 12h fasting, and serum was centrifuged (1150×g,10 min,4 ℃). The pancreas, liver and colon contents were stored to-80 ℃ for further analysis.
(2) Oral glucose tolerance test: after 6 weeks of treatment, an Oral Glucose Tolerance Test (OGTT) was performed after 12h of fasting as described previously.
(3) And (3) biochemical parameter measurement: fasting serum insulin (FINS) levels were determined using an ELISA kit (Tianjin, china). Liver alanine Aminotransferase (ALT) levels and liver aspartate transferase (AST) levels were measured as in example 8.
(4) Inflammatory factor determination: serum Lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta), interleukin 10 (IL-10) level assays were as in example 8. The results are shown in Table 7.
TABLE 7
From the results in Table 7, it can be seen that: in the OGTT test, severe glucose intolerance was observed in the T2DM group. Furthermore, the probiotic-intervened group effectively reduced higher levels of FINS in T2DM mice compared to the CTL group. Bacterial product LPS is one of the causes of inflammation, and probiotic intervention reduces the inflammation of T2DM mice, and the probiotic intervention group can well reduce the serum LPS level of the T2DM mice. Similarly, the concentration of IL-1. Beta. And TNF-a was effectively reduced and the concentration of IL-10 was increased in the probiotic-interfered mice as compared to the T2DM mice. LRa66 effectively improved serum IL-10 levels and inhibited TNF-a and IL-1β production. Compared to CTL group, liver ALT and AST levels were higher in T2DM group, LRa66 effectively reduced liver AST levels, suggesting that LRa66 increased liver protection in T2DM mice.
The applicant states that the present invention is described by the above embodiments as lactobacillus rhamnosus LRa66 with blood glucose and blood lipid lowering functions and applications thereof, but the present invention is not limited to the above embodiments, i.e. it does not mean that the present invention must be implemented depending on the above embodiments. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Claims (10)
1. The lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat is characterized in that the lactobacillus rhamnosus LRa66 with the functions of reducing blood sugar and blood fat is classified and named as lactobacillus rhamnosus Lactobacillus rhamnosus, the preservation number is CGMCC No.24282, and the preservation date is 2022, 1 month and 10 days.
2. A culture of lactobacillus rhamnosus LRa66 with blood glucose and blood lipid reducing functions, characterized in that the preparation method of the culture comprises the following steps: inoculating lactobacillus rhamnosus LRa66 of claim 1 into a culture medium and culturing at 30-37 ℃ for 18-24 h; the formula of the culture medium comprises the following components: peptone, beef extract, glucose, sodium acetate, yeast powder, diammonium hydrogen citrate, K 2PO4·3H2O、MgSO4·7H2O、MnSO4 and cysteine hydrochloride.
3. A probiotic with blood glucose and blood lipid reducing function, characterized in that the probiotic with blood glucose and blood lipid reducing function comprises lactobacillus rhamnosus LRa66 of claim 1.
4. The probiotic preparation with hypoglycemic and hypolipidemic functions according to claim 3, wherein in the probiotic preparation, the viable count of lactobacillus rhamnosus LRa66 is not lower than 1x10 8 CFU/mL or 1x10 8 CFU/g;
the probiotic preparation comprises freeze-dried powder, capsules, tablets and granules.
5. A probiotic with hypoglycemic and hypolipidemic functions according to claim 3, wherein the probiotic with hypoglycemic and hypolipidemic functions further comprises a protective and/or prebiotic;
the protective agent comprises skimmed milk powder;
The prebiotic comprises any one or a combination of at least two of xylooligosaccharide, fructooligosaccharide, galactooligosaccharide, mannooligosaccharide, inulin, trehalose, soybean oligosaccharide, resistant dextrin, spirulina, polydextrose, alpha-lactalbumin or lactoferrin.
6. The probiotic preparation with blood sugar and blood lipid reducing function according to claim 3, further comprising lactobacillus casei LC89, which is classified and named as lactobacillus casei Lactobacillus casei, with a preservation number of CGMCC No.15409 and a preservation date of 2018, 3 and 5 days.
7. The probiotic with blood glucose and blood lipid reducing function according to claim 6, wherein the ratio of viable count of lactobacillus casei LC89 to lactobacillus rhamnosus LRa66 is 3:1-1:3.
8. The probiotic preparation with blood sugar and blood lipid reducing function according to claim 3, further comprising lactobacillus plantarum Lp90, which is classified and named as lactobacillus plantarum Lactobacillus plantarum, with a preservation number of CGMCC No.10453 and a preservation date of 2015, 1 and 27.
9. The probiotic with blood sugar and blood lipid reducing function according to claim 8, wherein the ratio of viable count of lactobacillus plantarum Lp90 to lactobacillus rhamnosus LRa66 is 3:1-1:3.
10. Use of lactobacillus rhamnosus LRa66 according to claim 1 or the culture according to claim 2 or the probiotic according to any of claims 3-9 for the preparation of a medicament with hypoglycemic and hypolipidemic functions.
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