CN110777098A

CN110777098A - Lactobacillus with effect of preventing and treating diabetic encephalopathy and separation method and application thereof

Info

Publication number: CN110777098A
Application number: CN201911194149.7A
Authority: CN
Inventors: 方曙光; 郝沛妍; 朱建国; 陈珂可; 于惠; 程文; 蒋大成
Original assignee: Jiangsu Wecare Biotechnology Co ltd
Current assignee: WeCare Probiotics Co Ltd
Priority date: 2019-11-28
Filing date: 2019-11-28
Publication date: 2020-02-11
Anticipated expiration: 2039-11-28
Also published as: CN110777098B

Abstract

The invention relates to lactobacillus with the effect of preventing and treating diabetic encephalopathy and a separation method and application thereof. The Lactobacillus is named as Lactobacillus paracasei (Lactobacillus paracasei) LC86 strain, the preservation unit is China general microbiological culture Collection center, the preservation time is 12 months in 2013, the preservation number is CGMCC 1.12731, and the address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North. It can improve nitrogen monoxide content increase caused by diabetic encephalopathy, improve brain tissue antioxidase activity, and improve learning and memory ability. The compound can be applied to the medicines for preventing and treating the diabetic encephalopathy, has no toxic or side effect, and can be used for treating clinical patients together with other nutrients.

Description

Lactobacillus with effect of preventing and treating diabetic encephalopathy and separation method and application thereof

Technical Field

The invention belongs to the field of chemical medicine, and particularly relates to lactobacillus as well as a separation method and application thereof. In particular to lactobacillus with the effect of preventing and treating diabetic encephalopathy and a separation method and application thereof.

Background

The diabetes has high morbidity, multiple complications, high lethality and high disability rate, and most of the diabetes is type II diabetes. The long-term rise of blood sugar of the diabetic patients can not be controlled, and various chronic complications can be caused, such as coronary heart disease, hypertension, myocardial infarction, cerebral apoplexy, senile dementia, Parkinson, nephropathy, retinopathy and the like. Diabetic complications have become a leading cause of death and disability in diabetic patients, including diabetic encephalopathy, which apparently has a serious impact on the quality of life of diabetic patients.

Diabetic encephalopathy is easily overlooked and the research is relatively delayed. Diabetic encephalopathy is one of the most common concomitant diseases, is occured with secret and chronic progressive exacerbation, is usually ignored, but once obvious clinical symptoms appear, the diabetic encephalopathy can be developed into various diseases such as cerebral apoplexy injury, senile dementia and the like, the pathological injury of the diabetic encephalopathy is irreversible, and no effective measure is available so far to prevent the development of the diabetic encephalopathy. In recent years, the influence of diabetes on the central nervous system has attracted great attention, but the research on preventing and treating diabetic encephalopathy starts to be later than other diseases such as diabetic nephropathy and retinopathy, and it is very critical to find out the pathogenesis and find out a proper medicament for preventing and treating diabetic encephalopathy.

Diabetic encephalopathy is characterized by acquired cognitive behavioral deficits. Cognitive deficits can occur in both type i and type ii diabetics, affecting their learning, memory and complex information processing processes. Adult type i diabetes is often characterized by mild and moderate cognitive decline, with relatively few severe patients, and forms of cognitive deficits reported differently from one patient to another, which may be related to factors such as the degree of cognitive impairment, the population studied, and the degree of glycemic control. Patients with type II diabetes also have mild and moderate cognitive decline, which is mainly manifested by decline of speech, memory and complex information processing abilities, and has little influence on exercise response time and instant memory. Patients under 60 years of age often present with mild cognitive deficits, generally without significant impact on daily life, while elderly patients have a 2-fold increased risk of dementia than normal. The neurophysiological and neuroradiographic features of diabetic encephalopathy are very similar to brain aging, and these findings suggest that diabetic encephalopathy reflects in many ways the process of accelerated brain aging.

The damage of diabetes to the brain is mainly manifested as cerebrovascular diseases (mainly including thrombotic cerebral infarction, cerebral embolism, transient ischemic attack, lacunar infarction and hemorrhagic stroke) and diabetic encephalopathy (chronic complications of diabetes, which are characterized by acquired cognitive impairment, caused by cerebrovascular change due to metabolic disorder of diabetes, damage to central nervous system, pathological changes of brain in structure, neurophysiology and neuropsychiatry, etc.). Diabetic encephalopathy often has a low perfusion state, which is related to high sugar and high fat.

In diabetic encephalopathy, hyperglycemia can exacerbate cerebral ischemic injury through the toxic effects of NO. In early cerebral ischemia, eNOS-mediated NO improves blood supply to ischemic areas by dilating blood vessels, has a short-term protective effect, but with the massive production of NO, the neurotoxic effect mediated by iNOS rapidly dominates, mediating neurotoxic effects in the progressive and late cerebral ischemia, respectively, causing neuronal damage.

The probiotics are active microorganisms beneficial to human bodies, and have the effect of promoting nutrient absorption and keeping intestinal tract healthy by regulating the immune function of host mucous membranes and systems or regulating the balance of flora in the intestinal tract, so that the effect of benefiting the health of the host is generated. The important role of the intestinal flora in systemic diseases is of increasing interest to more and more researchers, but there are fewer studies on the prevention and treatment of diabetic encephalopathy by probiotics.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide lactobacillus and a separation method and application thereof. In particular to lactobacillus with the efficacy of preventing and treating diabetic encephalopathy and a separation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

on one hand, the invention provides Lactobacillus with the effect of preventing and treating diabetic encephalopathy, which is named as Lactobacillus paracasei (Lactobacillus paracasei) LC86 strain, the preservation unit is China general microbiological culture Collection center, the preservation time is 12 months in 2013, the preservation number is CGMCC 1.12731, and the address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

The invention separates a novel lactobacillus, namely lactobacillus paracasei (Lactobacillus paracasei) LC86 strain, and discloses the application of the lactobacillus in preventing and treating the diabetic encephalopathy for the first time, the lactobacillus paracasei can obviously improve the content rise of nitric oxide caused by the diabetic encephalopathy, and can improve the activity of brain tissue antioxidase, the resistance to nerve cell apoptosis and the learning and memory ability. The compound can be applied to the medicines for preventing and treating the diabetic encephalopathy, has no toxic or side effect, and can be used for the treatment or the adjuvant treatment of clinical patients together with other nutrients.

In the invention, the lactobacillus is obtained by screening from a yoghourt product. The lactobacillus paracasei LC86 strain related by the invention is separated from the yoghourt, and has high safety and good ecological mutual assistance after screening, domestication and verification.

The optimal conditions for culturing the lactobacillus related by the invention are as follows: the culture medium is facultative anaerobic, and has a growth temperature in the range of 5 to 53 ℃ (e.g., 5 ℃, 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 37 ℃, 40 ℃ or 53 ℃), an optimum growth temperature of 37 ℃, and an optimum pH of 6.2 to 6.8 (e.g., pH6.2, pH6.3, pH6.5, or pH 6.8).

In another aspect, the present invention provides a method for isolating lactobacillus having efficacy of preventing and treating diabetic encephalopathy as described above, comprising:

diluting the yoghourt product, and then performing anaerobic culture on a sterile MRS culture medium to obtain bacterial colonies; and selecting the colony with regular morphology, separating and purifying the colony on a sterile MRS culture medium to obtain a single pure colony, and screening the lactobacillus with the effect of preventing and treating the diabetic encephalopathy.

Preferably, the temperature of the anaerobic culture is 35-39 ℃, such as 35 ℃, 36 ℃, 37 ℃, 38 ℃ or 39 ℃ and the like; the anaerobic culture time is 40-50h, such as 40h, 42h, 44h, 45h, 47h, 48h or 50 h.

Preferably, the formula of the MRS culture medium is as follows: 10.0g/L of casein peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 5.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diamine citrate, 801.0 g/L of tween-phosphate, 2.0g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate, 20.0g/L of calcium carbonate, 20.0g/L of agar, pH6.8, and sterilization at 121 ℃ for 20 min.

Preferably, the separation and purification means: selecting colony with regular morphology, and streaking for 3-10 times (such as 3 times, 5 times, 8 times or 10 times) on sterile MRS culture medium to achieve the purpose of separation and purification.

Preferably, the screening is performed using 16S rDNA sequence homology analysis.

In another aspect, the invention provides an application of the lactobacillus with the effect of preventing and treating the diabetic encephalopathy in preparing a medicament with the effect of preventing and treating the diabetic encephalopathy.

In the present invention, the lactobacillus increases the activity of superoxide dismutase (SOD) in brain tissue.

In the present invention, the lactobacillus inhibits the content of Malondialdehyde (MDA) in brain tissue.

In the present invention, the lactobacillus inhibits the activity of acetylcholinesterase (AchE) in brain tissue.

In the present invention, the lactobacillus inhibits the activity of iNOS in hippocampus.

In the invention, the lactobacillus can remarkably improve the learning and memory ability of the diabetic encephalopathy patient.

In the invention, the lactobacillus can remarkably control the increase of the blood sugar value of a diabetic encephalopathy patient.

Preferably, the viable count of the lactobacillus is not less than 1 × 10 ⁷CFU/g, e.g. 1X 10 ⁷CFU/g、2×10 ⁷CFU/g、5×10 ⁷CFU/g、1×10 ⁸CFU/g or 1X 10 ⁹CFU/g, and the like.

Preferably, the medicament further comprises pharmaceutically acceptable auxiliary materials.

Preferably, the excipient includes any one of an excipient, diluent, carrier, flavoring agent, binder or filler, or a combination of at least two such as a diluent and carrier, flavoring agent and binder, binder and filler, and the like. Any other combination is not described in detail herein.

Preferably, the carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

The lactobacillus provided by the invention can be loaded on a common medicinal carrier to be used as a medicament for preventing and treating diabetic encephalopathy, and better biocompatibility, targeting property, biological safety and administration effect are realized.

The lactobacillus can also be used together with other medicines or nutritional preparations to realize better treatment effect on the diabetic encephalopathy.

Preferably, the dosage form of the medicine comprises tablets, capsules, granules, powder, sprays, films, suppositories, nasal drops or dropping pills. The medicament taking the lactobacillus related to the invention as the active ingredient can be prepared into any one of the above medicament dosage forms according to actual needs, and the medicaments of each dosage form can be prepared according to the conventional method in the pharmaceutical field.

The administration route can be any one of oral administration, sublingual administration, intraperitoneal injection, intramuscular injection, subcutaneous injection, nasal administration or transdermal administration according to actual needs.

Compared with the prior art, the invention has the following beneficial effects:

the invention creatively discovers the strain for preventing and treating the diabetic encephalopathy, which has good curative effect and no toxic or side effect, creatively takes the nitric oxide in the hippocampus as a treatment target point, and creates a new mode for preventing and treating the diabetic encephalopathy. Animal experiments prove that the strain can obviously improve the learning and memory ability of rats with diabetic encephalopathy, effectively improve the SOD activity of brain tissues, effectively reduce the MDA content, effectively inhibit the AchE activity and inhibit the iNOS activity in hippocampus. The traditional Chinese medicine composition is used for preventing and treating the diabetic encephalopathy or used for the adjuvant therapy of the diabetic encephalopathy, has obvious curative effect, does not discover toxic and side effects, is convenient to use, and can greatly relieve the pain of the diabetic encephalopathy.

Drawings

FIG. 1 is a diagram (1000 times magnification) showing the morphology of an individual of Lactobacillus with efficacy of preventing and treating diabetic encephalopathy in accordance with the present invention.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

And (3) separating and identifying lactobacillus paracasei.

The specific method comprises the following steps: taking Qinghai old yoghourt (product model 57338258454) as a separation sample, diluting the separation sample, coating the diluted separation sample on a sterile MRS culture medium by using a coating rod, and performing anaerobic culture at 37 ℃ for 48 hours to obtain a bacterial colony; selecting colony with regular morphology, performing streaking culture for 5 times on sterile MRS culture medium to separate and purify to obtain single pure colony, determining as lactobacillus by microscopic examination, and screening the lactobacillus with the effect of preventing and treating diabetic encephalopathy by 16S rDNA sequence homology analysis. The MRS culture medium comprises the following components in percentage by weight: 10.0g/L of casein peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 5.0g/L of glucose, 5.0g/L of sodium acetate, 2.0g/L of diamine citrate, 801.0 g/L of tween-801.0 g/L, 2.0g/L of dipotassium phosphate, 0.2g/L of magnesium sulfate heptahydrate, 0.05g/L of manganese sulfate heptahydrate, 20.0g/L of calcium carbonate and 20.0g/L of agar. Sterilizing at 121 deg.C for 20min at pH 6.8.

(1) And (3) carrying out 16S rDNA molecular biological identification on the lactobacillus obtained by screening. Picking single colony, mixing with sterile water, adding bacteria universal primer, performing colony PCR amplification, and determining gene sequence. The 16S rDNA base sequence of the lactobacillus with the effect of preventing and treating the diabetic encephalopathy is as follows:

TCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCGGTGGCGTAACCCTTTTAGGGAGCGAGCCGTCTAAGGTGGGACAAATGATTAGGGTGAAGTC。

(2) observing colony morphology and individual morphology of the lactobacillus obtained by screening, wherein the colony morphology is as follows: the color is white, the shape is round and convex, and the size is irregular; the morphology of the individual was gram-positive as observed in the optical microscope of FIG. 1, and the cells were rod-shaped and did not move.

(3) The results of physiological and biochemical characterization experiments of the selected lactobacilli are shown in table 1 ("-" indicates negative).

TABLE 1

According to the results of the physiological and biochemical experiments combined with the results of the colony morphological characteristics and the individual morphological characteristics, the lactobacillus can be finally determined.

Example 2

And (3) preparing lactobacillus paracasei powder.

The specific method comprises the following steps: selecting a single bacterial colony of lactobacillus paracasei LC86, inoculating the single bacterial colony in a 50mLMRS culture medium, performing anaerobic culture at the temperature of 37 ℃ for 24 hours to obtain a seed solution; inoculating the seed liquid into a new MRS culture medium by the inoculation amount of 2%, and carrying out anaerobic culture at 37 ℃ for 24h to obtain a fermentation liquid; centrifuging the fermentation liquor at 12000rpm for 10min, removing the fermentation supernatant, and separating out thallus; and freeze-drying the thalli to obtain the lactobacillus paracasei bacterial powder.

Example 3

Animal experiment research for preventing and treating diabetic encephalopathy.

First, experimental material

1. Lactobacillus paracasei LC86 powder with viable count of 1 × 10 ⁸CFU/g, preparation reference example 2.

2. STZ solution: 100mg of streptozotocin was weighed, 5mL of citric acid buffer was added, and streptozotocin was dissolved in ice bath and used within 30 min.

3. Experimental animals: wistar rats, male, 12 weeks old, body weight 230-.

Preparation of diabetic encephalopathy rat model

The rats are fed adaptively for one week, and the physiological characteristics of darkness and brightness are utilized, and the Y maze is applied to screen out the rats with similar learning and memory abilities for establishing the diabetic encephalopathy model. Wistar male rats were fasted for 12h and injected intraperitoneally with STZ solution at 60 mg/Kg. According to the habitual modeling method of diabetic rats, the fasting blood glucose value of each rat is detected by two tail blood sampling on the 3 rd day and the 7 th day of administration, and the blood glucose value is more than or equal to 11.1mmol/L, which can be considered as successful modeling.

Three, grouping and administration

Rats were divided into a blank group, a model group and a sample group, 10 per group. The rats that were not modelled and injected with ph4.6 citrate buffer were blank groups, and the modelled rats were randomly divided into model groups and sample groups, sample groups: 24 hours after the diabetes encephalopathy model is successfully established, the intragastric lactobacillus paracasei LC86 with the concentration of 1 multiplied by 10 is started ⁹CFU/day/rat, 1 time daily for 4 consecutive weeks.

Fourth, evaluation of drug efficacy and method

1. Blood glucose and body weight determination

The blood glucose and body weight changes 2h after administration were determined for each group of rats at the end of 0 and 4 weeks, respectively.

2. Spatial learning memory detection

After 4 weeks of drug intervention, the Morris water maze experiment was performed, animals were trained to be familiar with the environment of the test, and formal training began the next day. Including directional navigation and space exploration experiments. Directional sailing test: each rat is trained for 4 days in 1 round in the morning and afternoon every day for 120s every time, the interval between two times of training is 30s, a water inlet point is randomly selected during training, the rat is placed in water facing to the wall of the pool, and the time (escape latency) when the rat touches the platform for 120s is observed and recorded. Space exploration experiment: the platform was removed in the afternoon of the fifth day, and the rats were immersed in water in the contralateral quadrant of the original platform and observed for the percentage of residence time in the central zone and the number of passages through the original platform in 120 s. The experimental facility consists of a video acquisition system, a data analysis system and an annular water pool with four quadrants, and is used for detecting the space learning ability and the space memory ability of experimental animals.

3. Determination of SOD enzyme activity, MDA content and AchE enzyme activity of brain tissue

After the Morris water maze neuroethology test is finished, each group of rats are sacrificed, the hippocampus is quickly taken out on ice, homogenized, centrifuged at 2000rpm/min for 10min, and the supernatant is taken. SOD activity, MDA content and AChE activity were measured according to the instructions of the kit.

4. iNOS Activity assay

The iNOS concentration in rat hippocampal supernatant was determined using an enzyme linked immunosorbent assay kit according to the protocol.

5. Statistical analysis

The experimental data are expressed as mean ± standard deviation, statistical analysis of one-way anova was performed using SPSS 18.0 statistical software, the difference between groups was tested by q, and P < 0.05 indicated that there was a statistical difference.

Fifth, test results

1. Blood glucose and body weight comparison

The results are shown in Table 2, and it can be seen from the data in Table 2 that: at the beginning of the intervention, the blood glucose values of the model group and the sample group were significantly higher than those of the blank group (P < 0.01), while the body weight was significantly lower than that of the blank control group (P < 0.01). After four weeks of intervention, the blood glucose values of the sample group were significantly reduced (P < 0.05) and the body weight was increased (P < 0.05) compared to the model group.

TABLE 2

2. Comparison of spatial learning and memory abilities of diabetic encephalopathy rats

The results are shown in Table 3, and it can be seen from the data in Table 3 that: the escape latency of the model group was significantly increased (P < 0.01) compared to the blank group, and the escape latency of the sample group was significantly decreased (P < 0.01) compared to the model group. The percentage of residence time in the center region of the model group and the number of passes over the original platform position were significantly decreased (P < 0.01) compared to the blank group, and the percentage of residence time in the center region of the sample group and the number of passes over the original platform position were significantly increased (P < 0.01) compared to the model group. The results show that the lactobacillus paracasei has the effect of obviously improving the learning and memory abilities of rats with diabetic encephalopathy.

TABLE 3

3. Comparison of SOD (superoxide dismutase) Activity, MDA (malondialdehyde) content and AchE (AchE-cholinesterase) Activity in brain tissue

The results are shown in Table 4, and it can be seen from the data in Table 4 that: SOD activity in the model group hippocampal region is obviously lower than that in the blank group (P is less than 0.01), and after 4 weeks of dry prognosis, SOD activity in the sample group is obviously increased (P is less than 0.01); compared with the model group, the MDA content of the sample group is obviously reduced (P is less than 0.01); AchE activity in model group hippocampal region is obviously higher than that in blank group (P < 0.01), and after 4 weeks of dry prognosis, AchE activity in sample group is obviously inhibited (P < 0.01).

TABLE 4

4. Comparison of iNOS Activity

The results are shown in Table 4, and it can be seen from the data in Table 4 that: the iNOS activity in the hippocampus of the diabetic encephalopathy model group is obviously higher than that of a blank control group (P is less than 0.01), while the iNOS activity of a sample group intervened by lactobacillus paracasei is obviously reduced (P is less than 0.01), which indicates that the lactobacillus paracasei can obviously inhibit the iNOS activity in the hippocampus of a diabetic encephalopathy rat.

The applicant states that the lactobacillus having the effect of preventing and treating diabetic encephalopathy and the separation method and application thereof are illustrated by the above examples, but the invention is not limited by the above examples, i.e. the invention is not limited by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> Jiangsu microbial science and technology Limited

<120> lactobacillus with efficacy of preventing and treating diabetic encephalopathy, and separation method and application thereof

<130>2019

<160>1

<170>PatentIn version 3.3

<210>1

<211>1501

<212>DNA

<213> Lactobacillus paracasei (Lactobacillus paracasei) LC86 strain

<400>1

tcaggatgaa cgctggcggc gtgcctaata catgcaagtc gaacgagttc tcgttgatga 60

tcggtgcttg caccgagatt caacatggaa cgagtggcgg acgggtgagt aacacgtggg 120

taacctgccc ttaagtgggg gataacattt ggaaacagat gctaataccg catagatcca 180

agaaccgcat ggttcttggc tgaaagatgg cgtaagctat cgcttttgga tggacccgcg 240

gcgtattagc tagttggtga ggtaatggct caccaaggcg atgatacgta gccgaactga 300

gaggttgatc ggccacattg ggactgagac acggcccaaa ctcctacggg aggcagcagt 360

agggaatctt ccacaatgga cgcaagtctg atggagcaac gccgcgtgag tgaagaaggc 420

tttcgggtcg taaaactctg ttgttggaga agaatggtcg gcagagtaac tgttgtcggc 480

gtgacggtat ccaaccagaa agccacggct aactacgtgc cagcagccgc ggtaatacgt 540

aggtggcaag cgttatccgg atttattggg cgtaaagcga gcgcaggcgg ttttttaagt 600

ctgatgtgaa agccctcggc ttaaccgagg aagcgcatcg gaaactggga aacttgagtg 660

cagaagagga cagtggaact ccatgtgtag cggtgaaatg cgtagatata tggaagaaca 720

ccagtggcga aggcggctgt ctggtctgta actgacgctg aggctcgaaa gcatgggtag 780

cgaacaggat tagataccct ggtagtccat gccgtaaacg atgaatgcta ggtgttggag 840

ggtttccgcc cttcagtgcc gcagctaacg cattaagcat tccgcctggg gagtacgacc 900

gcaaggttga aactcaaagg aattgacggg ggcccgcaca agcggtggag catgtggttt 960

aattcgaagc aacgcgaaga accttaccag gtcttgacat cttttgatca cctgagagat 1020

caggtttccc cttcgggggc aaaatgacag gtggtgcatg gttgtcgtca gctcgtgtcg 1080

tgagatgttg ggttaagtcc cgcaacgagc gcaaccctta tgactagttg ccagcattta 1140

gttgggcact ctagtaagac tgccggtgac aaaccggagg aaggtgggga tgacgtcaaa 1200

tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggatggta caacgagttg 1260

cgagaccgcg aggtcaagct aatctcttaa agccattctc agttcggact gtaggctgca 1320

actcgcctac acgaagtcgg aatcgctagt aatcgcggat cagcacgccg cggtgaatac 1380

gttcccgggc cttgtacaca ccgcccgtca caccatgaga gtttgtaaca cccgaagccg 1440

gtggcgtaac ccttttaggg agcgagccgt ctaaggtggg acaaatgatt agggtgaagt 1500

c 1501

Claims

1. The Lactobacillus with the effect of preventing and treating the diabetic encephalopathy is named as Lactobacillus paracasei (Lactobacillus paracasei) LC86 strain, the preservation unit is China general microbiological culture Collection center, the preservation time is 12 months in 2013, the preservation number is CGMCC 1.12731, and the address is as follows: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

2. The lactobacillus having the effect of preventing and treating diabetic encephalopathy of claim 1, wherein the lactobacillus is screened from yogurt products.

3. The method for isolating lactobacillus having an effect of preventing and treating diabetic encephalopathy according to claim 1 or 2, wherein the method for isolating lactobacillus comprises:

4. The method for isolating lactobacillus according to claim 3, wherein the MRS medium has a formulation of: casein peptone 10.0g/L, beef extract 10.0g/L, yeast extract 5.0g/L, glucose 5.0g/L, sodium acetate 5.0g/L, diamine citrate 2.0g/L, Tween-801.0 g/L, dipotassium hydrogen phosphate 2.0g/L, magnesium sulfate heptahydrate 0.2g/L, manganese sulfate heptahydrate 0.05g/L, calcium carbonate 20.0g/L, agar 20.0 g/L; the pH value of the MRS culture medium is 6.8.

5. The method for separating Lactobacillus according to claim 3 or 4, wherein the separation and purification is: selecting colony with regular morphology to perform streak culture on a sterile MRS culture medium for 3-10 times to achieve the purpose of separation and purification;

6. Use of the lactobacillus having the effect of preventing and treating diabetic encephalopathy according to claim 1 or 2 in the preparation of a medicament having the effect of preventing and treating diabetic encephalopathy.

7. The use of claim 6, wherein the viable count of the lactobacillus is not less than 1 and is prepared10 ⁷CFU/g。

8. The use of claim 6 or 7, wherein the medicament further comprises a pharmaceutically acceptable excipient;

preferably, the excipient comprises any one of, or a combination of at least two of, an excipient, a diluent, a carrier, a flavoring agent, a binder, or a filler.

9. The use of any one of claims 6 to 8, wherein the carrier comprises a liposome, micelle, dendrimer, microsphere or microcapsule.

10. The use of any one of claims 6 to 9, wherein the medicament is in the form of a tablet, capsule, granule, powder, spray, film, suppository, nasal drop or drop pill.