CN109810912B - Lactobacillus plantarum LH-511 and application thereof - Google Patents

Abstract

The application discloses lactobacillus plantarum LH-511 and application thereof. The preservation number of the lactobacillus plantarum LH-511 is CGMCC No.14512. The lactobacillus plantarum LH-511 has high alpha-glucosidase activity inhibition rate, can regulate blood sugar, has excellent probiotics, has strong acid resistance and bile salt resistance, and can be stored for a long time; can be used for preparing various foods, health-care products, food additives or medicines for regulating blood sugar, and provides a new high-efficiency raw material for regulating blood sugar.

Description

Lactobacillus plantarum LH-511 and application thereof

Technical Field

The application relates to the field of lactobacillus plantarum, in particular to lactobacillus plantarum LH-511 and application thereof.

Background

With the development of socioeconomic performance, the incidence of metabolic syndromes such as diabetes and the total number of patients tend to increase year by year worldwide, and the disease becomes the third most chronic disease which endangers the life and health of human beings. Diabetes is a metabolic disease of multiple etiologies, and clinical symptoms are hyperglycemia, impaired glucose tolerance, abnormal insulin release, and the like. The main reason is postprandial hyperglycemia caused by reduced insulin secretion or reduced insulin sensitivity of pancreatic cells, which in turn inhibits insulin secretion by the pancreas, reduces the glucose uptake capacity of insulin-regulating peripheral tissues, and thus leads to disorders in glucose, fat and protein metabolism caused by insulin secretion and defective action.

The world health organization classifies diabetes as it is causative and pathogenesis: insulin-dependent type I diabetes, non-insulin-dependent type II diabetes, special type and gestational diabetes, wherein type II diabetes patients account for about 90% or more. Type II diabetes is mainly characterized by insulin resistance, i.e., the inability of cells to respond to insulin after its own production, and is accompanied by various complications such as coronary heart disease, atherosclerosis, nephropathy, neuropathy, retinopathy and foot lesions, and the like, and also by typical "three more or less" symptoms, i.e., polydipsia, polyuria, polyphagia and weight loss.

At present, four oral hypoglycemic drugs for treating type II diabetes are mainly used: (1) Alpha-glucosidase (alpha-glucosidase) inhibitor: such as acarbose, voglibose, etc.; (2) insulinotropic agent: such as sulfonylureas, repaglinide, nateglinide, and the like; (3) Biguanides and thiazolidinediones, etc. that inhibit hepatic glucose output; (4) glitazones for alleviating insulin resistance, and the like. Wherein, the alpha-glucosidase inhibitor can reversibly occupy the complex site of alpha-glucosidase and sugar, and inhibit the alpha-glucosidase from catalyzing the degradation of polysaccharide into monosaccharide, thereby delaying the absorption of intestinal carbohydrates. The alpha-glucosidase inhibitor can inhibit the activity of glucosidase to slow down the generation and absorption of glucose, reduce postprandial hyperglycemia and regulate blood glucose level, so as to reduce the stimulation of hyperglycemia to pancreas, raise insulin sensitivity, effectively prevent and improve diabetes complications, maintain the blood glucose of patients at a certain level and stabilize the glycemic index. The alpha-glucosidase inhibitor has the advantages of mild and durable effect, no toxicity or small side effect and the like, and is focused by a plurality of researchers at home and abroad, and the source of the alpha-glucosidase inhibitor also becomes a research bright spot. The original sources of glucosidase inhibitors extracted from actinomycetes and streptococci have been in various areas of the surrounding environment as research has progressed. Wherein, the inhibition of the alpha-glucosidase from lactic acid bacteria also becomes an important index for screening the blood glucose reducing function of the probiotics. Therefore, the screening of the novel probiotics with high alpha-glucosidase inhibition effect has important significance for the treatment of type II diabetes and the development of novel medicines or health products thereof.

Disclosure of Invention

The purpose of the application is to provide a novel lactobacillus plantarum LH-511 and application thereof.

The application adopts the following technical scheme:

one aspect of the application discloses a lactobacillus plantarum (Lactobacillus plantarum) LH-511 with a preservation number of CGMCC No.14512.

It should be noted that the lactobacillus plantarum LH-511 with the preservation number of CGMCC No.14512 is a newly discovered strain with good blood sugar regulating function, and the strain has good blood sugar reducing effect in vivo and in vitro and good tolerance to acid and bile, which means that the lactobacillus plantarum LH-511 can be eaten or orally taken for regulating blood sugar. Therefore, the lactobacillus plantarum LH-511 is specially preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at 8 and 10 of 2017, and the preservation number is CGMCC No.14512 and the preservation name is lactobacillus plantarum LH-511 of the national academy of sciences of China, including North Chen West Lu No.1 and North Sei, of the Korea of the Beijing area.

It should be further noted that Lactobacillus plantarum LH-511 of the present application differs from other prior art Lactobacillus plantarum in that it has been found that the in vitro activity inhibition of the present application Lactobacillus plantarum LH-511 on alpha-glucosidase is greater than 33%, whereas lactobacillus is typically under 10% and occasionally high, not more than 20%, which in principle explains the hypoglycemic effect of the present application Lactobacillus plantarum LH-511.

The application discloses application of lactobacillus plantarum LH-511 in preparing foods, health-care products, food additives, medicines or compositions for regulating blood sugar.

In another aspect of the present application, a composition is disclosed comprising Lactobacillus plantarum LH-511 of the present application.

The composition of the present application refers to other types of edible or pharmaceutical compositions containing lactobacillus plantarum LH-511 of the present application besides foods, health products, food additives, medicines, which can be used for regulating blood glucose.

The lactobacillus plantarum LH-511 has good blood sugar reducing function and can be eaten, so that the lactobacillus plantarum LH-511 can be completely prepared into various foods, health-care products, food additives or medicines for regulating blood sugar or playing a health-care role.

In another aspect of the present application, a food product is disclosed comprising Lactobacillus plantarum LH-511 of the present application.

Preferably, the food also contains a pharmaceutically acceptable additive or adjuvant.

More preferably, the food product of the present application is a lactic acid drink or a soy milk drink.

It should be noted that the Lactobacillus plantarum LH-511 can be made into various foods, such as lactic acid beverage, soybean milk beverage, etc., as in the existing Lactobacillus plantarum, and the prepared foods mainly have health care effect, therefore, the active bacterial amount or intake of Lactobacillus plantarum LH-511 in the foods is not particularly limited, and the application is practicalAnd can be flexibly selected according to the actual conditions. Studies in this application showed that 1.0X10 s of intake was taken daily ⁹ The active lactobacillus plantarum LH-511 of CFU unit can obviously reduce the blood sugar content in animal blood, and the active lactobacillus dosage can be used as a reference dosage or reference intake of food, health care products, food additives or medicines.

The food product of the present application is an edible product in any form in a broad sense, and is not limited to a lactic acid drink or a soybean milk drink, and may be, for example, a fermented food product, and the fermented food product includes an animal feed and the like.

The other side of the application discloses a health care product which contains lactobacillus plantarum LH-511.

It should be noted that the lactobacillus plantarum LH-511 has good blood sugar regulating function, so that the lactobacillus plantarum LH-511 can be added into various existing health-care products to enable the products to have good blood sugar regulating health-care functions, and only the other components of the health-care products and the lactobacillus plantarum LH-511 have no mutual inhibition or adverse side effects.

In another aspect of the present application, a food additive is disclosed comprising Lactobacillus plantarum LH-511 of the present application.

The Lactobacillus plantarum LH-511 can be eaten in combination with common food materials. For example, cereals including rice, flour, miscellaneous cereals, potatoes including potatoes, sweet potatoes, etc.; animal foods including meat, poultry, fish, milk, eggs, and the like; legumes and products thereof, including soybeans and other dried legumes; vegetables and fruits including fresh beans, rhizomes, leaf vegetables, solanaceous fruits, etc.; pure heat energy foods including animal and vegetable oils, starches, edible sugars, alcoholic beverages, and the like; therefore, the lactobacillus plantarum LH-511 can be independently used as a food additive or a modulator to be added into various food materials for direct eating, and has the health care function of regulating blood sugar.

Another aspect of the present application discloses a pharmaceutical product comprising Lactobacillus plantarum LH-511 of the present application.

Preferably, the medicament of the present application is for modulatingRegulating blood sugar, specifically inhibiting enzyme activity of alpha-glucosidase, and containing at least 1.0X10 in daily dosage of the medicine ⁹ CFU units of active lactobacillus plantarum LH-511.

Preferably, the medicine also contains a carrier or auxiliary material acceptable in medicine.

Preferably, the carrier or adjuvant is selected from at least one of glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water and physiological saline.

It will be appreciated that the pharmaceutical product is usually formulated with the addition of carriers or excipients, provided that the addition of carriers or excipients and lactobacillus plantarum LH-511 do not inhibit each other or have adverse side effects.

More preferably, the pharmaceutical product is at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, an emulsion, and a lyophilized preparation;

more preferably, the lactobacillus plantarum tablet.

More preferably, the Lactobacillus plantarum tablet comprises Lactobacillus plantarum LH-511, dietary fiber, sorbitol, microcrystalline cellulose and magnesium stearate.

In a preferred scheme of the application, the lactobacillus plantarum tablet comprises 20 weight percent of lactobacillus plantarum LH-511 lactobacillus freeze-dried powder, 60 weight percent of dietary fiber, 10 weight percent of sorbitol, 5 weight percent of microcrystalline cellulose and 5 weight percent of magnesium stearate, and is prepared into tablets after being uniformly mixed.

The medicine has good blood sugar regulating function because of containing the lactobacillus plantarum LH-511; the medicine can be various preparations of lactobacillus plantarum LH-511 with single active ingredients, and can also be matched with other active ingredients for use, so long as the activities are not affected.

It is understood that Lactobacillus plantarum LH-511 can be used as an active bacterial drug, and can be prepared into various existing dosage forms as long as the bacterial strain activity is not affected. In the medicines of the application, the medicines or the dosage forms can further comprise auxiliary materials commonly used in medicines or dosage forms, such as stabilizing agents, wetting agents, emulsifying agents, adhesives, isotonic agents and the like.

The content of the lactobacillus plantarum LH-511 active bacteria in the medicine, or the medicine amount is not particularly limited, and in practical application, the medicine can be flexibly selected according to the health condition of an administration object. However, the study of the present application showed that 1.0X10 s of intake per day ⁹ The active lactobacillus plantarum LH-511 of CFU unit can obviously reduce the blood sugar content in animal blood, and the dosage can be used as the content of the active lactobacillus plantarum LH-511 in medicines or the reference of the administration dosage.

When the lactobacillus plantarum LH-511 is combined with other probiotics or probiotic materials, as long as the components have no inhibition effect or adverse reaction, it is understood that the components can have functional complementation or promotion effect, for example, the lactobacillus plantarum LH-511 can be prepared into a composite probiotic tablet, and by combining with other probiotics, better or more active functions can be achieved, for example, besides regulating blood sugar, the lactobacillus plantarum LH-511 can be combined with other probiotics, and the functions of regulating gastrointestinal functions, improving immunity and the like can be achieved, and the invention is not limited herein.

The beneficial effects of this application lie in:

the lactobacillus plantarum LH-511 has high alpha-glucosidase activity inhibition rate, can regulate blood sugar, has excellent probiotics, has strong acid resistance and bile salt resistance, and can be stored for a long time; can be used for preparing various foods, health-care products, food additives or medicines for regulating blood sugar, and provides a new high-efficiency raw material for regulating blood sugar.

Drawings

FIG. 1 is a graph showing the growth of Lactobacillus plantarum LH-511 in vitro in an anti-bile salt experiment, as described in the examples of the present application.

The Lactobacillus plantarum LH-511 is classified and named as Lactobacillus plantarum Lactobacillus plantarum, and is preserved in China general microbiological culture Collection center (China Committee) for culture Collection of microorganisms in the period of 08 and 10 of 2017, wherein the preservation unit is China center for type 3 microbiological study (CGMCC) with the preservation number of CGMCC No.14512 in the North Sei Hirudo No.1 of the Korean area of Beijing.

Detailed Description

The lactobacillus plantarum LH-511 is an active bacterium which is newly separated and discovered and has a good blood sugar regulating function. The lactobacillus plantarum LH-511 is in a spherical bacillus form under an optical microscope in an MRS culture medium, has the diameter of about 1 mu m, is free of spores, and has a complete cell wall structure; the plasmid is not contained, the metabolites do not produce toxic substances such as D-lactic acid, indole and the like, the nitroreductase is not expressed, and the capability of producing biogenic amine is not provided; the minimum inhibitory concentration of 13 antibiotics such as Gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, amplicilin, neomycin, trimethoprim, ciprofoxacin, rifampicin, vancomycin also meets the regulation of European food and drug administration (abbreviated EFSA).

The lactobacillus plantarum LH-511 strain has good resistance to simulated artificial gastric juice environment, has higher alpha-glucosidase activity inhibition rate in vitro and in vivo, and has obvious blood sugar reducing effect; therefore, the lactobacillus plantarum LH-511 newly separated and discovered by the application has the requisite condition for being medicinal bacteria or edible fungi, and can be used for developing new medicines, foods, health care products, food additives or other edible or medicinal compositions.

The present application is described in further detail below by way of specific examples. The following examples are merely illustrative of the present application and should not be construed as limiting the present application.

Example 1

In the example, the traditional fermented food is taken as a sample as a study object, suspected bacterial colonies of the lactobacillus are separated and screened from the sample, and then the selected suspected bacterial strains are identified by adopting 16s DNA amplification. The details are as follows:

(1) Culture medium formula

Optimized MRS solid media and MRS media were prepared in this example.

MRS solid culture medium, MRS culture medium for short, 1L formula is: 10.0g/L of casein peptone, 10.0g/L of beef extract, 5.0g/L of yeast extract, 20.0g/L of glucose, 2.0g/L of dipotassium hydrogen phosphate, 1.0g/L of tween 80, 2.0g/L of tri-ammonium citrate, 5.0g/L of sodium acetate, 0.1g/L of magnesium sulfate, 0.05g/L of manganese sulfate and 17.5g of agar.

The formulation of MRS broth, i.e., MRS broth, is the same as that of MRS broth except that no agar was added.

The pH of the prepared culture medium and culture solution is adjusted to 6.5, sterilization is carried out for 20min at 121 ℃, and then refrigeration is carried out at 4 ℃ for standby.

(2) Sources and identification of Lactobacillus plantarum LH-511 Strain

Taking fermented food of inner Mongolian farmhouse yoghurt as a sample, carrying out 10-time gradient dilution by using sterile normal saline, and diluting the sample to 10 ^-3 The strain is coated on the surface of MRS culture medium prepared in this example, and cultured in an incubator at 37 ℃ for 24-48 hours, and colony morphological characteristics are observed. The suspected lactic acid bacteria were picked from the culture medium and streaked onto a new MRS medium, and after overnight culture at 37℃the streaked bacteria were again streaked. And (3) picking a plurality of single colonies from the culture medium separated by the second streak by using a sterile toothpick, and numbering the single colonies respectively. Of these 20 single colonies were selected for 16s DNA sequencing.

The primer sequences of the DNA amplification of the example 16s are shown as SEQ ID NO.1 and SEQ ID NO. 2.

SEQ ID NO.1：5’-AGAGTTTGATCATGGCTCAG-3’

SEQ ID NO.2：5’-TAGGGTTACCTTGTTACGACTT-3’

The primer is synthesized by Shenzhen Hua big Gene research institute.

The PCR reaction system was 50. Mu.L of the reaction solution comprising: 10mM dNTPs 1. Mu.L, 10 Xbuffer 5. Mu.L, 10mM upstream and downstream primers 1. Mu.L, bacterial liquid PCR template 1. Mu.L, 5U/. Mu.L Taq enzyme 1. Mu.L, and ddH was supplemented ₂ O was replenished to 50. Mu.L.

The PCR reaction conditions were: pre-denatured at 94 ℃ for 5min, then enter 35 cycles: 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min, and after the circulation is finished, the temperature is 72 ℃ for 5min, and the standby is 4 ℃.

And performing gel cutting recovery on the PCR amplification product, and sequencing the recovered PCR product. In this example, the kit TaKaRa MiniBEST agarose GeL DNA Extraction kit is used for glue cutting recovery, and the detailed steps are described in the kit instruction. The PCR products of this example were sequenced by Shenzhen big Gene research institute.

The sequencing result shows that the strain with the number of LH-511 has the sequence shown as SEQ ID NO.3 as the sequencing result of 16s rDNA.

SEQ ID NO.3：

5’-GAGGGCGCAGCTATACATGCAGTCGAACGAACTTCCGTTAATTGATTATGACGTACTTGTACTGATTGAGATTTTAACACGAAGTGAGTGGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCAGAAGTAGGGGATAACACCTGGAAACAGATGCTAATACCGTATAACAGAGAAAACCGCATGGTTTTCTTTTAAAAGATGGCTCTGCTATCACTTCTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAAAGGCTCACCAAGGCAGTGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACGTGGGTAAGAGTAACTGTTTACCCAGTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTCTTTTAAGTCTAATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGATTACTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGTAATCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAAGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGACAGTCTAAGAGATTAGAGGTTCCCTTCGGGGACAGAATGACAGTGTGCATGATGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTAT-3’

The comparison in NCBI database by BLAST tool shows that LH-511 strain has closest affinity with lactobacillus plantarum Lactobacillus plantarum and similarity reaches 99%, therefore, LH-511 strain is identified as lactobacillus plantarum, named lactobacillus plantarum LH-511, which is obtained by screening in this example.

Single colonies of Lactobacillus plantarum LH-511 were transferred to MRS broth for pure culture at 37℃for 18h for subsequent use.

In addition, the result shows that the lactobacillus plantarum LH-511 is in a spherical bacillus form, has the diameter of about 1 mu m, has no spores and complete cell wall structure and accords with the morphological structure of the lactobacillus plantarum when being observed under an optical microscope in an MRS culture medium.

The lactobacillus plantarum LH-511 obtained by screening in the example is preserved in China general microbiological culture Collection center (China Committee for culture Collection) on 10 th of 2017, wherein the preservation unit is provided by China national academy of sciences of China, including North Chen West Lu No.1 and 3 of the Korean area of Beijing, and the preservation number is provided by CGMCC No.14512.

(3) Bacterial safety

1) Antibiotic susceptibility testing

The resistance of Lactobacillus plantarum LH-511 to 13 antibiotics such as Gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, amplicilin, neomycin, trimethoprim, ciprofoxacin, rifampicin, vancomycin was tested in this example. The specific test method is as follows:

the 13 antibiotics were diluted with a series of 2-fold ratios to give diluted drugs having concentrations of 64. Mu.g/mL, 32. Mu.g/mL, 16. Mu.g/mL, 8. Mu.g/mL, 4. Mu.g/mL, 2. Mu.g/mL, 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, and 0.125. Mu.g/mL, respectively.

The diluted antibiotics are respectively added into corresponding holes of a 96-well plate, then MRS culture solution of lactobacillus plantarum LH-511 is added into the corresponding holes, after uniform mixing, the culture is carried out for 48 hours at 37 ℃, and the culture result is observed. The sensitivity of Lactobacillus plantarum LH-511 was characterized in this example by the minimum inhibitory concentration (abbreviated MIC) of the antibacterial agent per well and the test results are shown in Table 1.

TABLE 1 determination of the resistance of Lactobacillus plantarum LH-511 to 13 antibiotics

Antibiotics	MIC(μg/mL)	EFSA specifies a security value
			Gentamicin	2	16
Kanamycin	16	64
			Streptomycin	16	NR
Tetracycline
		8	32
Erythromycin				0.25	1
	Clindamycin	0.25	2
Chloramphenicol				2	8
	Amplicilin	0.125	2
Neomycin				0.5	8
	Trimethoprim	64	128
Ciprofoxacin				16	64
	Rifampicin	2	4
Vancomycin				8	NR

In Table 1, NR indicates that European food and drug administration is not required for this, and the results in Table 1 show that the minimum inhibitory concentration of Lactobacillus plantarum LH-511 to Gentamicin, kanamycin, tetracycline, erythromycin, clindamycin, chloramphenicol, amplicilin, neomycin, trimethoprim, ciprofoxacin, rifampicin obtained by the separation in this example is in accordance with the regulation of European food and drug administration (abbreviated EFSA) and is far less than the safety value specified by it; the minimum inhibitory concentrations of Lactobacillus plantarum LH-511 on Streptomyces and Vancomycin are 16 mug/mL and 8 mug/mL respectively, and although the two antibiotics are not required in EFSA, the measured values of the two antibiotics are basically in line with the expectations, and the Lactobacillus plantarum LH-511 has better sensibility to the antibiotics.

In addition, the TIANGEN plasmid miniprep kit DP103 was used to extract plasmids from the Lactobacillus plantarum LH-511 of this example, which showed that Lactobacillus plantarum LH-511 was not found to carry any plasmids, indicating that Lactobacillus plantarum LH-511 has no possibility of gene level transfer.

2) Metabolite toxicity detection

Lactic acid optical rotation detection: the detection was performed using the D-/L-lactic acid detection kit from Ireland Megazyme. The results show that Lactobacillus plantarum LH-511 of this example does not produce D-lactic acid.

Nitrate reductase activity detection: the Lactobacillus plantarum LH-511 of this example was transferred into MRS liquid medium, cultured at 37℃for 24 hours, and under aseptic conditions, the activated strain was transferred into nitrate medium at 37℃for 5 days in an amount of 3% of the transferred amount, and then 10 drops of potassium iodide solution and starch solution were added dropwise, respectively, and experimental results were observed. Positive control tests were also performed. The experimental result shows that the bacterial liquid does not turn blue, and is a negative reaction, and the positive control turns blue, which indicates that the lactobacillus plantarum LH-511 of the example does not express nitroreductase.

Indole experiment: under the aseptic condition, the activated strain is inoculated into peptone water culture medium according to the inoculation amount of 3 percent, the culture is carried out for 72 hours at 37 ℃, then 8 to 10 drops of indole reagent is added, and the experimental result is observed. Positive control tests were also performed. The experimental result shows that the bacterial liquid does not have red circular rings, and the positive control has red color, which shows that the metabolism of the lactobacillus plantarum LH-511 does not produce indole.

Amino decarboxylase activity assay: bacteria having an amino acid decarboxylase, which are capable of decomposing amino acids to decarboxylate them to amines and carbon dioxide, and making the medium alkaline, for example, to decompose lysine to cadaverine, ornithine to putrescine, and arginine to spermine; an indicator, such as bromocresol purple, is added dropwise, and the yellow color is negative and the purple color is positive. Thus, in this example, the activated strain was inoculated into a peptone water medium at a inoculation amount of 3% under aseptic conditions, cultured at 37℃for 72 hours, and then bromocresol purple was added in an amount of 8 to 10 drops, and the experimental result was observed. The titration result is yellow and negative, which indicates that the lactobacillus plantarum LH-511 of the example does not have the capability of producing biogenic amine.

Example two

In this example, the Lactobacillus plantarum LH-511 isolated in example I was subjected to an in vitro inhibition of the enzyme activity of alpha-glucosidase. The method comprises the following steps:

the 205. Mu.L system includes: 50. Mu.L of PB having a concentration of 0.1mol/L and a pH=6.8S solution, 50. Mu.L of p-nitrophenol-alpha-D-glucopyranoside (abbreviated PNPG, purchased from Sigma) solution at a concentration of 20mmol/L, and 25. Mu.L of a sample to be tested, incubating the mixture at 37℃for 10min, adding 30. Mu.L of alpha-glucosidase solution at a concentration of 20U/mL for further reaction for 20min, and adding 50. Mu.L of Na at a concentration of 1mol/L ₂ CO ₃ As reaction stopping solution, measuring the light absorption value of the reaction solution at 405nm, wherein the light absorption value is in direct proportion to the free quantity of p-nitrophenol PNP, adopting PBS solution with pH value of 6.8 and concentration of 0.1mol/L as alpha-glucosidase solution and blank control of a sample to be tested in the reaction system, and calculating the inhibition of lactobacillus on alpha-glucosidase enzyme activity after the reaction, wherein the inhibition rate calculation formula is bacterial alpha-glucosidase enzyme activity inhibition rate= [1- (A-B)/(C-D)]×100％。

Wherein A, B, C, D is the absorbance value measured by four sample groups to be measured, specifically, A is the absorbance value of positive group, namely lactobacillus plantarum LH-511 thallus solution and alpha-glucosidase solution; b is the absorbance measured for the group containing only Lactobacillus plantarum LH-511 cell solution; c is the absorbance of the control group, i.e., only the alpha-glucosidase solution; d is the absorbance of the blank, i.e., the solution does not contain Lactobacillus plantarum LH-511 strain nor alpha-glucosidase.

Meanwhile, this example uses commercial strain 299V as a control, and its enzyme activity inhibition rate against alpha-glucosidase was measured by the same method.

The results show that the inhibition of the alpha-glucosidase of lactobacillus plantarum LH-511 obtained in the first example was 33.7% with a very remarkable inhibition compared to only 14.2% for the control commercial strain 299V. As can be seen, the Lactobacillus plantarum LH-511 obtained by the separation in the example has good inhibition rate of the activity of alpha-glucosidase.

Example III

The in vitro acid and bile resistance of Lactobacillus plantarum LH-511 isolated in example I was tested in this example as follows:

preparing simulated gastric fluid SGF:2.0g NaCl,3.2g pepsin, 7mL concentrated HCl, water was added to adjust to pH3.0, and then the mixture was filtered through a 0.2 μm microporous filter membrane to obtain the SGF solution of this example for use.

The lactobacillus plantarum LH-511 bevel strain is selected and cultured in MRS culture solution for 16 to 18 hours at 37 ℃. Centrifuging the bacterial suspension for 15min at 4 000r/min, removing the supernatant, weighing the wet weight of the bacterial suspension, re-suspending the bacterial suspension in normal saline according to the proportion of 0.1g/mL, adding the bacterial suspension into SGF liquid according to the proportion of 1:10, fully and uniformly mixing, placing the bacterial suspension in a constant temperature incubator at 37 ℃ for culturing for 2h, and respectively counting cells for 0h and 2 h. The cell count results are shown in Table 2.

TABLE 2 results of viable count of Lactobacillus plantarum LH-511 tolerant to Artificial gastric juice SGF

Strain	0h count	Counting 2h	Survival rate
				LH-511	2.56×10 9	2.55×10 9	99.6％

The results in Table 2 show that the viable count of Lactobacillus plantarum LH-511 after 2h of culture is maintained at one order of magnitude, and the survival rate is as high as 99.6%, which indicates that Lactobacillus plantarum LH-511 has relatively strong acid resistance in vitro.

Further, the activated bacterial solution was inoculated in an amount of 1% into 10mL of sterilized MRS medium containing 0.3% bile salt by weight, and the growth curve of Lactobacillus plantarum LH-511 in the medium was measured, and the test was repeated 4 times in this example, and the result is shown in FIG. 1. In FIG. 1, each curve is a growth curve of 4 repetitions, and the results show that Lactobacillus plantarum LH-511 can normally grow in a bile salt culture medium, and demonstrate that Lactobacillus plantarum LH-511 of the first embodiment has a strong bile resistance in vitro.

Example IV

In this example, the lactobacillus plantarum LH-511 isolated in example one was tested for in vitro regulatory cytokines as follows:

taking human THP-1 cell line cells in good growth state and logarithmic growth phase, centrifuging to extract culture medium, discarding waste liquid, adding appropriate amount of complete culture medium, blowing and beating uniformly-mixed and stripped cells with a pipette to prepare cell suspension, and regulating cell count density to 5×10 ⁵ Inoculating cfu/mL to 24-well plate by sucking 0.9mL cell suspension, sucking the above prepared bacterial suspension after 2 hr, and adding into corresponding well to give lactic acid bacteria suspension concentration of 10 in each well ⁶ cfu/mL concentration, three times, after culturing for 24 hours, absorbing supernatant, centrifuging for 10min at 3000r/min, taking supernatant, and performing ELISA test: interleukin-1 beta (IL-1 beta), IL-6, tumor necrosis factor-alpha (TNF-alpha) and IL-10, the experimental results are shown in Table 3, wherein each group P<0.05。

TABLE 3 in vitro cytokine control test results of Lactobacillus plantarum LH-511

Group of	IL-1β/(ng/mL)	IL-6/(ng/mL)	TNF-α(pg/mL)	IL-10/(ng/mL)
					Blank control	40.15	89.28	15.68	19.29
LH-511	35.31	61.54	12.93	96.56

The results in Table 3 show that Lactobacillus plantarum LH-511 of example I has downregulation effects on pro-inflammatory factors such as IL-1β, IL-6, TNF- α, etc. of THP-1 cell lines and upregulation effects on anti-inflammatory factors such as IL-10, indicating that Lactobacillus plantarum LH-511 does have anti-inflammatory effects.

Example five

In vivo efficacy studies were performed on Lactobacillus plantarum LH-511 isolated in example one, which was tested in vivo in Sprague-Dawley 5 week old rats, as follows:

1. preparation of experimental strains

Inoculating activated Lactobacillus plantarum LH-511 into MRS liquid culture medium, culturing at 37deg.C for 18h, centrifuging at 6000r/m for 10min, washing with sterilized normal saline, and collecting thallus. Then, 0.85% physiological saline was added to adjust the cell count to 1.0X10 ⁹ CFU/mL, and then subpackaging the live bacteria into 15mL centrifuge tubes according to daily usage amount, wherein the taking dosage is 2 mL/per day, and the total dosage is 6 groups of 10 per group, and the live bacteria are fed for 28 days.

2. Grouping and raising mode for experimental animals

In this example, sprague-Dawley rats of 5 weeks old were induced with streptozotocin in combination with high-fat feed to induce SD rats of 5 weeks old, and the rats were subjected to model formation, fed to day 28, and divided into 6 groups of 10 animals each. The feeding of each group was continued as follows:

a model group fed with high calorie feed, and distilled water;

a positive group fed with high calorie diet, and metformin, supplied at 0.3mg/g body weight;

LH-511 group fed with high calorie feed, and lactobacillus plantarum LH-511;

299v, which feeds high calorie feeds, and probiotics 299v;

a group Sc52 fed with high calorie feed, and probiotic Sc52;

normal group, which is fed standard feed, and distilled water.

The feed was fed in the same amounts, and each 2mL of distilled water, metformin, lactobacillus plantarum LH-511, probiotic 299v and probiotic Sc52 were intragastric in each group daily for 28 days.

Wherein, the high-calorie feed is prepared by self, fat or high-fat substances are added on the basis of purchased standard feed, and the specific formula is as follows: 78.8% of standard feed, 10% of lard, 10% of yolk powder, l% of cholesterol and 0.2% of bile salt. The standard feed is a special feed for mice purchased in Henan Tianchi laboratory animal feed factories. The probiotics 299v is a commercial strain and can be purchased commercially; the probiotics Sc52 is purchased from CGMCC, the preservation number is CGMCC No.11027 and is recorded in patent CN 201510581420.8.

3. Sample collection and analysis testing

Blood collection was performed before the official test and at the 28 th day. The blood sampling method is to take blood from femoral vein of rat after eating for one night, and separate serum by centrifugation at 4000r/m for 10min after coagulation. The liver tissue SOD value was measured on day 28 by measuring the contents of fasting blood glucose, postprandial 2h blood glucose, glycosylated hemoglobin, adiponectin and TNF-alpha.

4. Experimental results

The experimental results are shown in table 4, where each group P <0.05.

TABLE 4 animal test results

The results in Table 4 show that the 28 th sky abdominal blood sugar index of the stomach of the rats in the LH-511 group is reduced by 24.0% compared with the model group, the 28 th day postprandial blood sugar index of the LH-511 group is reduced by 37.1% compared with the model group, which shows that the Lactobacillus plantarum LH-511 has the function of reducing blood sugar in vivo, and other indexes show that the Lactobacillus plantarum LH-511 also has the effect of relieving the inflammatory environment in vivo caused by hyperglycemia. In addition, the Lactobacillus plantarum LH-511 has better functions than other control strains 299v (P > 0.05) and Sc52 (12.6%, P < 0.05).

Example six

The lactobacillus plantarum LH-511 obtained by separation in the first embodiment is respectively prepared into common medicines and foods in the embodiment, and the common medicines and foods are specifically as follows:

(1) Lactobacillus plantarum tablet

The formula comprises the following components: CFU10 ⁹ 20% of freeze-dried powder of lactobacillus plantarum LH-511, 60% of dietary fiber, 10% of sorbitol, 5% of microcrystalline cellulose and 5% of magnesium stearate; the components are all weight parts, and after being uniformly mixed, the components are pressed into tablets. Wherein the freeze-dried powder of the lactobacillus plantarum LH-511 is prepared by freezing, vacuumizing and drying bacterial suspension of the lactobacillus plantarum LH-511 in a sterile environment by adopting a freeze dryer.

(2) Lactobacillus plantarum fermented yogurt

Mixing milk powder and water, homogenizing, sterilizing at 121deg.C for 300s, cooling to 42deg.C, and inoculating activated starter: the total amount of the lactobacillus bulgaricus and lactobacillus thermophilus bacterial powder is 0.4 percent by weight. Fermenting at 42deg.C for 10 hr, cooling, adding 1% CFU10 by weight ⁸ Is prepared from Lactobacillus plantarum LH-511 by stirring and packaging.

Wherein, the milk powder is the milk powder conventionally used in the preparation of the yoghourt.

(3) Pharmacodynamic test of the above drugs and foods

In this example, the same method as in example five was used to perform in vivo hypoglycemic tests on the lactobacillus plantarum tablets and lactobacillus plantarum fermented yogurt of this example.

Wherein lactobacillus plantarum tablet is smashed and added with sterile water to prepare suspension, and the bacterial count is adjusted to 1.0x10 ⁹ CFU/mL. Plant milkThe bacillus fermented acidophilus milk is also added with a suspension prepared by sterile water, and the bacterial count is regulated to 1.0x10 ⁹ CFU/mL. The viable bacteria suspension in example five was replaced by this.

The feeding method and the subsequent blood sampling and measuring method are the same as those of the fifth embodiment.

The results show that, blood is drawn and detected after the high-fat feed rats are fed with the medicines and the foods of the lactobacillus plantarum LH-511 for 28 days, the blood sugar of the rats fed with the lactobacillus plantarum fermented yogurt is reduced by 24.5 percent compared with the blood sugar of the rats on the 0 th day, the P is less than 0.05, the fasting blood sugar concentration on the 0 th day is 4.7mmol/L, and the fasting blood sugar concentration after the 28 th day is 3.55mmol/L; rats fed lactobacillus plantarum tablets had comparable effects to rats fed lactobacillus plantarum fermented yogurt, with a decrease in fasting blood glucose concentration of about 24.8% after 28 days, with P <0.05. Therefore, the medicines and the foods added with the lactobacillus plantarum LH-511 have the effect that the lactobacillus plantarum LH-511 is fixedly planted in the rat body, and the medicines and the foods added with the lactobacillus plantarum LH-511 have the effect of reducing blood sugar in the body.

The foregoing is a further detailed description of the present application in connection with the specific embodiments, and it is not intended that the practice of the present application be limited to such descriptions. It will be apparent to those skilled in the art to which the present application pertains that several simple deductions or substitutions may be made without departing from the spirit of the present application.

SEQUENCE LISTING

<110> Shenzhen Huada Sanshengyuan science and technology Co., ltd

<120> Lactobacillus plantarum LH-511 and application thereof

<130> 17I25288

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 20

<212> DNA

<213> artificial sequence

<400> 1

agagtttgat catggctcag 20

<210> 2

<211> 22

<212> DNA

<213> artificial sequence

<400> 2

tagggttacc ttgttacgac tt 22

<210> 3

<211> 1110

<212> DNA

<213> 16s rDNA sequencing results of Lactobacillus plantarum LH-511

<400> 3

gagggcgcag ctatacatgc agtcgaacga acttccgtta attgattatg acgtacttgt 60

actgattgag attttaacac gaagtgagtg gcgaacgggt gagtaacacg tgggtaacct 120

gcccagaagt aggggataac acctggaaac agatgctaat accgtataac agagaaaacc 180

gcatggtttt cttttaaaag atggctctgc tatcacttct ggatggaccc gcggcgtatt 240

agctagttgg tgaggtaaag gctcaccaag gcagtgatac gtagccgacc tgagagggta 300

atcggccaca ttgggactga gacacggccc agactcctac gggaggcagc agtagggaat 360

cttccacaat ggacgcaagt ctgatggagc aacgccgcgt gagtgaagaa gggtttcggc 420

tcgtaaagct ctgttgttaa agaagaacgt gggtaagagt aactgtttac ccagtgacgg 480

tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 540

aagcgttatc cggatttatt gggcgtaaag cgagcgcagg cggtctttta agtctaatgt 600

gaaagccttc ggctcaaccg aagaagtgca ttggaaactg ggagacttga gtgcagaaga 660

ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga acaccagtgg 720

cgaaggcggc tgtctggtct gcaactgacg ctgaggctcg aaagcatggg tagcgaacag 780

gattagatac cctggtagtc catgccgtaa acgatgatta ctaagtgttg gagggtttcc 840

gcccttcagt gctgcagcta acgcattaag taatccgcct ggggagtacg accgcaaggt 900

tgaaactcaa aagaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 960

agctacgcga agaaccttac caggtcttga catcttctga cagtctaaga gattagaggt 1020

tcccttcggg gacagaatga cagtgtgcat gatgtcgtca gctcgtgtcg tgagatgttg 1080

ggttaagtcc cgcaacgagc gcaaccctat 1110

Claims (13)

1. Lactobacillus plantarum LH-511 has a collection number of CGMCC No.14512.

2. Use of lactobacillus plantarum LH-511 according to claim 1 for the preparation of a food or a pharmaceutical product for regulating blood glucose.

3. A food product characterized by: the food contains Lactobacillus plantarum LH-511 according to claim 1.

4. A food according to claim 3, wherein the food further comprises a food-acceptable additive or adjuvant.

5. A food according to claim 3, characterized in that the food is a lactic acid drink or a soy milk drink.

6. A health product, characterized in that: the health product contains the lactobacillus plantarum LH-511 as claimed in claim 1.

7. A pharmaceutical product, characterized in that: the medicine contains the lactobacillus plantarum LH-511 as claimed in claim 1.

8. The pharmaceutical product of claim 7, wherein: the medicine is used for regulating blood sugar, and contains at least 1.0X10 in daily dosage ⁵ CFU units of active lactobacillus plantarum LH-511.

9. The pharmaceutical product according to claim 7 or 8, wherein: the medicine also contains a carrier or an auxiliary material which is acceptable in medicine.

10. The pharmaceutical product according to claim 9, wherein the carrier or adjuvant is selected from at least one of glucose, lactose, sucrose, starch, mannitol, dextrin, fatty acid glyceride, polyethylene glycol, hydroxyethyl starch, ethylene glycol, polyoxyethylene sorbitan fatty acid ester, amino acid, gelatin, albumin, water and physiological saline.

11. The pharmaceutical product of claim 9, wherein the pharmaceutical product is at least one of a tablet, a granule, a powder, a capsule, a solution, a suspension, an emulsion, and a lyophilized formulation.

12. The pharmaceutical product according to claim 9, characterized in that it is a lactobacillus plantarum tablet.

13. The pharmaceutical product according to claim 12, wherein the lactobacillus plantarum tablet comprises lactobacillus plantarum LH-511, dietary fiber, sorbitol, microcrystalline cellulose and magnesium stearate.

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