CN113637603B - Lactobacillus entericus and application thereof - Google Patents
Lactobacillus entericus and application thereof Download PDFInfo
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- CN113637603B CN113637603B CN202110786852.8A CN202110786852A CN113637603B CN 113637603 B CN113637603 B CN 113637603B CN 202110786852 A CN202110786852 A CN 202110786852A CN 113637603 B CN113637603 B CN 113637603B
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- lactobacillus
- lactobacillus acidophilus
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- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 1
- DNDNWOWHUWNBCK-NMIPTCLMSA-N indolylmethylglucosinolate Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1S\C(=N\OS(O)(=O)=O)CC1=CNC2=CC=CC=C12 DNDNWOWHUWNBCK-NMIPTCLMSA-N 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 244000000074 intestinal pathogen Species 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 229960004125 ketoconazole Drugs 0.000 description 1
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 208000037841 lung tumor Diseases 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 235000019369 penicillin G sodium Nutrition 0.000 description 1
- 229940056360 penicillin g Drugs 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000000529 probiotic effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- GNBVPFITFYNRCN-UHFFFAOYSA-M sodium thioglycolate Chemical compound [Na+].[O-]C(=O)CS GNBVPFITFYNRCN-UHFFFAOYSA-M 0.000 description 1
- 229940046307 sodium thioglycolate Drugs 0.000 description 1
- 235000014347 soups Nutrition 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960002385 streptomycin sulfate Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 108010058651 thioglucosidase Proteins 0.000 description 1
- 238000000844 transformation Methods 0.000 description 1
- YWYZEGXAUVWDED-UHFFFAOYSA-N triammonium citrate Chemical compound [NH4+].[NH4+].[NH4+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O YWYZEGXAUVWDED-UHFFFAOYSA-N 0.000 description 1
- 239000001393 triammonium citrate Substances 0.000 description 1
- 235000011046 triammonium citrate Nutrition 0.000 description 1
- 230000004614 tumor growth Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses lactobacillus entericus for endowing food ingredients with anticancer efficacy and application thereof. The invention relates to an intestinal lactobacillus strain endowed with anticancer efficacy of food ingredients, which is preserved in the national emphasis laboratory of medical biotechnology of university of Nanjing. The 16SrDNA gene sequence of the lactobacillus enterica strain endowing the food ingredient with anticancer efficacy is a nucleotide sequence shown as SEQ ID No. 1. The lactobacillus enteris taken together with the I3C pure product or the vegetables containing the I3C, and can play a role in preventing and treating tumors such as lung cancer, melanoma, leukemia and the like. The enterobacteria can be planted in the alimentary canal of mammals, maintain the lower pH microenvironment of the enterobacteria, and promote the conversion of I3C in food into DIM and LTr1 with better antitumor activity, thereby endowing or obviously improving the anticancer effect of oral I3C.
Description
Technical Field
The invention relates to the technical field of microbial pharmacy, in particular to lactobacillus entericus and application thereof.
Background
The cruciferous vegetables such as broccoli, green vegetables, chinese cabbage, radishes and the like are food materials widely eaten worldwide and are frequently eaten and are necessary to be healthful. Most cruciferous vegetables contain indole glucosinolates, which can be further degraded by acid or myrosinase to produce indole-3-methanol (I3C). Although I3C has biological activities such as anti-tumor, I3C can be further metabolized into indole-3-carbaldehyde (I3A), indole-3-carboxylic acid (I3 CA), indolocarbazole (ICZ), DIM, LTr and the like in vivo. Given the chemical instability of I3C, its antitumor activity is likely to be due to its metabolites. The stomach and small intestine of mammals are unique in physiological structure and biochemically constitute a special acidic channel which also inhabits a large number of intestinal microorganisms. Thus, I3C (as a vegetable ingredient or commercially pure product) must undergo complex structural and functional transformations in the gastrointestinal tract after oral administration. However, the course of this transformation and its contribution to "I3C anti-tumor" are not known.
Thus, first an in vivo conversion product of I3C was studied, revealing that I3C can be converted in the gastrointestinal tract of mammals, yielding the main products DIM and LTr1.DIM has been marketed as a health product with antitumor potential; meanwhile, as an antitumor drug, DIM has also entered phase III clinical studies. LTr1 has little research on the biological functions. Thus, the antitumor activity of I3C and its metabolites DIM and LTr1 were compared in the same experimental background using various tumor cell lines and tumor models. Meanwhile, the transformation capacity of the intestinal bacteria to I3C is also screened by separating the intestinal bacteria in the feces of healthy volunteers. The results show that the anti-tumor activity of LTr is superior to that of DIM and I3C, and that the lactobacillus enterica can convert the I3C into LTr and DIM to the greatest extent.
A large number of lactobacilli inhabit the intestinal tract of healthy people, and many probiotics are still studied. Lactobacillus belongs to gram-positive bacillus, and the tail end of a rod is round and is distributed in human small intestine. Lactobacillus usually releases small molecules with strong penetrability such as lactic acid, acetic acid and the like, and has antagonism to harmful bacteria coexisting (or 'mixed') in intestinal tracts, but has weaker inhibition/sterilization effect, and plays an important role in adjusting and maintaining the balance of intestinal flora. In the human intestinal tract, some lactobacillus with higher abundance, such as lactobacillus acidophilus, not only antagonizes pathogenic microorganisms, but also secretes antibiotic-like substances such as acidophilin (acidophilin), and lactobacillus (1 aeetocidin), and also has inhibitory or antagonistic effect on intestinal pathogens.
Three meals a day, the meals are varied, of which I3C is a typical representative, without acid sensitive food ingredients. The following problems exist in the oral administration of I3C to exert an antitumor effect: (1) The antitumor effect of oral administration of I3C alone or crucifers is still not ideal. (2) The structure of the I3C is unstable, and preliminary experiments show that the I3C can be converted into various compounds in weak acid aqueous solution; however, the effective components which actually play roles are not clear so far; (3) The transformation process of I3C in vivo is unclear and the answer is urgently needed: whether the intestinal bacteria can convert I3C or not. What, if any, bacteria can be transformed? I3C is converted into what compounds. Whether these transformation products are "true killers" of tumors? If so, how to increase the conversion of "I3C to anticancer molecules".
At present, there is an urgent need for an intestinal lactobacillus and its use.
Disclosure of Invention
In view of the above problems/deficiencies, an object of the present invention is to provide an enterolactobacillus and its use.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: the invention relates to an intestinal lactobacillus strain which is preserved in China Center for Type Culture Collection (CCTCC), and the preservation name is as follows: lactobacillus acidophilus, the preservation address is the university of Chinese Wuhan; preservation date: 2021, 07, 12; preservation number: cctccc M2021863.
The invention relates to application of lactobacillus enteriae in preparing food-borne antitumor drugs.
Further, the 16S rDNA gene sequence of the lactobacillus enterica strain is a nucleotide sequence shown as SEQ ID No. 1.
The invention relates to an intestinal lactobacillus microbial agent prepared from intestinal lactobacillus.
Further, the active ingredients are at least one of the following (a), (b) and (c):
(a) A fermentation culture of the enterolactobacillus;
(b) Ultrasonic lysis supernatant of the obtained intestinal lactobacillus cells;
(c) Ultrasonic lysis precipitation of the obtained intestinal lactobacillus cells.
The invention relates to application of lactobacillus in intestinal tract in preparing health care products.
Further, the medicine is an oral medicine, and the oral medicine is powder, granule, tablet or capsule containing lactobacillus.
Furthermore, the oral medicine is powder, granule, tablet or capsule prepared by mixing lactobacillus with crucifer extract or component I3C thereof, other probiotics, vitamins and other acid sensitive components in any proportion.
Further, the preparation method of the oral medicine comprises the following steps: inoculating lactobacillus into MRS liquid culture medium plate, culturing for 18-24 hr under facultative anaerobic condition at 37+ -3deg.C with 1% -5% inoculum size, centrifuging MRS, and vacuum freeze drying to obtain lactobacillus powder.
Further, uniformly mixing lactobacillus powder with proper auxiliary materials or vitamins or other bacteria powder according to a proportion, and granulating to obtain granules; filling the granules into capsules to obtain capsules, and tabletting the granules to obtain tablets.
The beneficial effects are that: the lactobacillus enteris taken together with the I3C pure product or the vegetables containing the I3C, and can play a role in preventing and treating tumors such as lung cancer, melanoma, leukemia and the like. The enterobacteria can be planted in the alimentary canal of mammals, maintain the lower pH microenvironment of the enterobacteria, and promote the conversion of I3C in food into DIM and LTr1 with better antitumor activity, thereby endowing or obviously improving the anticancer effect of oral I3C.
Compared with the prior art, the invention has the following advantages: (1) The lactobacillus is separated from the digestive tract of healthy people, and comprises indigenous intestinal bacteria and lactobacillus which colonizes the intestinal tract through feeding (such as yoghurt drinking) way.
(2) The specific product provided by the invention is a mixed preparation of oral lactobacillus enterica, cruciferous vegetable extract and I3C or a subpackaged powder/granule/tablet or capsule, and is convenient to store, carry and eat. Lactobacillus is a well-known probiotic, and cruciferous vegetables such as broccoli, green vegetables, chinese cabbage and the like are rich in I3C. Lactobacillus enterica includes both indigenous inhabitants inherent in the digestive tract and excellent immigration recruited from foods (e.g. yogurt); the vegetables rich in I3C are eaten by people; the I3C is easily available in pure form, so that different dosages can be designed according to the requirements and taken together with lactobacillus to exert the therapeutic effect similar to (or at least not inferior to) that of the common anticancer drugs. These advantages suggest that the present invention has great significance in tumor prevention and treatment.
(3) The lactobacillus enteris very unique, and can convert vegetable components, namely indole-3-methanol (I3C for short), into dimeric indole (3, 3 '-diindolmethane for short) and LTr1 (2- (input-3-ylmethyl) -3,3' -diindolmethane for short) with stronger anti-tumor activity in the intestinal tract, so that the anti-tumor effect of the vegetables is improved.
Drawings
FIG. 1 is a graph showing the different enterobacteria' ability to convert I3C to DIM and LTr1.
FIG. 2 is a graph showing that the Lactobacillus entericus promotes the conversion of I3C into DIM and LTr1 by acidogenesis according to the present invention.
FIG. 3 is a graph of the acid-promoted conversion of I3C to LTr1 in accordance with the present invention. (A) pH value in MRS Medium: with or without the addition of I3C or Lactobacillus entericus. (B) I3C transformation LTr1 optimum pH was found.
FIG. 4 is a graph showing the effect verification of antibiotic sterilization and intestinal lactobacillus colonization according to the present invention. Nude mice were randomly divided into normal group (I), antibiotic clearance group (II) and lactobacillus enterica colonization group (III). After one week of antibiotic treatment or three days of intestinal lactobacillus colonization, the mouse feces are taken for relevant detection. (A) Colony status in feces was detected with LB and MRS solid medium plates, respectively. (B) cecum morphology.
FIG. 5 is a graph showing the effect of antibiotic sterilization and Lactobacillus colonization by 16S rRNA verification according to the present invention. Nude mice were randomly divided into normal group (I), antibiotic clearance group (II) and lactobacillus enterica colonization group (III). After one week of antibiotic treatment or three days of intestinal lactobacillus colonization, the mouse feces were taken for 16S rRNA detection.
FIG. 6 is a diagram showing the process of promoting the conversion of I3C to LTr1 at the small intestine by the Lactobacillus intestinal bacteria. (A) protocol. Four-week-old nude mice were randomly divided into three groups (I-III groups, n=5). Group I, I3C administered by gavage. Group II, intestinal microorganisms were removed with complex antibiotics (ampicillin + colistin + streptomycin) and then administered I3C by gavage. Group III, which was sterilized with the combination antibiotic (first week), followed by colonization with Lactobacillus enterica and gavage with I3C (from the second week). (B) qPCR quantitatively analyzes the abundance of lactobacillus in each intestinal segment in the feces of the mice planted with the intestinal lactobacillus group. (C-J) levels of DIM and LTr1 at different gut sites in three groups of mice.
FIG. 7 is a graph showing that intestinal lactobacillus colonization of the present invention can significantly reduce the pH of the intestinal microenvironment;
FIG. 8 shows comparison of cytotoxic activity of I3C and its metabolites according to the present invention (X represents IC 50 >10 μm) plot;
fig. 9 is a graph showing that the anti-tumor activity of LTr1 is better than that of I3C and DIM according to the naked murine model of a549 cell engraftment of the present invention.
(A) Experimental protocol: four-week-old nude mice were randomly divided into four groups (I-IV, n=6). Blank solvent (100 μl of corn oil with 2% DMSO), I3C, DIM and LTr1, 150 mg/kg/day, were administered daily by gavage, respectively, for four weeks. Four weeks after (B-D) administration, the tumor volume sizes of the transplanted tumors were compared, and the antitumor activity of LTr was found to be superior to I3C and DIM. The indicators (E and F) Ki67 immunohistochemistry and expression levels thereof indicate that LTr1 has better antitumor activity than I3C and DIM (E, scale, ki67 immunohistochemistry 20 μm; boxed in upper panel, enlarged to lower panel; F, each data point represents the mean value of five fields in one mouse tumor section: p <0.05, p <0.01, p <0.001, p <0.0001, mean value of 6 biological samples.+ -. SEM compared to the corresponding control.
FIG. 10 is Kras of the present invention G12D The mouse model showed LTr1 activity superior to I3C, DIM and pemetrexed disodium. (A) test protocol: five week old Kras G12D Mice were randomly divided into five groups (I-V, n=6). Groups I-IV were administered by daily intragastric administration. Group I: blank solvent (100 μl, corn oil with 2% dmso); group II-IV: the stomachs I3C, DIM and LTr1 were filled each day with a dose of 150 mg/kg/day. Group V: permitrexed disodium is injected intraperitoneally, 150 mg/kg/day, twice a week. All groups were dosed for thirteen weeks continuously. (B-D) comparing the antitumor Activity of I3C, DIM, LTr1 with pemetrexed disodium. According to the index: lung morphology, microCT image, H&E staining (scale, 2000 μm; enlarged in the upper box into the lower box (scale, 100 μm)); ki67 immunohistochemical positive cells of lung tissue (scale, 100 μm; boxed in upper panel to lower panel (scale, 20 μm; tumor area quantification of mouse lung (C) and Ki67 immunohistochemical positive rate statistics (D). One dot represents the average of five fields in a mouse lung section. Student's t test, p:<0.05，****p<0.0001, data are mean ± SEM of 6 biological replicates vs. corresponding control.
Detailed Description
In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the test examples of the present invention will be clearly and completely described below in connection with the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
Example 1
The invention relates to an intestinal lactobacillus strain which is preserved in China Center for Type Culture Collection (CCTCC), and the preservation name is as follows: lactobacillus acidophilus, the preservation address is the university of Chinese Wuhan; preservation date: 2021, 07, 12; preservation number: cctccc M2021863. The invention relates to application of lactobacillus enteriae in preparing food-borne antitumor drugs.
The invention relates to an intestinal lactobacillus microbial agent prepared from intestinal lactobacillus. The active ingredients are at least one of the following (a), (b) and (c):
(a) A fermentation culture of the enterolactobacillus;
(b) Ultrasonic lysis supernatant of the obtained intestinal lactobacillus cells;
(c) Ultrasonic lysis precipitation of the obtained intestinal lactobacillus cells.
The invention relates to application of lactobacillus in intestinal tract in preparing health care products.
The medicine is an oral medicine, and the oral medicine is powder, granule, tablet or capsule containing lactobacillus.
The oral medicine is powder, granule, tablet or capsule prepared by mixing lactobacillus with crucifer extract or component I3C thereof, other probiotics, vitamins and other acid sensitive components in any proportion.
The preparation method of the oral medicine comprises the following steps: inoculating lactobacillus into MRS liquid culture medium plate, culturing for 24 hr under facultative anaerobic condition at 40deg.C, centrifuging MRS, and vacuum freeze drying to obtain lactobacillus powder.
Mixing lactobacillus powder with appropriate adjuvant or vitamins or other bacteria powder, and granulating; filling the granules into capsules to obtain capsules, and tabletting the granules to obtain tablets.
Example 2
Example 2 differs from example 1 in that:
the preparation method of the oral medicine comprises the following steps: inoculating lactobacillus into MRS liquid culture medium plate, culturing for 18 hr under facultative anaerobic condition at 34 deg.C, centrifuging MRS, and vacuum freeze drying to obtain lactobacillus powder.
Example 3
Example 3 differs from example 1 in that:
the preparation method of the oral medicine comprises the following steps: lactobacillus is inoculated into a MRS liquid culture medium plate, 1 percent of inoculum size is cultured for 22 hours in a facultative anaerobic mode at 37 ℃, and the MRS is subjected to centrifugation and vacuum freeze drying to prepare lactobacillus bacteria powder.
Test example 1
Intestinal bacteria separation screening for efficient conversion of I3C
Strain sources: food metabolism residue (commonly referred to as faeces) from healthy adult volunteers.
Plate medium:
2.1 brain heart infusion Medium formulation (BHI) (g/L): 10.0 parts of peptone, 12.5 parts of dehydrated calf brain extract powder, 5.0 parts of dehydrated calf heart extract powder, 5.0 parts of sodium chloride, 2.0 parts of glucose, 2.5 parts of disodium hydrogen phosphate and 20.0 parts of agar.
2.2 bifidobacterium BS media formulation (g/L): 10.0 parts of peptone, 5.0 parts of liver extract powder, 3.0 parts of beef extract powder, 5.0 parts of yeast extract powder, 8.0 parts of tryptone, 0.5 parts of soluble starch, 1.0 part of sodium chloride, 1.0 part of dipotassium hydrogen phosphate, 1.0 part of potassium dihydrogen phosphate, 10.0 parts of glucose and FeSO 4 .7H 2 O 0.01，MnSO 4 0.005, L-cysteine 0.5 and agar 20.0.
2.3MRS liquid Medium formulation (g/L): peptone 10, beef extract 8.6, yeast extract 5, K 2 HPO 4 2. Triammonium citrate 2, sodium acetate 5, glucose 20, tween 80ml, mgSO 4 ·7H 2 O 0.58、MnSO 4 ·4H 2 O0.25 and agar 20.0.
2.4 Formula (g/L) of LB medium: tryptone 10, yeast extract 5, sodium chloride 10 and agar 20.0.
2.5 Formulation of GAM Medium (g/L): soybean peptone 3, koutone 10, digested serum powder 13.5, yeast extract 5, beef extract 2.2, beef liver extract (powder) 1.2, glucose 3, KH 2 PO 4 2.5, soluble starch 5, L-cysteine 0.3, sodium thioglycolate 0.3, broth (beef heart soup) 1000ml and agar 20.0.
3. Isolation of strains
The inventor uses a sterile excrement collector to collect the excrement of volunteers, samples are taken out, then the surfaces of the samplers are wiped by 75% alcohol, 50g of samples are weighed, 100ml of sterile water is injected into an ultra-clean workbench, ten-time gradient dilution is carried out on the sample liquid after uniform mixing, and 10 gradients are respectively takenmu.L of each of the above five solid plate media was uniformly coated with 6 plates each, divided into two groups of three plates, and then one group was placed in an anaerobic incubator (Whitley DG250 Anaerobic Workstation, UK) (gas composition 20% CO) 2 、10％H 2 、70％N 2 ) The other group was placed in a normal incubator and incubated at 37℃for 24 hours.
The next day shows that colonies with different shapes and sizes grow on the flat plate, colonies with different shapes are selected for secondary streak purification treatment, and after the flat plate is continuously cultured for 24 hours, the obtained strain is identified by 16S rRNA.
Blast analysis shows that the isolated strain has 99% homology with lactobacillus acidophilus DNA sequence. The length of the identified sequence is 1471bp, and the 16S rDNA gene sequence of the lactobacillus enterica strain is the nucleotide sequence shown as SEQ ID No. 1.
Test example 2
Screening of I3C converting ability of the separated intestinal strains
Separating to obtain 20 intestinal bacteria (Table 1), performing in vitro liquid phase culture, inoculating into corresponding culture medium, and performing in vitro culture when the concentration of thallus reaches OD 600nm At=2.50, 10ml each was taken, I3C (0.15 mg/ml) was added, co-cultured under the same conditions for 24 hours, 100 μl each was taken, an equal volume of methanol was added, vortexed, centrifuged at 13000rpm for 15 minutes, and the supernatant was taken for HPLC analysis. The results showed that both lactobacillus strains had the strongest ability to convert I3C to DIM and LTr1 (fig. 1). Human intestinal bacteria for screening for I3C conversion capacity are shown in table 1:
TABLE 1
| Sequence number | Strain name | Sequence number | Strain name |
| 1 | Bacteroides fragilis | 11 | Clostridium clostridioforme |
| 2 | Bacteroides thetaiotaomicron | 12 | Clostridium perfringen |
| 3 | Bacteroides vulgatus | 13 | Enterococcus avium |
| 4 | Bifidobacterium adolescentis | 14 | Enterococcus casseliflavus |
| 5 | Bifidobacterium angulatum | 15 | Escherichia coli |
| 6 | Bifidobacterium bifidum | 16 | Enterococcus faecalis |
| 7 | Bifidobacterium breve | 17 | Lactobacillus acidophilus |
| 8 | Bifidobacterium catenulatum | 18 | Lactobacillus gasseri |
| 9 | Bifidobacterium longum subsp.longum | 19 | Lactobacillus johnsonii |
| 10 | Clostridium butyricum | 20 | Streptococcus salivarius |
Test example 3
Acid dependency study of in vitro conversion of Lactobacillus entericus to I3C
The MRS culture solution of the above lactobacillus enteris (OD 600 nm=2.50) was divided into 6 parts, 20ml each, boiled 0 (i.e. not boiled, used as a control), 1, 5, 10, 30 and 60 minutes and sterilized by moist heat at 120 ℃ for 30 minutes. Then divided into 3 equal parts of 6ml each, and the pH was adjusted to 4.4, 7.2 and 8.0, respectively. Split into 2ml centrifuge tubes, 1ml of each tube, add I3C (0.15 mg/ml), incubate at 37 ℃ for 2 and 24 hours, sample 100 μl each, add equal volume of methanol, vortex, centrifuge at 13000rpm for 15 minutes, and take the supernatant for HPLC analysis. The results indicate that lactic acid secreted by lactobacillus enterica significantly promoted conversion of I3C to DIM and LTr1 (fig. 2).
Test example 4
The conversion of I3C to LTr1 is also acid dependent
Respectively are provided withInoculating Lactobacillus entericus to MRS culture medium to OD of culture solution 600nm When the value is equal to or close to 2.50, the mixture is divided into 6 parts, the 6 parts are respectively packed into 2ml centrifuge tubes, 1ml of each tube is added or not added with I3C (0.15 mg/ml), shake culture is carried out under the aerobic condition at 37 ℃, and the pH values are respectively sampled and detected at 2, 4, 8, 12, 24 and 36 hours. The results showed that the enterobacteria can lower the pH in the culture to maintain it between 4.0 and 4.5 (FIG. 3A). MRS culture solution (OD) containing Lactobacillus entericus 600nm =2.50) was divided into 22 parts, each 100ml, and the pH was adjusted to 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.4, 4.92, 5.29, 6.24, 6.64, 6.92, 7.38, 7.73, 8.04, 8.67, 9.3, 9.82, 10.91, 11.60 and 12.1, respectively. Each pH solution was split into 3 parts, 1ml of each part, and 0.15mg/ml of I3C solution was added thereto, followed by shaking culture at 37℃under aerobic conditions for 24 hours. 100 μl each was sampled, an equal volume of methanol was added, vortexed, centrifuged at 13000rpm for 15 minutes, and the supernatant was taken for quantitative HPLC analysis. The results indicate that the process of I3C to DIM and LTr1 is also related to acidic media; at a pH of about 4.0, the conversion efficiency was highest (FIG. 3B).
FIG. 3 is an acid-promoted I3C to LTr1 conversion of the present invention. (A) pH value in MRS Medium: with or without the addition of I3C or Lactobacillus entericus. (B) I3C transformation LTr1 optimum pH was found.
Test example 5
Effect of in vivo colonization of Lactobacillus entericus on I3C conversion to DIM and LTr1
1. Grouping and pretreatment of mice:
four-week-old nude mice were randomly divided into 3 groups of 25 mice each, group I being normal, group II being antibiotic, and group III being constant. Antibiotic groups a sterile aqueous solution of the compound antibiotic (ampicillin 1mg/ml + colistin 1mg/ml + streptomycin 5 mg/ml) was given to nude mice 2 weeks in advance to replace drinking water. Antibiotics and valuing effect were detected and evaluated by plate method, cecum morphology, H & E staining of cecum wall, and 16S rRNA or qPCR method. The results show that the combined antibiotics have good bactericidal effect (figure 4). In addition, after colonization of intestinal lactobacilli, the abundance of the intestinal lactobacilli was greatly increased (fig. 5 and 6B).
FIG. 4 shows the effect verification of antibiotic sterilization and intestinal lactobacillus colonization according to the present invention. Nude mice were randomly divided into normal group (I), antibiotic clearance group (II) and lactobacillus enterica colonization group (III). After one week of antibiotic treatment or three days of intestinal lactobacillus colonization, the mouse feces are taken for relevant detection. (A) Colony status in feces was detected with LB and MRS solid medium plates, respectively. (B) cecum morphology.
FIG. 5 shows the effect of antibiotic sterilization and Lactobacillus colonization by 16S rRNA. Nude mice were randomly divided into normal group (I), antibiotic clearance group (II) and lactobacillus enterica colonization group (III). After one week of antibiotic treatment or three days of intestinal lactobacillus colonization, the mouse feces were taken for 16S rRNA detection.
2. Sampling process
Mice were gavaged for I3C, and sacrificed 5, 10, 20, 40 and 60 minutes after gavage, taking stomach, duodenum, jejunum, ileum, cecum and colon, respectively. The contents were weighed separately, diluted 5-fold with sterile water and the pH was checked. Simultaneously, the contents were taken at 100mg each, extracted with 5 volumes of ethyl acetate containing 50ng/l ketoconazole, vortexed, centrifuged, the solvent removed in supernatant vacuum, redissolved with 200 μl of chromatographic methanol, centrifuged, and 50 μl each was sent for LC-MS/MS analysis. The results indicate that after antibiotic sterilization, the yield of conversion of I3C to DIM and LTr1 is significantly reduced; intestinal lactobacillus colonization can significantly reduce pH in the intestinal microenvironment (fig. 7), and the conversion of I3C to DIM and LTr1 yields are significantly improved (fig. 6G-J).
FIG. 6 shows the process of promoting the conversion of I3C to LTr1 at the small intestine by the Lactobacillus intestinal colonization of the present invention. (A) protocol. Four-week-old nude mice were randomly divided into three groups (I-III groups, n=5). Group I, I3C administered by gavage. Group II, intestinal microorganisms were removed with complex antibiotics (ampicillin + colistin + streptomycin) and then administered I3C by gavage. Group III, which was sterilized with the combination antibiotic (first week), followed by colonization with Lactobacillus enterica and gavage with I3C (from the second week). (B) qPCR quantitative analysis of the abundance of each intestinal tract of Lactobacillus in the colonized intestinal tract Lactobacillus group mice. (C-J) content of DIM and LTr1 in the contents of different digestive tract parts of three groups of mice.
Test example 6
Comparison of cytotoxic Activity of I3C and its metabolites
Human tumor cells purchased from Qiao Xin boat biotechnology limited in Shanghai included: non-small cell lung cancer cell A549, colon cancer cell SW480, liver cancer cell HepG2, melanoma cell A375, breast cancer subcontracting MCF-7 and ovarian cancer cell Caov-3 were cultured with DMEM medium (containing 10% fetal bovine serum, 100units/L penicillin G sodium and 100. Mu.g/L streptomycin sulfate), inoculated into 96-well plates, and inoculated with cell amounts of about 10000 cells/well, 200. Mu.l medium per well volume, 37℃C, 5% CO 2 Culturing in an incubator for 24 hours. All samples to be tested (I3A, I3CA, ICZ, I3C, DIM, LTr1, doxorubicin) were prepared with DMSO as a stock solution at a final concentration of 10 mM. Diluted to different concentrations in DMSO, and added to 96-well plates. Meanwhile, DMSO was used as a blank. After further culturing for 48 hours, 20. Mu.l of MTT (3- (4, 5-dimethyl-2-thiazolyl) -2,5-diphenyl-2-H-tetrazolium bromide) solution (5 mg/ml) was added, and the culturing was continued for 4 hours. The culture was terminated, the supernatant was carefully removed, 150. Mu.l of DMSO was added to each well to dissolve the crystals, and absorbance was measured on a microplate reader (490 nm). Calculating half inhibition rate IC 50 Values. The results indicate that among the series of metabolites of I3C, IC of I3A, I3CA, ICZ, I3C 50 Are all greater than 10 mu M; LTr1 has the best cytotoxic activity in vitro, better than I3C and DIM (FIG. 8).
Test example 7
LTr1 the effect of resisting non-small cell lung cancer in vivo is better than that of I3C and DIM
1. Cell inoculation:
100 μl (6X10) of a549 cell Phosphate Buffer (PBS) suspension 6 ) The right armpit of female nude mice will be injected separately. All test animals were monitored for viability, physiological status, body weight and tumor growth every three days. Tumor volume was 1/2 XLXW 2 Formula calculation, wherein "L" and "W" are the long and short diameters (units: cm) of the tumor, respectively. When the size of transplanted tumor is 20X 40mm 2 (designated "day 0"), mice were randomized into treatment and control groups.
2. Evaluation of antitumor Activity:
(A) Test protocol: four-week-old nude mice were randomly divided into four groups (I-IV, n=6). Blank solvent (100 μl of corn oil with 2% DMSO), I3C, DIM and LTr1, 150 mg/kg/day, were administered daily by gavage, respectively, for four weeks.
(B-F) evaluation of antitumor Activity: after four weeks of administration, the antitumor activity of I3C, DIM and LTr was achieved by: tumor volume and weight, ki67 immunohistochemistry, and expression level were evaluated. The results showed that the antitumor activity of LTr was superior to I3C and DIM in nude mouse model of non-small cell lung cancer cell A549 colonization (FIG. 9)
Fig. 9 is a graph showing that the anti-tumor activity of LTr1 is better than that of I3C and DIM according to the naked murine model of a549 cell engraftment of the present invention. Experimental protocol (a): four-week-old nude mice were randomly divided into four groups (I-IV, n=6). Blank solvent (100 μl of corn oil with 2% DMSO), I3C, DIM and LTr1, 150 mg/kg/day, were administered daily by gavage, respectively, for four weeks. Four weeks after (B-D) administration, the tumor volume sizes of the transplanted tumors were compared, and the antitumor activity of LTr was found to be superior to I3C and DIM. The indicators (E and F) Ki67 immunohistochemistry and expression levels thereof indicate that LTr1 has better antitumor activity than I3C and DIM (E, scale, ki67 immunohistochemistry 20 μm; boxed in upper panel, enlarged to lower panel; F, each data point represents the mean value of five fields in one mouse tumor section: p <0.05, p <0.01, p <0.001, p <0.0001, mean value of 6 biological samples.+ -. SEM compared to the corresponding control.
Test example 8
Kras G12D The in-situ lung cancer mouse model shows that the anticancer effect of LTr1 is better than that of I3C, DIM and pemetrexed disodium
Kras G12D Mice and adenovirus infection and identification:
with the following primer pair C57BL/6kras G12D Mice were identified:
forward primer: wild mice 5'-tgtctccagagt-3'; mutant mice
5′-GCAGGTCG-agggacctata-3′。
Reverse primer: 5' -CTGCATAGTACGCTATACCCTGT-3.
After anesthesia with 8% chloral hydrate, kras G12D Mice were given 15 μl 2.7X10 by nasal administration 7 Mu.g/ml AdCre virus (pAAV-CMV-bGlobal-Cre-eGFP, shanghai Ji Kai Gene chemical technology Co., ltd., china) produced endogenous lung tumors.
2. Evaluation of Activity:
(A) Experimental protocol: five week old Kras G12D Mice were randomly divided into five groups (I-V, n=6). Groups I-IV were administered by daily intragastric administration. Group I: blank solvent (100 μl, corn oil with 2% dmso); group II-IV: the stomachs I3C, DIM and LTr1, 150 mg/kg/day, respectively, were irrigated daily. Group V: permitrexed disodium (positive control) was injected intraperitoneally, 150 mg/kg/day twice a week. All groups were dosed for thirteen weeks continuously.
(B) Evaluation of antitumor Activity:
the efficacy of the drug administration was evaluated by CT image analysis 13 weeks after the drug administration. After 15 minutes of the last administration, the orbit was bled, the lungs were collected, a portion was snap frozen with liquid nitrogen and a portion was formalin fixed. Evaluation of antitumor Activity of I3C, DIM, LTr1 and pemetrexed disodium: according to lung morphology, microCT image and H&E staining, ki67 immunohistochemistry of lung tissues, quantitative tumor area of the lungs of mice, positive rate of Ki67 immunohistochemistry and the like. The results show that in Kras G12D The activity of LTr1 was superior to I3C, DIM and pemetrexed disodium in the in situ lung cancer mouse model (fig. 10).
FIG. 10 Kras of the present invention G12D The mouse model showed LTr1 activity superior to I3C, DIM and pemetrexed disodium profile. (A) test protocol: five week old Kras G12D Mice were randomly divided into five groups (I-V, n=6). Groups I-IV were administered by daily intragastric administration. Group I: blank solvent (100 μl, corn oil with 2% dmso); group II-IV: the stomachs I3C, DIM and LTr1 were filled each day with a dose of 150 mg/kg/day. Group V: permitrexed disodium is injected intraperitoneally, 150 mg/kg/day, twice a week. All groups were dosed for thirteen weeks continuously. (B-D) comparing the antitumor Activity of I3C, DIM, LTr1 with pemetrexed disodium. According to the index: lung morphology, microCT image, H&E staining (scale, 2000 μm; enlarged in the upper box into the lower box (scale, 100 μm)); ki67 immunohistochemical positive cells of lung tissue (scale, 100 μm; boxed magnification in the upper panel)Into the lower layer graph (scale, 20 μm); tumor area quantification of mice lung (C) and Ki67 immunohistochemical positive rate statistics (D). One point represents the average of five fields in a mouse lung slice. Student's t test, ×p<0.05，****p<0.0001, data are mean ± SEM of 6 biological replicates vs. corresponding control.
Test example 9
Preparation of intestinal lactobacillus powder, granule, capsule and tablet
The preparation method of the freeze-dried bacterial powder comprises the following steps: inoculating lactobacillus enteris into MRS liquid culture medium plate, performing facultative anaerobic culture for 18-24 hours at 37+ -3deg.C with 1% -5% inoculum size, centrifuging MRS, vacuum freeze drying to obtain lactobacillus enteris powder, and granulating to obtain granule; filling the granules into capsules to obtain capsules, and further tabletting the granules to obtain tablets.
Test example 10
Compound preparation of lactobacillus powder and crucifer extract
The lactobacillus powder and crucifer extract are taken and respectively or mixed to prepare powder, granules, capsules or tablets (mixed according to the proportion of 1-100 to 1-100) for separate or simultaneous administration.
Test example 11
Intestinal lactobacillus powder and I3C composite preparation
The lactobacillus enteris powder and I3C are taken and respectively or mixed to prepare (mixed according to the proportion of 1-100 to 1-100) powder, granule, capsule or tablet which are taken respectively or simultaneously.
Test example 12
Preparation of intestinal lactobacillus powder
The lactobacillus johnsonii powder and the lactobacillus gasseri Lactobacillus gasseri and the lactobacillus johnsonii Lactobacillus johnsonii powder are singly or mixed to prepare (mixed according to the proportion of 1-100 to 1-100) powder.
Test example 13
Preparation of intestinal lactobacillus powder
The lactobacillus johnsonii Lactobacillus johnsonii powder is taken and singly or mixed with the lactobacillus johnsonii Lactobacillus gasseri and then mixed with auxiliary materials such as magnesium stearate, silicon dioxide and the like.
Test example 14
Preparation of intestinal lactobacillus powder
Mixing lactobacillus johnsonii powder and lactobacillus gasseri Lactobacillus gasseri, lactobacillus johnsonii Lactobacillus johnsonii powder according to a certain proportion, adding appropriate vitamin E, B2, etc., and mixing with adjuvants such as magnesium stearate, silicon dioxide, etc.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the foregoing test examples and descriptions are merely illustrative of the principles of the present invention and that various changes and modifications may be made therein without departing from the spirit and scope of the invention, the scope of which is defined in the appended claims, specification and their equivalents.
Sequence listing
<110> university of Nanjing
<120> Lactobacillus entericus for imparting anticancer efficacy to food ingredients and use thereof
<130> 2010
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1471
<212> DNA
<213> Artificial sequence (16S rRNA of Strain)
<400> 1
gcgcggtgcg cgtgctatac tgcagtcgag cgagctgaac caacagattc acttcggtga 60
tgacgttggg aacgcgagcg gcggatgggt gagtaacacg tggggaacct gccccatagt 120
ctgggatacc acttggaaac aggtgctaat accggataag aaagcagatc gcatgatcag 180
cttataaaag gcggcgtaag ctgtcgctat gggatggccc cgcggtgcat tagctagttg 240
gtagggtaac ggcctaccaa ggcaatgatg catagccgag ttgagagact gatcggccac 300
attgggactg agacacggcc caaactccta cgggaggcag cagtagggaa tcttccacaa 360
tggacgaaag tctgatggag caacgccgcg tgagtgaaga aggttttcgg atcgtaaagc 420
tctgttgttg gtgaagaagg atagaggtag taactggcct ttatttgacg gtaatcaacc 480
agaaagtcac ggctaactac gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt 540
ccggatttat tgggcgtaaa gcgagcgcag gcggaagaat aagtctgatg tgaaagccct 600
cggcttaacc gaggaactgc atcggaaact gtttttcttg agtgcagaag aggagagtgg 660
aactccatgt gtagcggtgg aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg 720
ctctctggtc tgcaactgac gctgaggctc gaaagcatgg gtagcgaaca ggattagata 780
ccctggtagt ccatgccgta aacgatgagt gctaagtgtt gggaggtttc cgcctctcag 840
tgctgcagct aacgcattaa gcactccgcc tggggagtac gaccgcaagg ttgaaactca 900
aaggaattga cgggggcccg cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg 960
aagaacctta ccaggtcttg acatctagtg caatccgtag agatacggag ttcccttcgg 1020
ggacactaag acaggtggtg catggctgtc gtcagctcgt gtcgtgagat gttgggttaa 1080
gtcccgcaac gagcgcaacc cttgtcatta gttgccagca ttaagttggg cactctaatg 1140
agactgccgg tgacaaaccg gaggaaggtg gggatgacgt caagtcatca tgccccttat 1200
gacctgggct acacacgtgc tacaatggac agtacaacga ggagcaagcc tgcgaaggca 1260
agcgaatctc ttaaagctgt tctcagttcg gactgcagtc tgcaactcga ctgcacgaag 1320
ctggaatcgc tagtaatcgc ggatcagcac gccgcggtga atacgttccc gggccttgta 1380
cacaccgccc gtcacaccat gggagtctgc aatgcccaaa gccggtggcc taaccttcgg 1440
gaaggagccg tctaagtcag tcaggtctca a 1471
Claims (8)
1. A lactobacillus acidophilus strain characterized in that: the lactobacillus acidophilus strain is preserved in China Center for Type Culture Collection (CCTCC), and the preservation name is:Lactobacillus acidophilusthe preservation address is the university of Chinese Wuhan; preservation date: 2021, 07, 12; preservation number: cctccc M2021863.
2. An intestinal lactobacillus bacterial agent prepared by lactobacillus acidophilus as claimed in claim 1.
3. The lactobacillus acidophilus agent of claim 2, wherein the active ingredient is at least one of the following (a) (b) (c):
(a) A fermentation culture of lactobacillus acidophilus as claimed in claim 1;
(b) An ultrasonically lysed supernatant of lactobacillus acidophilus cells as obtained in claim 1;
(c) An ultrasonically lysed pellet of lactobacillus acidophilus cells as obtained in claim 1.
4. Use of lactobacillus acidophilus as claimed in claim 1 in the manufacture of a food-borne antitumor drug, characterized in that: promoting the conversion of I3C into antitumor components LTr1 and DIM.
5. The use of lactobacillus acidophilus as claimed in claim 1 in the manufacture of a health product.
6. The use according to claim 4, characterized in that: the medicine is an oral medicine, and the oral medicine is powder, granule, tablet or capsule containing lactobacillus.
7. The use according to claim 4, characterized in that: the preparation method of the oral medicine comprises the following steps: inoculating lactobacillus into MRS liquid culture medium plate, culturing for 18-24 hr under facultative anaerobic condition at 37+ -3deg.C, centrifuging MRS, and vacuum freeze drying to obtain lactobacillus acidophilus powder.
8. The use according to claim 7, characterized in that: mixing lactobacillus acidophilus powder with proper auxiliary materials or vitamins or other bacteria powder according to a certain proportion, and granulating to obtain granules; filling the granules into capsules to obtain capsules, and tabletting the granules to obtain tablets.
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