CN112592872A - Bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals and application thereof - Google Patents
Bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals and application thereof Download PDFInfo
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- CN112592872A CN112592872A CN202110227019.XA CN202110227019A CN112592872A CN 112592872 A CN112592872 A CN 112592872A CN 202110227019 A CN202110227019 A CN 202110227019A CN 112592872 A CN112592872 A CN 112592872A
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- bifidobacterium longum
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- 238000004321 preservation Methods 0.000 claims abstract 4
- 239000000843 powder Substances 0.000 claims description 21
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- 239000007788 liquid Substances 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 9
- DLRVVLDZNNYCBX-RTPHMHGBSA-N isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
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- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 6
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 6
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- 241000282414 Homo sapiens Species 0.000 description 5
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- 238000002835 absorbance Methods 0.000 description 4
- 238000004140 cleaning Methods 0.000 description 4
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
- 229930003427 Vitamin E Natural products 0.000 description 3
- 238000009835 boiling Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
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- 235000019154 vitamin C Nutrition 0.000 description 3
- 239000011718 vitamin C Substances 0.000 description 3
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- 239000011709 vitamin E Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 2
- 102000019197 Superoxide Dismutase Human genes 0.000 description 2
- 108010012715 Superoxide dismutase Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000007760 free radical scavenging Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 241000186016 Bifidobacterium bifidum Species 0.000 description 1
- 241000186012 Bifidobacterium breve Species 0.000 description 1
- 241000186011 Bifidobacterium catenulatum Species 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
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- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000012894 fetal calf serum Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
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- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
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Images
Classifications
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- 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
- A61K35/745—Bifidobacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
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- 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
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/533—Longum
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Abstract
The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals and application thereof, wherein the bifidobacterium longum JBLC-141 is preserved in China general microbiological culture Collection center in 7-8.7.2019, the preservation address is No. 3 of Xilu No. 1 Beichen of the sunward area in Beijing, the preservation number is CGMCC number 18094, and the bifidobacterium longum is classified and named as bifidobacterium longumBifidobacterium longum(ii) a The Bifidobacterium longumThe strain JBLC-141 can be used for preparing products for eliminating in-vivo free radicals and/or relieving body fatigue. The bifidobacterium longum JBLC-141 can remove redundant free radicals in a body, prevent the loss of the free radicals to body cells, play a certain role in protecting oxidative damage of the cells and relieve body fatigue.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals and application thereof.
Background
The bifidobacterium is gram positive, is a strictly anaerobic bacterium which is widely existed in the habitats of human and animals such as digestive tracts and the like, is one of important constituent members of human and animal intestinal flora, and plays an important role in maintaining the micro-ecological balance of human bodies. The shape of the bifidobacterium is variable, and the bifidobacterium presents different shapes under the influence of different species, different ages and different growth environments, such as short-rod cells with more regular or slender rod-shaped interstitial ends, spherical cells, rod-shaped cells, spoon-shaped cells or various branches and bifurcations, and the like. The colonies of bifidobacteria are smooth, convex, complete in edge, white in cream color, shiny and soft in texture. Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium fusiform and Bifidobacterium catenulatum are all Bacillus bifidus strains closely related to human beings.
Therefore, the bifidobacterium longum capable of eliminating free radicals in vivo and the application thereof have very important significance for developing and utilizing the bifidobacterium, improving the body health and improving the quality of life of human beings.
Disclosure of Invention
Aiming at the problem that the prior art lacks a strain capable of eliminating free radicals in vivo, the invention provides bifidobacterium longum JBLC-141 capable of eliminating free radicals in vivo and application thereof, wherein the bifidobacterium longum JBLC-141 can eliminate redundant free radicals in vivo, prevent the loss of free radicals to cells of an organism, relieve the reduction of SOD activity and the surge effect of GPx activity, simultaneously play a certain role in protecting cells from oxidative damage, and relieve the fatigue of the organism.
In a first aspect, the invention provides bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals, wherein the bifidobacterium longum JBLC-141 is deposited in China General Microbiological Culture Collection Center (CGMCC) in 7-8.2019 at the position of No. 3 Hospital No. 1 of the south China Committee for Culture Collection of microorganisms, the deposition address is No. 3 of the national institute of Microbiological Culture Collection, the deposition number is CGMCC number 18094, and the classification name is bifidobacterium longumBifidobacterium longum。
In a second aspect, the invention provides the application of the bifidobacterium longum JBLC-141 in preparing products for eliminating free radicals in vivo and/or relieving body fatigue.
Further, the product comprises bifidobacterium longum JBLC-141 powder and isomaltooligosaccharide.
Further, the preparation of the product comprises the following steps:
(1) preparing bifidobacterium longum JBLC-141 powder;
(2) compounding the bifidobacterium longum JBLC-141 powder with isomaltooligosaccharide.
Further, the bifidobacterium longum JBLC-141 powder is prepared by the following steps:
(a) inoculating Bifidobacterium longum JBLC-141 to MRS liquid culture medium, and anaerobically culturing at 37 deg.C to obtain MRS bacterial liquid;
(b) and centrifuging the MRS bacterial liquid, and carrying out vacuum freeze drying to obtain Bifidobacterium longum JBLC-141 bacterial powder.
Further, the inoculation amount of Bifidobacterium longum JBLC-141 in the step (a) is 1%, and the strain is anaerobic (N) at 37 ℃2：H2：CO2= 85: 10: 5) and culturing for 24 h.
Further, the MRS liquid medium includes:
peptone 10g, beef powder 5g, K2HPO4·7H2O2 g, triammonium citrate 2g, CH3COONa·3H2O5 g, glucose 20g, Tween 801 mL, MgSO4·7H2O 0.2g、MnSO4·4H20.05g of O and 1000mL of distilled water.
Further, the step (2) is to mix and compound the bifidobacterium longum JBLC-141 powder and the isomaltooligosaccharide into products with the viable bacteria number of 50 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g and 1000 hundred million/g.
The beneficial effect of the invention is that,
the invention provides bifidobacterium longum JBLC-141 capable of eliminating free radicals in vivo and application thereof, and experiments prove that the bifidobacterium longum JBLC-141 has good capability of eliminating hydroxyl free radicals, superoxide anion free radicals and DPPH free radicals, remarkable capability of relieving Caco-2 cell oxidative damage and capability of relieving body fatigue.
Drawings
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings used in the description of the embodiments or prior art will be briefly described below, and it is obvious for those skilled in the art that other drawings can be obtained based on these drawings without creative efforts.
FIG. 1 is a bar graph of hydroxyl radical scavenging activity;
FIG. 2 is a histogram of superoxide anion radical scavenging activity;
FIG. 3 is a bar graph of DPPH radical scavenging activity.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the technical solution in the embodiment of the present invention will be clearly and completely described below with reference to the drawings in the embodiment of the present invention, and it is obvious that the described embodiment is only a part of the embodiment of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 isolation and purification of bacterial species
(1) The Guangxi Bama takes a sample of the local healthy adult excrement by using a sterilized sampling spoon, prevents other parts from contacting the sample in the sampling process so as to prevent pollution, and immediately stores the sample in a cold storage box after sampling and records the sample number;
(2) weighing 1g of a feces sample, dissolving the feces sample in 10ml of normal saline, uniformly mixing, diluting the sample liquid by ten times of gradient, uniformly coating 100 mu L of each gradient on an MRS plate culture medium, then taking out and placing the sample liquid in an anaerobic tank, and carrying out anaerobic culture at 37 ℃ overnight;
(3) the next day, colonies with different shapes and sizes are grown on the plate, a plurality of characteristic colonies (white, glossy, regular edges, round and convex in the center) of bifidobacterium longum are selected for the second-generation streak purification treatment, and the plate is subjected to anaerobic activation for 1 day to obtain a culture. And then strain identification is carried out.
Example 2 species identification
(1) Identification unit
Biometrics (Shanghai) Ltd.
(2) Primer and method for producing the same
27F：5'-AGAGTTTGATCMTGGCTCAG-3'；
1492R：5'-GGTTACCTTGTTACGACTT-3'。
(3) Identification of sequences
The identification sequences are shown below:
ggatgtaatc cgagcctcac cttagacggc tccatcccac aaggggttag gccaccggct 60
tcgggtgctg cccactttca tgacttgacg ggcggtgtgt acaaggcccg ggaacgcatt 120
caccgcgacg ttgctgattc gcgattacta gcgactccgc cttcacgcag tcgagttgca 180
gactgcgatc cgaactgaga ccggttttca gggatccgct ccgcgtcgcc gcgtcgcatc 240
ccgttgtacc ggccattgta gcatgcgtga agccctggac gtaaggggca tgatgatctg 300
acgtcatccc caccttcctc cgagttaacc ccggcggtcc cccgtgagtt cccggcataa 360
tccgctggca acacggggcg agggttgcgc tcgttgcggg acttaaccca acatctcacg 420
acacgagctg acgacgacca tgcaccacct gtgaacccgc cccgaaggga agccgtatct 480
ctacgaccgt cgggaacatg tcaagcccag gtaaggttct tcgcgttgca tcgaattaat 540
ccgcatgctc cgccgcttgt gcgggccccc gtcaatttct ttgagtttta gccttgcggc 600
cgtactcccc aggcgggatg cttaacgcgt tagctccgac acggaacccg tggaacgggc 660
cccacatcca gcatccaccg tttacggcgt ggactaccag ggtatctaat cctgttcgct 720
ccccacgctt tcgctcctca gcgtcagtaa cggcccagag acctgccttc gccattggtg 780
ttcttcccga tatctacaca ttccaccgtt acaccgggaa ttccagtctc ccctaccgca 840
ctcaagcccg cccgtacccg gcgcggatcc accgttaagc gatggacttt cacaccggac 900
gcgacgaacc gcctacgagc cctttacgcc caataattcc ggataacgct tgcaccctac 960
gtattaccgc ggctgctggc acgtagttag ccggtgctta ttcaacgggt aaactcactc 1020
tcgcttgctc cccgataaaa gaggtttaca acccgaaggc ctccatccct cacgcggcgt 1080
cgctgcatca ggcttgcgcc cattgtgcaa tattccccac tgctgcctcc cgtaggagtc 1140
tgggccgtat ctcagtccca atgtggccgg tcgccctctc aggccggcta cccgtcgaag 1200
ccacggtggg ccgttacccc gccgtcaagc tgataggacg cgaccccatc ccataccgcg 1260
aaagctttcc cagaagacca tgcgatcaac tggagcatcc ggcattacca cccgtttcca 1320
ggagctattc cggtgtatgg ggcaggtcgg tcacgcatta ctcacccgtt cgccactctc 1380
accaccaagc aagctaatcc c 1401
EXAMPLE 3 free radical scavenging experiments
(1) Cell culture
At 37 ℃ 5% CO2Under the condition of using the mass fractionCaco-2 cells (cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences) are cultured in high-glucose DMEM culture solution containing 10% fetal calf serum and 1% non-essential amino acids by mass fraction.
(2) Hydroxy radical scavenging test
The sample groups included the JBLC-141 group and the inactivated group,
for JBLC-141 group, 1mL of phenanthroline (0.75 mmol/L), 1mL of FeSO4(0.75 mmol/L) and 2mL PBS (pH = 7.4) were mixed well and 1mL H was added2O2(the mass fraction is 0.12%), then adding 1mL of Bifidobacterium longum JBLC-141 bacterial liquid, uniformly mixing, and incubating for 90min at 37 ℃, wherein the Bifidobacterium longum JBLC-141 bacterial liquid is prepared by dissolving 1g of Bifidobacterium longum powder after vacuum drying in 1mL of PBS.
For the inactivated group, 1mL of phenanthroline (0.75 mmol/L) and 1mL of FeSO4(0.75 mmol/L) and 2mL PBS (pH = 7.4) were mixed well and 1mL H was added2O2(mass fraction is 0.12%), adding 1mL of inactivated thallus, uniformly mixing, and incubating for 90min at 37 ℃, wherein the preparation method of the inactivated thallus comprises the steps of treating the JBLC-141 bacterial liquid in a boiling water bath for 20min, washing for 3 times by using sterile PBS, and centrifuging for 10min at 6000r/min and 4 ℃;
the control group comprises 1mL of phenanthroline (0.75 mmol/L) and 1mL of FeSO4(0.75 mmol/L) and 2mL PBS (pH = 7.4) were mixed well, and 1mL H was added2O2(mass fraction 0.12%) and mixing;
the blank group is prepared by mixing 1mL of phenanthroline (0.75 mmol/L) and 1mL of FeSO4(0.75 mmol/L) and 2mL PBS (pH = 7.4) were mixed well;
the absorbance was measured at 536nm, and the hydroxyl radical scavenging activity was calculated by the following formula,
hydroxyl radical scavenging activity = (a)s-Ac）/（Ab-Ac）×100%，
Wherein A iss: the light absorption value of the sample group is,
Ac: control group absorbance (including phenanthroline and FeSO)4、H2O2And PBS),
Ab: blank light absorption value (including phenanthroline and FeSO)4、PBS）；
As shown in FIG. 1, the results of the hydroxyl radical scavenging test showed that the hydroxyl radical scavenging ability of JBLC-141 group (65.07. + -. 0.025) > the inactivation group (13.09. + -. 0.036) was demonstrated in FIG. 1.
(3) Superoxide anion radical scavenging experiment
The sample groups include JBLC-141 group and inactivation group, the sample groups are incubated at 37 deg.C for 5min,
for JBLC-141 group, each 1mL of the reaction solution contained 200. mu.L of PBS (20 mmol/L, pH = 7.4), 250. mu.L of NBT (50. mu. mol/L), 250. mu.L of NADH (75. mu. mol/L), 250. mu.L of PMS (15. mu. mol/L) and 50. mu.L of Bifidobacterium longum JBLC-141 bacterial solution (50. mu.L of PBS +50mg of dried Bifidobacterium longum powder),
for the inactivated group, the reaction solution contained 200. mu.L of PBS (20 mmol/L, pH = 7.4), 250. mu.L of NBT (50. mu. mol/L), 250. mu.L of NADH (75. mu. mol/L), 250. mu.L of PMS (15. mu. mol/L) and 50. mu.L of inactivated cells (obtained by boiling the above Bifidobacterium longum solution, washing and centrifuging) per 1mL of the reaction solution;
the control group was distilled water;
the absorbance was measured at 560nm, and the superoxide anion radical scavenging activity was calculated by the following formula,
superoxide anion radical scavenging activity = [ (a)s-Ac）/As]×100%，
Wherein A iss: the light absorption value of the sample group is,
Ac: the light absorption value of the control group;
as shown in FIG. 2, it can be seen from FIG. 2 that the superoxide anion radical scavenging ability (46.65. + -. 0.016) > of the JBLC-141 group was higher than that of the inactivation group (19.53. + -. 0.052).
(4) DPPH free radical scavenging experiment
The sample groups include JBLC-141 group and inactivation group, the sample groups are incubated at 37 deg.C for 5min,
for JBLC-141 group, DPPH was dissolved in methanol to prepare a DPPH solution with a concentration of 0.1mmol/L, 2mL of the solution was then mixed with 1mL of Bifidobacterium longum JBLC-141 (prepared by dissolving 1g of dried Bifidobacterium longum powder in 1mL of PBS, and the mixture was mixed well and incubated at room temperature in the dark for 30 min.
For the inactivated group, dissolving DPPH in methanol to prepare DPPH solution with concentration of 0.1mmol/L, then taking 2mL of solution to be uniformly mixed with 1mL of inactivated thallus, incubating for 30min at room temperature in a dark place, and preparing the inactivated thallus by treating the JBLC-141 bacterial solution in a boiling water bath for 2min, washing for 3 times with sterile PBS, and centrifuging for 10min at 4 ℃ at 6000 r/min;
the control group was distilled water;
the absorbance was measured at 517nm, the DPPH radical scavenging activity was calculated by the following formula,
DPPH radical scavenging Activity = [ (A)s-Ac）/As]×100%，
Wherein A iss: the light absorption value of the sample group is,
Ac: the light absorption value of the control group;
as shown in FIG. 3, it can be seen from FIG. 3 that the DPPH radical scavenging ability of the JBLC-141 group (36.32. + -. 0.042) > is higher than that of the inactivated group (4.25. + -. 0.039).
Example 4 Caco-2 cell oxidative damage alleviating ability test
For the JBLC-141 group, Caco-2 cells were cultured at 3X 105The concentration per well was inoculated in 6-well plates and cultured continuously for 18 days, and 3mL of H prepared in serum-free and double-antibody-free DMEM (purchased from Gibco Co.) at a concentration of 500. mu. mol/L was added to each well2O2Incubation for 30min and then H aspiration2O2Cleaning, adding 3mL DMEM into each well to prepare viable count of 108continuously culturing the JBLC-141 bacterial suspension of cfu/mL for 4 h;
for positive control group, Caco-2 cells were cultured at 3X 105The concentration per well was inoculated in a six-well plate and cultured continuously for 18 days, and 3mL of H prepared in serum-free and double-antibody-free DMEM (purchased from Gibco Co.) at a concentration of 500. mu. mol/L was added to each well2O2Incubation for 30min and then H aspiration2O2Cleaning, adding 3mL of Vc with the mass fraction of 0.01% into each hole, and continuously culturing for 4 h;
for the model group, Caco-2 cells were cultured at 3X 105The concentration per well was inoculated in 6-well plates and cultured continuously for 18 days, 3mL of DMEM (serum-free and double-antibody-free) was added to each wellFrom Gibco) at a concentration of 500. mu. mol/L2O2Incubation for 30min and then H aspiration2O2Cleaning, adding 3mL of DMEM into each hole, and continuously culturing for 4 h;
for the blank, Caco-2 cells were plated at 3X 105Inoculating the concentration of the/hole to a 6-hole plate, culturing for 48h, adding 3mL of DMEM into each hole, and continuously culturing for 4 h;
rapidly cleaning a six-hole plate 3 times by using cold sterile PBS, collecting cells by trypsinization, centrifuging at 4 ℃ for 10min at 2000g, discarding supernatant, washing the cells by using 1mL of cold PBS, centrifuging at 4 ℃ for 10min at 2000g, adding 1mL of 1% Tritonx-100, uniformly mixing, centrifuging at 4 ℃ for 15min at 4000g, collecting supernatant, and respectively measuring SOD activity and GPx activity in the supernatants of different experimental groups.
The test results are shown in table 1 below for SOD activity: compared with the blank group, the SOD activity of the model group is obviously reduced (P is less than 0.05), which indicates that Caco-2 cells are subjected to oxidative damage and an oxidative damage model is established; compared with the model group, the SOD activities of the positive control group and the JBLC-141 group are obviously improved, and the JBLC-141 group is closer to the blank group than the positive control group.
For GPx activity: the GPx activity was significantly increased in the model group compared to the blank group (P)<0.05), indicating that Caco-2 cells are at low concentration H2O2The GPx activity tends to increase in the environment to relieve the oxidative damage to the GPx; compared with the model group, the GPx activity of the positive control group and the JBLC-141 group is remarkably reduced, the JBLC-141 group is closer to the blank group than the positive control group, and the GPx activity is recovered to be not remarkably different from that of the blank group (P)>0.05）。
TABLE 1 influence of JBLC-141 on antioxidant Activity
| Experimental group | SOD Activity/% (with)Blank set phase ratio) | GPx Activity/% (vs blank) |
| Blank group | 100 | 100 |
| Model set | 68.13±0.3* | 216.32±1.3* |
| Positive control group | 87.12±2.6# | 118.36±3.6# |
| Group JBLC-141 | 92.36±3.1# | 105.45±2.1# |
Note: differences were significant compared to blank group: p < 0.05; significant difference # compared to model group: p < 0.05.
Example 5A product having effects of scavenging free radicals and relieving fatigue
A product with the functions of eliminating free radicals in vivo and relieving body fatigue comprises bifidobacterium longum JBLC-141 powder and isomaltooligosaccharide, and the preparation method comprises the following steps:
(1) preparation of bifidobacterium longum JBLC-141 powder:
(a) inoculating Bifidobacterium longum JBLC-141 to MRS liquid medium at 1%, anaerobic (N) at 37 ℃2：H2：CO2= 85: 10: 5) and culturing for 24h to obtain MRS bacterial liquid, wherein the MRS liquid culture medium comprises:
peptone 10g, bovineMeat meal 5g, K2HPO4·7H2O2 g, triammonium citrate 2g, CH3COONa·3H2O5 g, glucose 20g, Tween 801 mL, MgSO4·7H2O 0.2g、MnSO4·4H20.05g of O and 1000mL of distilled water;
(b) centrifuging the MRS bacterial liquid, and freeze-drying in vacuum to obtain Bifidobacterium longum JBLC-141 bacterial powder;
(2) the Bifidobacterium longum JBLC-141 powder and isomaltooligosaccharide are mixed to prepare products with viable bacteria number of 50 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g and 1000 hundred million/g.
Example 6 fatigue-relieving Effect of Bifidobacterium longum JBLC-141
42 students (male 23, female 19) of the track and field major were selected and randomly divided into a control group and a test group, each of which was 21. Each volunteer carries out routine exercise training every day, and in the early morning before and after the test, venous blood is taken on an empty stomach for measuring physiological and biochemical indexes, and the influence of the bifidobacterium longum JBLC-141 on the numerical values of serum malondialdehyde MDA, superoxide dismutase SOD and urea nitrogen BUN is analyzed.
The control group takes vitamin E and vitamin C, 50 mg/vitamin E tablet and 100 mg/vitamin C tablet each time, 3 times daily for 60 days;
the test group takes the product of example 5, 150 hundred million viable bacteria each time, 3 times daily, and 60 days continuously.
The test results are shown in table 2, and it can be seen that after the treatment, the SOD in the body of the two groups is significantly increased, MDA and BUN are significantly decreased, and the effect of bifidobacterium longum is better than that of vitamin E + vitamin C. This indicates that Bifidobacterium longum can relieve fatigue by increasing the activity of superoxide dismutase, reducing oxidative damage in the body, and scavenging oxygen radicals.
TABLE 2 Effect of Bifidobacterium longum JBLC-141 on Biochemical index
P <0.05 compared to pre-treatment; compared to the control group, # P < 0.05.
Although the present invention has been described in detail by referring to the drawings in connection with the preferred embodiments, the present invention is not limited thereto. Various equivalent modifications or substitutions can be made on the embodiments of the present invention by those skilled in the art without departing from the spirit and scope of the present invention, and these modifications or substitutions are within the scope of the present invention/any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
SEQUENCE LISTING
<110> Shandong Zhongke Jiayi bioengineering Co., Ltd
<120> Bifidobacterium longum JBLC-141 capable of eliminating free radicals in vivo and application thereof
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 1401
<212> DNA
<213> Bifidobacterium longum JBLC-141 (Bifidobacterium longum JBLC-141)
<400> 1
ggatgtaatc cgagcctcac cttagacggc tccatcccac aaggggttag gccaccggct 60
tcgggtgctg cccactttca tgacttgacg ggcggtgtgt acaaggcccg ggaacgcatt 120
caccgcgacg ttgctgattc gcgattacta gcgactccgc cttcacgcag tcgagttgca 180
gactgcgatc cgaactgaga ccggttttca gggatccgct ccgcgtcgcc gcgtcgcatc 240
ccgttgtacc ggccattgta gcatgcgtga agccctggac gtaaggggca tgatgatctg 300
acgtcatccc caccttcctc cgagttaacc ccggcggtcc cccgtgagtt cccggcataa 360
tccgctggca acacggggcg agggttgcgc tcgttgcggg acttaaccca acatctcacg 420
acacgagctg acgacgacca tgcaccacct gtgaacccgc cccgaaggga agccgtatct 480
ctacgaccgt cgggaacatg tcaagcccag gtaaggttct tcgcgttgca tcgaattaat 540
ccgcatgctc cgccgcttgt gcgggccccc gtcaatttct ttgagtttta gccttgcggc 600
cgtactcccc aggcgggatg cttaacgcgt tagctccgac acggaacccg tggaacgggc 660
cccacatcca gcatccaccg tttacggcgt ggactaccag ggtatctaat cctgttcgct 720
ccccacgctt tcgctcctca gcgtcagtaa cggcccagag acctgccttc gccattggtg 780
ttcttcccga tatctacaca ttccaccgtt acaccgggaa ttccagtctc ccctaccgca 840
ctcaagcccg cccgtacccg gcgcggatcc accgttaagc gatggacttt cacaccggac 900
gcgacgaacc gcctacgagc cctttacgcc caataattcc ggataacgct tgcaccctac 960
gtattaccgc ggctgctggc acgtagttag ccggtgctta ttcaacgggt aaactcactc 1020
tcgcttgctc cccgataaaa gaggtttaca acccgaaggc ctccatccct cacgcggcgt 1080
cgctgcatca ggcttgcgcc cattgtgcaa tattccccac tgctgcctcc cgtaggagtc 1140
tgggccgtat ctcagtccca atgtggccgg tcgccctctc aggccggcta cccgtcgaag 1200
ccacggtggg ccgttacccc gccgtcaagc tgataggacg cgaccccatc ccataccgcg 1260
aaagctttcc cagaagacca tgcgatcaac tggagcatcc ggcattacca cccgtttcca 1320
ggagctattc cggtgtatgg ggcaggtcgg tcacgcatta ctcacccgtt cgccactctc 1380
accaccaagc aagctaatcc c 1401
<210> 2
<211> 20
<212> DNA
<213> Artificial Synthesis
<400> 2
<210> 3
<211> 19
<212> DNA
<213> Artificial Synthesis
<400> 3
ggttaccttg ttacgactt 19
Claims (8)
1. The Bifidobacterium longum JBLC-141 capable of eliminating in-vivo free radicals is characterized in that the Bifidobacterium longum JBLC-141 is preserved in China general microbiological culture Collection Center (CCM) at 7.8.2019, the preservation address is No. 3 of Siro No. 1 Hospital in the area of the rising of Beijing, the preservation number is CGMCC number 18094, and the Bifidobacterium longum JBLC-141 is classified and named as Bifidobacterium longumBifidobacterium longum。
2. Use of bifidobacterium longum JBLC-141 as claimed in claim 1 for the preparation of a product for scavenging free radicals in the body and/or alleviating fatigue in the body.
3. The use according to claim 2, wherein the product comprises bifidobacterium longum JBLC-141 fungal powder and isomaltooligosaccharide.
4. Use according to claim 3, wherein the preparation of said product comprises the following steps:
(1) preparing bifidobacterium longum JBLC-141 powder;
(2) compounding the bifidobacterium longum JBLC-141 powder with isomaltooligosaccharide.
5. The use of claim 4, wherein the powder of Bifidobacterium longum JBLC-141 is prepared by:
(a) inoculating Bifidobacterium longum JBLC-141 to MRS liquid culture medium, and anaerobically culturing at 37 deg.C to obtain MRS bacterial liquid;
(b) and centrifuging the MRS bacterial liquid, and carrying out vacuum freeze drying to obtain Bifidobacterium longum JBLC-141 bacterial powder.
6. Such asThe use according to claim 5, wherein the Bifidobacterium longum JBLC-141 is inoculated in step (a) in an amount of 1% and is anaerobically cultured at 37 ℃ for 24 hours under N2：H2：CO2=85：10：5。
7. The use of claim 5, wherein the MRS liquid medium comprises:
peptone 10g, beef powder 5g, K2HPO4·7H2O2 g, triammonium citrate 2g, CH3COONa·3H2O5 g, glucose 20g, Tween 801 mL, MgSO4·7H2O 0.2g、MnSO4·4H20.05g of O and 1000mL of distilled water.
8. The use according to claim 4, wherein the step (2) is to mix the powder of Bifidobacterium longum JBLC-141 and isomaltooligosaccharide to obtain the product with viable bacteria number of 50 hundred million/g, 150 hundred million/g, 250 hundred million/g, 500 hundred million/g and 1000 hundred million/g.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112940989A (en) * | 2021-04-16 | 2021-06-11 | 吉林大学 | Complex microbial inoculant with intelligence-developing function and preparation method and application thereof |
| CN112940989B (en) * | 2021-04-16 | 2021-09-03 | 吉林大学 | Complex microbial inoculant with intelligence-developing function and preparation method and application thereof |
| CN118001305A (en) * | 2024-04-09 | 2024-05-10 | 山东中科嘉亿生物工程有限公司 | Application of bifidobacterium longum JBLC-141 in preparation of medicine for improving paraneoplastic thrombocytosis |
| CN118001305B (en) * | 2024-04-09 | 2024-06-11 | 山东中科嘉亿生物工程有限公司 | Application of bifidobacterium longum JBLC-141 in preparation of medicine for improving paraneoplastic thrombocytosis |
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