CN105969680B - One plant of norcholesterol, the Lactobacillus pentosus and its screening technique for dropping nitrite - Google Patents
One plant of norcholesterol, the Lactobacillus pentosus and its screening technique for dropping nitrite Download PDFInfo
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- CN105969680B CN105969680B CN201610168004.XA CN201610168004A CN105969680B CN 105969680 B CN105969680 B CN 105969680B CN 201610168004 A CN201610168004 A CN 201610168004A CN 105969680 B CN105969680 B CN 105969680B
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- nitrite
- lactobacillus pentosus
- lactobacillus
- cholesterol
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- 241000186684 Lactobacillus pentosus Species 0.000 title claims abstract description 28
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title description 14
- 238000012216 screening Methods 0.000 title description 8
- 238000000855 fermentation Methods 0.000 claims abstract description 6
- 230000004151 fermentation Effects 0.000 claims abstract description 6
- 235000012149 noodles Nutrition 0.000 claims abstract description 4
- 235000021404 traditional food Nutrition 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 24
- 210000000941 bile Anatomy 0.000 claims description 4
- 150000002972 pentoses Chemical class 0.000 claims description 3
- 239000006071 cream Substances 0.000 claims description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 abstract description 56
- 235000012000 cholesterol Nutrition 0.000 abstract description 23
- 241000186660 Lactobacillus Species 0.000 abstract description 6
- 229940039696 lactobacillus Drugs 0.000 abstract description 6
- 230000015556 catabolic process Effects 0.000 abstract description 5
- 238000006731 degradation reaction Methods 0.000 abstract description 5
- 229940079593 drug Drugs 0.000 abstract description 3
- 239000003814 drug Substances 0.000 abstract description 3
- 239000000796 flavoring agent Substances 0.000 abstract description 3
- 235000019634 flavors Nutrition 0.000 abstract description 3
- 230000036039 immunity Effects 0.000 abstract description 3
- 235000013402 health food Nutrition 0.000 abstract description 2
- 230000000968 intestinal effect Effects 0.000 abstract description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N nitrate group Chemical group [N+](=O)([O-])[O-] NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 30
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 239000012530 fluid Substances 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- 229940054441 o-phthalaldehyde Drugs 0.000 description 9
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 9
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 229940099352 cholate Drugs 0.000 description 8
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 8
- 239000004310 lactic acid Substances 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
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- 102000016938 Catalase Human genes 0.000 description 7
- 108010053835 Catalase Proteins 0.000 description 7
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 239000001569 carbon dioxide Substances 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
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- 229960000935 dehydrated alcohol Drugs 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
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- 108020004465 16S ribosomal RNA Proteins 0.000 description 3
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
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- LPXPTNMVRIOKMN-UHFFFAOYSA-M sodium nitrite Substances [Na+].[O-]N=O LPXPTNMVRIOKMN-UHFFFAOYSA-M 0.000 description 3
- 239000012086 standard solution Substances 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- MWUXSHHQAYIFBG-UHFFFAOYSA-N Nitric oxide Chemical compound O=[N] MWUXSHHQAYIFBG-UHFFFAOYSA-N 0.000 description 2
- 241000209094 Oryza Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
- 108010080698 Peptones Proteins 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
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- LECSHJWIACEDPZ-UHFFFAOYSA-N ethane-1,2-diamine naphthalene hydrochloride Chemical compound C(CN)N.C1=CC=CC2=CC=CC=C12.Cl LECSHJWIACEDPZ-UHFFFAOYSA-N 0.000 description 2
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- GQPLMRYTRLFLPF-UHFFFAOYSA-N nitrous oxide Inorganic materials [O-][N+]#N GQPLMRYTRLFLPF-UHFFFAOYSA-N 0.000 description 2
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- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- FRKYZIHMQZATAH-MVIOUDGNSA-N (2r,3s,4r,5r)-n-acetyl-2,3,4,5,6-pentahydroxyhexanamide Chemical compound CC(=O)NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FRKYZIHMQZATAH-MVIOUDGNSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- VBUYCZFBVCCYFD-JJYYJPOSSA-M 2-dehydro-D-gluconate Chemical class OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)C([O-])=O VBUYCZFBVCCYFD-JJYYJPOSSA-M 0.000 description 1
- HVBSAKJJOYLTQU-UHFFFAOYSA-N 4-aminobenzenesulfonic acid Chemical compound NC1=CC=C(S(O)(=O)=O)C=C1 HVBSAKJJOYLTQU-UHFFFAOYSA-N 0.000 description 1
- IZSRJDGCGRAUAR-MROZADKFSA-M 5-dehydro-D-gluconate Chemical class OCC(=O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O IZSRJDGCGRAUAR-MROZADKFSA-M 0.000 description 1
- 201000001320 Atherosclerosis Diseases 0.000 description 1
- 108010062877 Bacteriocins Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- OCUCCJIRFHNWBP-IYEMJOQQSA-L Copper gluconate Chemical class [Cu+2].OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O OCUCCJIRFHNWBP-IYEMJOQQSA-L 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 150000000736 D-arabinoses Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-SOOFDHNKSA-N D-ribopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@@H]1O SRBFZHDQGSBBOR-SOOFDHNKSA-N 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- SRBFZHDQGSBBOR-HWQSCIPKSA-N L-arabinopyranose Chemical compound O[C@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-HWQSCIPKSA-N 0.000 description 1
- 150000000992 L-arabinoses Chemical class 0.000 description 1
- SRBFZHDQGSBBOR-OWMBCFKOSA-N L-ribopyranose Chemical compound O[C@H]1COC(O)[C@@H](O)[C@H]1O SRBFZHDQGSBBOR-OWMBCFKOSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-N Nitrous acid Chemical compound ON=O IOVCWXUNBOPUCH-UHFFFAOYSA-N 0.000 description 1
- 235000008331 Pinus X rigitaeda Nutrition 0.000 description 1
- 235000011613 Pinus brutia Nutrition 0.000 description 1
- 241000018646 Pinus brutia Species 0.000 description 1
- 208000005374 Poisoning Diseases 0.000 description 1
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
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- 231100000570 acute poisoning Toxicity 0.000 description 1
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
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- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
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- 239000000594 mannitol Substances 0.000 description 1
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- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 235000021110 pickles Nutrition 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
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- 235000013311 vegetables Nutrition 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- 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
- C12N1/205—Bacterial isolates
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- 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
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Abstract
One plant of norcholesterol, drop nitrite Lactobacillus pentosus, the Lactobacillus pentosus be it is isolated from the traditional food fermentation rice-flour noodles of Guizhou, Strain Designation isLactobacillus pentousGUFHSL-69 is preserved in China typical culture collection center on January 4th, 2016, depositary institution address: China, Wuhan, Wuhan University, deposit number are as follows: CCTCC M 2016001.Lactobacillus pentosus GUFHSL-69 is a kind of functional lactobacillus, has the superperformance for reducing cholesterol and nitrite, and the removal rate to cholesterol is 25.66%, and culture is afterwards for 24 hours 94.45% to nitrite degradation rate.Health food or drug are developed into, taking for a long time improves intestinal flora, reduces serum cholesterol, improves body immunity；It can reduce nitrate residue in fermented product, shorten fermentation period, improve product special flavour.
Description
Technical field
The present invention relates to microorganisms, furthermore, it is understood that be related to Bacillus acidi lactici, in particular to not only there is norcholesterol ability but also
There is the lactic acid bacteria of degrading nitrite ability.
Background technique
According to medical research, serum cholesterol levels are increased, it is considered to be induce the painstaking effort such as coronary heart disease, atherosclerosis
The important risk factor of pipe disease.The World Health Organization (WHO) predicts that cardiovascular disease is still dead master until the year two thousand thirty
Reason is wanted, 2,003 million peoples are influenced.Clinical research shows to be more than normal serum cholesterol levels (> 5.2mmol) 1mmol, suffers from
The probability increase by 35% of coronary heart disease, meanwhile, reduce by 1% serum cholesterol level for the risk of reduction by 2%~3%.
The study found that nitrite acute poisoning leads to methemoglobinemia, slow poisoning has carcinogenic and teratogenesis wind
Dangerous, nitrate residue is higher in pickled vegetable product, but nitrite has maintenance human body nitric oxide balance, promotes angiocarpy
The effect of health.How a difficult problem that content of nitrite in pickles be food processing is controlled.
Probiotics is widely distributed in nature, since it is beneficial to host health, thus is widely used.It is many
All contain profitable probliotics in functional food, they can improve intestinal microflora, reduce the quantity of harmful bacteria, prevent abdomen
It rushes down；Improve immunity of organisms；Reduce serum cholesterol；Improve the oxidation resistance of body, anti-aging and preventing cancer function etc..
Therefore people constantly explore and find the probiotics that can reduce cholesterol and nitrite, especially lactobacillus,
Wish that it is widely applied in food, develop various functional foods, this has the nutrition of the mankind and health important
Meaning.
At present in Chinese patent database, the patent application for being related to Lactobacillus pentosus is few, and only No. ZL02820204X
A kind of " comprising Lactobacillus pentosus strain composition and application thereof ", No. ZL031262252 " Lactobacillus pentosus strain and with the bacterial strain
The application of manufactured leavening and the leavening in meat product ", a kind of No. ZL2005101059855 " Lactobacillus pentosus bacterium
Element and its special preparing strain and application ", No. ZL2008102473358 " a kind of Lactobacillus pentosus, active fresh-keeping flavor intensifier and its
Using ", No. ZL2010800331767 " generate the Lactobacillus pentosus of bacteriocin and its answering in food and pharmaceutical composition
With ", No. ZL2011100489071 " a kind of Lactobacillus pentosus and the tunning of the lactobacillus and the purposes of tunning ",
No. ZL2012104063212 " Lactobacillus pentosus ", No. ZL2013102336309 " one plant is used for lactic acid fermented Lactobacillus pentosus
LacticUVC-02 and its application method " etc..These Lactobacillus pentosus have certain practical application, but so far, there is no both
There is the application part of the Lactobacillus pentosus of degrading nitrite ability again with norcholesterol ability.
Summary of the invention
The present invention is intended to provide the Lactobacillus pentosus of one plant of norcholesterol, drop nitrite, to be developed into health care
Food or drug, or it is used for fermented product, enable the functional food with norcholesterol.
It is yet another object of the invention to provide above-mentioned norcholesterol, drop nitrite Lactobacillus pentosus screening technique,
It is set to have specific industrial use.
In order to achieve the above objectives, the Lactobacillus pentosus of inventor provides one plant of norcholesterol, drop nitrite is from expensive
Isolated Lactobacillus pentosus, Strain Designation are in the traditional food fermentation rice-flour noodles of stateLactobacillus pentous
GUFHSL-69 is preserved in China typical culture collection center on January 4th, 2016, referred to as；CCTCC, depositary institution
Location: China, Wuhan, Wuhan University, deposit number are as follows: CCTCC M 2016001.
Above-mentioned bacterial strains GUFHSL-69 carries out 16S rRNA gene sequencing, and measurement result compares on NCBI, identification knot
Fruit be Lactobacillus pentosus (Lactobacillus pentous).
Inventor provide norcholesterol, drop nitrite Lactobacillus pentosus screening technique the following steps are included:
(1) bacterial strain is acquired from Guizhou Southern Guizhou ethnic group traditional food fermentation rice-flour noodles, after being cultivated, being isolated and purified
Screening obtains lactic acid bacteria, and slant preservation is spare；
(2) pass through Gram's staining, catalase experiment, thalli morphology and 16S rRNA Sequence Identification system, screening
A strains of lactic acid bacteria is obtained, is GUFHSL-69 to strain number；
(3) there is norcholesterol by the bacterial strain that o-phthalaldehyde colorimetric method, hydrochloric acid-naphthalene-ethylenediamine method evaluation and screening obtain
Ability and degrading nitrite ability；
(4) secondary screening is carried out by the inspection of acid-fast ability, bile tolerance ability, determines whether bacterial strain is resistant to gastrointestinal tract environment,
Reach prebiotic effect；There is the pentose cream that the bacterial strain compared with strong tolerance is preparation with acid resistance, simultaneously to 0.3% cholate
Bacillus.
In (1) step of the above method, the acquisition bacterial strain is cultivated, the way that isolates and purifies is: acquisition rice flour system
Product take 10g sample to be put into the sterile peptone water of 90mL, in shaking 30min on shaking table, take 200 μ L in superclean bench
Supernatant is coated on CaCO3It on-MRS agar plate, is placed in carbon dioxide incubator and cultivates 48h at 37 DEG C, it is bright to select generation
The single colonie for showing molten calcium circle is purified.
In (2) step of the above method, the way that the Gram's staining, catalase are tested is: bacterial strain is flat in MRS
It is cultivated on plate for 24 hours, observes and records colonial morphology, color；Bacterium colony on picking plate carry out Gram's staining smear, fixation,
Crystal violet just redye by dye, mordant dyeing, decoloration, washing, sarranine, dry, microscopy；Bacterium to be measured uses capillary prior to exposure 20min in air
Pipe draws a small amount of 15%H2O2Directly drop carries out catalase experiment in the bacterium colony that planar surface is grown, if not producing bubble
For negative catalase.
In (3) step of the above method, the way of the o-phthalaldehyde colorimetric method is: strain inoculated is consolidated in containing gallbladder
Determining alcohol is to cultivate that measure its gallbladder afterwards for 24 hours solid in 37 DEG C of carbon dioxide incubator in the MRS fluid nutrient medium of 100 μ g/mL
Alcohol Scavenging activity: accurate cholesterol standard solution of drawing is in clean tube, and adding dehydrated alcohol makes its volume 1mL, so
OPA reagent 4mL is added in each test tube afterwards, is placed at room temperature for 10min.The 4mL concentrated sulfuric acid is slowly added to thereto again, immediately with oscillation
Device vibration 20s, places 15min under room temperature dark condition after being sufficiently mixed, blank control replaces cholesterol molten with 1mL dehydrated alcohol
Liquid surveys its light absorption value at 550nm, and using cholesterol concentration as abscissa, absorbance value is that ordinate does standard curve, carries out
Colorimetric；The way of the hydrochloric acid-naphthalene-ethylenediamine method is: by strain inoculated in the MRS liquid for being 100 μ g/mL containing cholesterol concentration
In culture medium, is cultivated at 37 DEG C in carbon dioxide incubator and measure its cholesterol Scavenging activity afterwards for 24 hours；The degradation nitrous acid
The identification way of salt ability is: the bacterial strain for cultivating 18~20h 3% is inoculated into containing NaNO by volume2MRS Liquid Culture
In base, then measure 0h, 12h, for 24 hours when MRS fluid nutrient medium in NaNO2Content confirms.
In (4) step of the above method, the inspection way of the acid-fast ability is: the strain inoculated of fresh cultured is arrived
In the PBS buffer solution of pH3.0, viable count is measured respectively in 0h, 3h, with log CFU/mL note, calculates viable bacteria rate;It is described resistance to
The inspection way of cholate ability is: the bacterial strain GUFHSL-69 of fresh cultured is inoculated in the MRS culture medium of 0.3% cholate,
The MRS fluid nutrient medium of cholate is not added as control group, respectively in 0h, measure viable count for 24 hours, with log CFU/mL note, calculate
Viable bacteria rate.
Lactobacillus pentosus GUFHSL-69 of the invention is a kind of functional lactobacillus, is especially reducing cholesterol and nitrous
There is good characteristic in terms of hydrochlorate.Removal rate to cholesterol is 25.66%, and culture is to nitrite degradation rate afterwards for 24 hours
94.45%.Such as by lactic acid bacteria GUFHSL-69 exploitation of the invention at health food or drug, taking for a long time improves intestinal flora,
Serum cholesterol is reduced, immunity of organism Lee is improved；Or put into the present invention in fermented product, nitrous in fermented product can be reduced
Hydrochlorate residual quantity shortens fermentation period, improves product special flavour, develops the functional food with norcholesterol.
Detailed description of the invention:
Fig. 1 is the colonial morphology of bacterial strain GUFHSL-69 of the present invention；
Fig. 2 is the thalli morphology of bacterial strain GUFHSL-69 of the present invention；
Fig. 3 indicates the phylogenetic tree of bacterial strain GUFHSL-69 of the present invention；
Fig. 4 is cholesterol standard curve；
Fig. 5 is nitrite standard curve.
Specific embodiment
Embodiment screens Lactobacillus pentosus GUFHSL-69 bacterial strain:
1. the acquisition of sample and isolating and purifying for bacterial strain
Rice flour produts are acquired from Guizhou Province Southern Guizhou, take 10g sample to be put into the sterile peptone water of 90mL, in shaking table
Upper concussion 30min takes 200 μ L supernatants to be coated on CaCO3-MRS agar plate in superclean bench, is placed in 37 DEG C of dioxies
Change in carbon incubator and cultivate 48h, the single colonie for selecting the obvious molten calcium circle of generation is further purified, and then slant preservation is spare.
2. colonial morphology identification, Physiology and biochemistry identification and molecular biology identification
2.1 bacterial strains observe and record colonial morphology, color in cultivating for 24 hours on MRS plate.
2.2 Gram's staining: the bacterium colony on picking plate carries out smear, fixation, crystal violet just dye, mordant dyeing, decoloration, water
It washes, sarranine is redyed, dry, microscopy.
The experiment of 2.3 catalases: bacterium to be measured draws a small amount of 15%H prior to exposure 20min in air, with capillary2O2
Directly drip the bacterium colony grown in planar surface.It is negative catalase if not producing bubble.Final preservation Gram-positive,
Catalase negative strain is GUFHSL-69 to strain number, then carries out next step experiment:
The colonial morphology for visually observing bacterial strain is special, observation as a result, the bacterial strain colony colour be it is faint yellow, bacterium colony moistens side
Edge is neat, rounded protrusion, 1~2mm of colony diameter, after Gram's staining, in optical microscopy oil microscopic observation cellular morphology
Feature is nonspore-bearing Gram-positive bacillus, amphimicrobian.
2.4 physiological and biochemical property
The physiological and biochemical property of GUFHSL-69 is as shown in table 1
1 bacterial strain GUFHSL-69 physio-biochemical characteristics of table -- acid is produced using carbon source
| Reagent strip respective tube/substrate | Testing result | Reagent strip respective tube/substrate | Testing result |
| 0 control | - | 25 aesculins | + |
| 1 glycerol | + | 26 salicins | + |
| 2 red tinea alcohol | - | 27 cellobioses | + |
| 3 D-arabinoses | - | 28 maltose | + |
| 4 L-arabinoses | + | 29 lactose | + |
| 5 ribose | + | 30 melibioses | + |
| 6 D- xyloses | + | 31 sucrose | + |
| 7 L- xyloses | - | 32 trehaloses | + |
| 8 adonites | - | 33 synanthrin | - |
| 9 Beta-methyl-D- xylosides | - | 34 pine, three sugar | - |
| 10 galactolipins | + | 35 gossyposes | w |
| 11 glucose | + | 36 starch | - |
| 12 fructose | + | 37 glycogens | - |
| 13 mannoses | + | 38 xylitols | - |
| 14 sorboses | - | 39 hold together ox sugar | + |
| 15 rhamnoses | w | 40 D- turanoses | - |
| 16 melampyrins | - | 41 D- lyxoses | - |
| 17 inositols | - | 42 D-Tags | - |
| 18 mannitol | + | 43 D- rock sugar | - |
| 19 sorbierites | + | 44 L- rock sugar | - |
| 20 Alpha-Methyls-D-MANNOSE glucoside | - | 45 D-arabinose alcohol | - |
| 21 Alpha-Methyls-D-Glucose glucoside | - | 46 L-arabinose alcohol | - |
| 22 N- acetyl-aminoglucose | + | 47 gluconates | w |
| 23 amygdalins | + | 48 2- keto-D-gluconate salt | - |
| 24 Arbutins | w | 49 5- keto-D-gluconate salt | - |
Remarks: "+" indicates positive reaction, and "-" indicates negative reaction, and " W " indicates that reaction is weaker.
2.5 molecular biological characteristics
16S rRNA gene sequencing is carried out to bacterial strain GUFHSL-69, measurement result compares on NCBI, qualification result
For Lactobacillus pentosus (Lactobacillus pentous), construct the phylogenetic tree of GUFHSL-69.
3. using o-phthalaldehyde colorimetric method for determining bacterial strain norcholesterol ability
By strain inoculated in the MRS fluid nutrient medium for being 100 μ g/mL containing cholesterol concentration, in 37 DEG C of titanium dioxide
It is cultivated in carbon incubator and measures its cholesterol Scavenging activity afterwards for 24 hours.
3.1 o-phthalaldehyde methods (OPA) Specification Curve of Increasing
For accurate cholesterol standard solution of drawing in clean tube, adding dehydrated alcohol makes its volume 1mL, then exists
OPA reagent 4mL is added in each test tube, is placed at room temperature for 10min.It is slowly added to the 4mL concentrated sulfuric acid thereto again, is shaken immediately with oscillator
20s places 15min under room temperature dark condition after being sufficiently mixed.Blank control replaces cholesterol solution with 1mL dehydrated alcohol,
Its light absorption value at 550nm is finally surveyed, using cholesterol concentration as abscissa, absorbance value is that ordinate does standard curve, is counted
Calculating its equation of linear regression is y=0.0051x-0.0009, coefficient R2=0.9988。
The measurement of 3.2 pentose lactic acid bacteria norcholesterol abilities
The bacterial strain of slant preservation is passed on 2~3 times in the flat lining out of MRS, is inoculated into identical item in liquid MRS culture medium
After carrying out enrichment culture 20h under part, with 4000r/min, it is centrifuged 10min under the conditions of 4 DEG C, collects thallus.Then it is buffered with PBS molten
Liquid dilutes thallus, and bacterium solution to be measured is adjusted to 3.0 × 1083% inoculum concentration is inoculated in cholesterol level and is by volume after CFU/mL
In the MRS fluid nutrient medium of 100 μ g/mL, 37 DEG C, cultivated for 24 hours in carbon dioxide incubator.Bacterium solution is with 12000r/min, 4 DEG C of items
It is centrifuged 20min under part, retains supernatant liquor as prepare liquid, for measuring cholesterol level, with nonvaccinated cholesterol culture
Base is as blank control group.Then the cholesterol clearance rate of lactic acid bacteria is surveyed using o-phthalaldehyde (OPA) method.
Cholesterol clearance rate X is calculated as follows:
X=(1-A/B) × 100%
Note: X-cholesterol clearance rate；
Cholesterol concentration in A-experimental group fermented supernatant fluid, μ g/mL；
Cholesterol concentration in B-blank group fermented supernatant fluid, μ g/mL.
Using cholesterol clearance rate as index, the cholesterol clearance rate of bacterial strain GUFHSL-69 is 25.66%.
4. the measurement of lactic acid bacteria degrading nitrite ability
The measurement of 4.1 nitrite standard curves
Draw various concentration nitrite standard solution be added 50mL colorimetric cylinder in, then plus 40mL water, mix, add
2mL p-aminobenzene sulfonic acid solution, mix, be protected from light stand 5min, then plus 1mL hydrochloric acid how ethylenediamine, add water to scale, mix,
It is protected from light and stands 15min, Yu Bochang 538nm measurement, using nitrite concentration as abscissa, absorbance value is ordinate, and standard is bent
Line such as Fig. 5, equation of linear regression y=0.0158x-0.0011, coefficient R2=0.9995。
The measurement of 4.2 strains for degrading nitrite abilities
The assay of nitrite is referring to GB-50093-2010.Bacterial strain 3% inoculation by volume of 18 ~ 20h will be cultivated
To containing NaNO2MRS fluid nutrient medium in, then measure 0h, 12h, for 24 hours when MRS fluid nutrient medium in NaNO2Content.Measurement
The result shows that bacterial strain GUFHSL-69 is for 24 hours to the degradation rate of nitrite 94.45%, the NaNO of 88.61 μ g/mL of degradation amount2。
5. bacterial strain is acidproof, bile tolerance ability
5.1 acid-fast ability
By in the PBS buffer solution of the strain inoculated of fresh cultured to pH3.0, viable count is measured respectively in 0h, 3h, with
Log CFU/mL note, calculates viable bacteria rate, and bacterial strain GUFHSL-69 viable bacteria rate is up to 102.0%.
5.2 bile tolerance abilities
The bacterial strain GUFHSL-69 of fresh cultured is inoculated in the MRS culture medium of 0.3% cholate, cholate is not added
MRS fluid nutrient medium be control group, respectively in 0h, measure viable count for 24 hours, with log CFU/mL note, calculate viable bacteria rate, bacterial strain
GUFHSL-69 under the conditions of 0.3% cholate viable bacteria rate up to 98.25%.
Bacterial strain GUFHSL-69 has with acid resistance, simultaneously to 0.3% cholate compared with strong tolerance, for the target of preparation
Lactobacillus pentosus.
Claims (1)
1. one plant of norcholesterol, acidproof and bile tolerance the Lactobacillus pentosus for dropping nitrite, it is characterised in that the pentose cream bar
Bacterium is isolated Lactobacillus pentosus from the traditional food fermentation rice-flour noodles of Guizhou, and Strain Designation is
Lactobacilluspentous GUFHSL-69 is preserved in China typical culture collection center on January 4th, 2016, letter
Claim；CCTCC, depositary institution address: China, Wuhan, Wuhan University, deposit number are as follows: CCTCC M 2016001.
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