CN106636448A - Method for fast identifying generation of EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria - Google Patents
Method for fast identifying generation of EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria Download PDFInfo
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses a method for fast identifying the generation of an EC (ethyl carbamate) precursor of citrulline from lactic acid bacteria, and belongs to the technical field of microbiological detection. By the method, whether a lactic acid bacterium strain has an arginine deiminase path or not and whether citrulline can be formed by using arginine or not can be judged, so that whether the lactic acid bacterium strain in the fermented food and beverage has the possibility to generate a cancerogen of EC or not can be determined. The method disclosed by the invention is fast, simple, convenient and accurate; the degenerate primer specificity is high; the universality is high; the lactic acid bacteria of different species from different food can be detected; good food safety detection prospects are realized.
Description
Technical field
The present invention relates to a kind of method that Rapid identification lactic acid bacteria produces urethanes precursor citrulling, belongs to micro-
Technical field of biological.
Background technology
Lactic acid bacteria is a kind of micro- life being widely present in fermented food or beverage (such as soy sauce, ferment sausage, yellow rice wine etc.)
Thing, the lactic acid that its metabolism is produced is a kind of flavor substance, therefore fermentation is had a positive effect.But, research both domestic and external recently
It was found that, part lactic acid bacteria can decompose arginine and produce citrulling, and citrulling is be widely present in fermented food
A kind of important precursor of carcinogenic substance-urethanes (EC) is planted, it is especially more in consumer's daily consumption (drinking)
In soy sauce and yellow rice wine, the content of EC is of a relatively high.Citrulling is by lactic acid bacteria Jing arginine deiminases approach degraded arginine shape
Into, but and not all lactic acid bacteria all has the approach, its presence shows as strain specificity, i.e. different strains degraded arginine
Or the ability of generation citrulling is not quite similar.
The encoding gene of lactic acid bacteria arginine deiminase approach, except arcA, arcB, arcC, arcD gene, part breast
Sour bacterium such as Lactobacillus saki also has arcR (controlling gene), PTP (transport proteins that may be relevant with citrulling secretion and reabsorption
Encoding gene), different lactic acid bacteria strains arc gene clusters put in order may be different with integrality.Research has shown that, each breast
Sour bacteria strain degraded arginine and produce the capacity variance of citrulling and have its source in the polymorphism of arc gene clusters.
At present, detect lactic acid bacteria whether produce urethanes precursor citrulling method be mainly it is pure after strain isolation
Culture, then growth a period of time (for example ferments in arginic MRS culture mediums are added in containing arginic culture medium
3 days), centrifuging and taking supernatant using the extracellular arginine of high performance liquid chromatography detection and the content of citrulling, and then determines that the bacterium is
The no ability with product citrulling.The method is time-consuming longer, not only needs to prepare mobile phase, in addition it is also necessary to do calibration curve, detects
In about 1 week or so cycle, be not a quick detection method.Another kind of method be with degenerate primer amplification arcA, arcB,
The partial sequence of arcC genes, the method is fast and convenient, but universality is poor, is mainly used for detecting Lactobacillus hilgardii, short breast
The lactic acid bacteria in grape wine such as bacillus, Oenococcus Oeni, Pediococcus pentosaceus, Leuconostoc mesenteroides, for from yellow rice wine point
From lactic acid bacteria Detection results it is very poor, when being mainly shown as with the primer detection, many can produce the lactic acid bacterias of citrulling all
ArcA and arcB can not be amplified, these primers having been reported have sizable limitation in practical application.
The content of the invention
In order to solve the above problems, the present invention provides a kind of side of quick detection lactic acid bacteria arginine deiminase approach
Method, methods described is, with lactic acid bacteria genomic DNA as template, to enter performing PCR with primer pair A and primer pair B respectively, if it is possible to
To respectively 200～300bp, the PCR primer of 450bp～550bp, then the lactic acid bacteria has arginine deiminase approach,
Otherwise the lactic acid bacteria does not have arginine deiminase approach.
Wherein primer pair A is classified as SEQ ID NO.1 by nucleotides sequence and nucleotides sequence is classified as the nucleotides of SEQ ID NO.2
Fragment is constituted, or two cores for carrying out being obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.1 and SEQ ID NO.2
Acid fragments are constituted；Primer pair B is classified as SEQ ID NO.3 by nucleotides sequence and nucleotides sequence is classified as SEQ ID NO.4's
Nucleotide fragments are constituted, or carry out what is obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.3 and SEQ ID NO.4
Two nucleotide fragments compositions.
In one embodiment, the lactic acid bacteria includes Lactococcus lactis, lactobacillus curvatus, lactobacillus fermenti, plant
Lactobacillus, lactobacillus, Lactobacillus brevis, Wei Si Salmonellas, Lactobacillus delbrueckii, Lactobacillus casei, Lactobacillus coryniformis, the bright beading of lemon
Bacterium, leuconostoc pseudomesenteroides, Leuconostoc mesenteroides.
In one embodiment, the 200～300bp specifically refers to 250bp；Use 450bp～550bp is specifically
Refer to 500bp.
In one embodiment, the reaction system of the PCR is：DNA profiling<500ng, upstream primer and downstream primer
Each 0.2～1 μM, the μ L of 10 × Ex Taq Buffer 5, dNTP4 μ L, sterile purified water is mended to 50 μ L.
In one embodiment, the reaction condition of the PCR is：Denaturation, 94 DEG C, 4min；Denaturation, 94 DEG C, 35s；
Annealing, 50 DEG C, 35s extends, 72 DEG C, 40s；Denaturation, annealing, extension carry out 30 circulations, continue to extend, 72 DEG C, 10min, most
Afterwards 10 DEG C are maintained up to taking-up.
In one embodiment, the detection of the PCR primer is to use agarose gel electrophoresis to detect amplified production.
Second object of the present invention is to provide a kind of combination primer pair, including primer pair A and primer pair B；Wherein primer
SEQ ID NO.1 are classified as by nucleotides sequence to A and nucleotides sequence is classified as the nucleotide fragments of SEQ ID NO.2 and constitutes, or
Two nucleotide fragments for carrying out being obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.1 and SEQ ID NO.2 are constituted；Institute
State that primer pair B is classified as SEQ ID NO.3 by nucleotides sequence and nucleotides sequence is classified as the nucleotide fragments of SEQ ID NO.4 and constitutes,
Or two nucleotide fragments for carrying out being obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.3 and SEQ ID NO.4
Composition.
The present invention is also claimed the application of combination primer pair lactic acid bacteria context of detection in food.
In one embodiment, the food includes fermented food or beverage.
In one embodiment, the food is soy sauce, ferment sausage, soy sauce, yellow rice wine etc..
Advantages of the present invention and effect：
Primer specificity disclosed by the invention is high, versatility is good, and detection method is sensitive, accurate, convenient, can detect difference
The lactic acid bacteria of food sources different genera, with good food safety detection prospect.
Description of the drawings
Fig. 1 is the lactic acid bacteria arginine deiminase that yellow wine fermentation liquid is derived from using the degenerate primer detection of present invention design
The gel images (A, B are respectively the product for entering performing PCR amplification using two pairs of primers) of enzymatic pathway, wherein duct M is 2000bp
Marker (from top to bottom be respectively 2000,1000,750,500,250,100bp), duct 1-33 is followed successively by yellow wine fermentation liquid
Detached Lactococcus lactis 1-20, lactobacillus curvatus 1-21, lactobacillus fermenti 2-1, lactobacillus fermenti 2-17, lactobacillus fermenti 2-
18, Lactobacillus plantarum 2-19, lactobacillus curvatus 2-20, lactobacillus 2-22, Lactobacillus plantarum 2-24, lactobacillus fermenti 2-28 are short
Lactobacillus 2-29, lactobacillus fermenti 2-32, Lactobacillus brevis 2-34, lactobacillus fermenti 2-36, lactobacillus fermenti 2-1-1, acidified milk
Bacillus 2-1-17, lactobacillus 2-1-19, Wei Si Salmonella 2-1-26, Wei Si Salmonella 4-15, Wei Si Salmonella 4-19, Lactobacillus brevis 4-
22, Lactobacillus plantarum 7-6, Lactobacillus delbrueckii 7-13, lactobacillus fermenti 7-14, Lactobacillus casei 14-1, lactobacillus fermenti 14-6,
Lactobacillus plantarum 14-8, Lactobacillus coryniformis 14-9, lemon leukonid 14-16, the bright beading of leuconostoc pseudomesenteroides 1-5, lemon
Bacterium 1-6, Leuconostoc mesenteroides 1-15.
Specific embodiment
It is presented herein below and the present invention is specifically described.
Embodiment 1：
1st, the extraction of lactic acid bacteria genomic DNA：(work is given birth to using Ezup pillar bacterial genomes DNA extraction agent boxes in Shanghai
Bioengineering Co., Ltd), the genomic DNA of detached lactic acid bacteria strains in yellow wine fermentation liquid is extracted according to specification.
2nd, design of primers and PCR are expanded：
Degenerate primer is devised to A and degenerate primer to B；Wherein the nucleotide sequence of the upstream and downstream primer of primer pair A divides
Not Wei 5 '-AACCAYGCHATGATGCAYYT-3 '/5 '-TTKGABCCRTCRTTCCATTG-3 ', for detecting arcA genes, produce
Thing length is 250bp；The nucleotide sequence of the upstream and downstream primer of primer pair B be respectively 5 '-AYTCHGGKGTDGTTTGGAA-3 '/
5 '-TCVGTMACTTCCATTTCHGT-3 ', for detecting arcB genes, product length is 500bp；Wherein, Y=C/T, K=G/
T, R=A/G, B=G/T/C, H=A/C/T, D=A/G/T.
Lactic acid bacteria strains genomic DNA template<500ng, upstream primer and downstream primer it is each 0.2～1 μM, 10 × Ex Taq
The μ L of Buffer5 μ L, dNTP 4, sterile purified water is mended to 50 μ L.PCR amplification condition be：Denaturation, 94 DEG C, 4min；Denaturation,
94 DEG C, 35s；Annealing, 50 DEG C, 35s extends, 72 DEG C, 40s；Denaturation, annealing, extension carry out 30 circulations, continue to extend, 72
DEG C, 10min, last 10 DEG C are maintained up to taking-up.
3rd, agarose gel electrophoresis detection pcr amplification product：If expanded using primer pair A and primer pair B, respectively
There is the band of 250bp, 500bp size, then illustrate that the lactic acid bacteria strains have arginine deiminase approach, can be using essence
Propylhomoserin produces citrulling, and it just has in fermented food or beverage and produces a kind of possibility of carcinogenic substance-urethanes (EC)；
If without correspondingly sized band, illustrating that the lactic acid bacteria does not have arginine deiminase approach, it is impossible to utilize arginine
Produce citrulling.
Embodiment 2：The validation verification of the inventive method
With reference to the detection example of lactic acid bacteria in yellow wine fermentation process, the invention will be further described, but the guarantor of the present invention
Shield scope is not limited to this, it is also possible to be generalized to the detection of lactic acid bacteria in other Foods or drinkses.
As shown in figure 1, detached 33 strains of lactic acid bacteria is (including lactobacillus fermenti, Lactobacillus plantarum, curved from yellow wine fermentation liquid
The lactic acid of the kinds such as bent lactobacillus, Lactobacillus brevis, Lactobacillus delbrueckii, Lactobacillus coryniformis, lemon leukonid, Lactococcus lactis
Bacterium), processed using the method for embodiment 1, there are 30 plants can amplify correspondingly sized band, there are 3 plants cannot detect
Corresponding band.
Then, these lactic acid bacterias are fermented and using high performance liquid chromatography detection lactic acid bacteria degraded arginine, product melon
The ability of propylhomoserin, specifically：Glycerol stocks bacterium activates 2 times to OD in MRS culture mediums600=0.8-1.0, is transferred to MRS-Arg
(the arginic MRS culture mediums of addition 5g/L, pH5.0), 30 DEG C of quiescent culture 72h, then 10000rpm centrifugations 5min, takes supernatant
Liquid, the albumen in diluting 1 times to precipitate supernatant with 5%TCA solution, then with arginine in high performance liquid chromatography detection sample and
Citrulling content.The method of HPLC detection amino acid is as follows：Using the high performance liquid chromatography of Agilent 1260, chromatographic column is ODS
HYPERSIL C-18column (250 × 4.6mm, 5 μm), mobile phase A be 0.8% (w/v) sodium acetate, 0.5% (v/v) tetrahydrochysene
The aqueous solution of furans, Mobile phase B is sodium acetate：Water：Methyl alcohol：Acetonitrile=1:50:100:100.Elution program is 0min, A：B=
92:8；15-22min, A：B=40:60；22-25min, A：B=92:8.Flow velocity keeps 1mL/min, qualitative with retention time, with
Peak area quantification.
Measurement result shows that previous step detects this 30 strains of lactic acid bacteria that can amplify correspondingly sized band in fermentation
The citrulling of 5g/L can be utilized after 72h, and has produced a certain amount of citrulling (the busy difference of different strains yield, most bacterium
Strain yield about 100mg/L)；And previous step detects the 3 plants of bacterium that cannot amplify correspondingly sized band, it is impossible to produce melon ammonia
Acid.
In addition, inventor have also been attempted the detection of the lactic acid bacteria in a large amount of other sources, as a result find, if using this
The primer pair of invention carries out expanding the lactic acid bacteria of the band that 250bp, 500bp or so size can respectively occur, fermented checking
There is using arginine afterwards and produce the performance of citrulling really；And after testing without correspondingly sized band lactic acid bacteria, it is fermented
Using citrulling and/or citrulling cannot be produced really after checking.
Data above explanation, the degenerate primer of the present invention, with stronger versatility, specificity and accuracy.
Embodiment 3：Primer sequence is to detection validity, the impact of accuracy
Inventor additionally use have been reported from Detection ofarc Genes Related with the
Primer in Ethyl Carbamate Precursors in Wine Lactic Acid Bacteria documents enters performing PCR expansion
Increase to detect that can detached lactic acid bacteria produce citrulling from yellow rice wine, as a result show much true by high performance liquid chromatography detection
Surely the strain gene group of citrulling can be produced can not amplify corresponding PCR primer, illustrate the primer reported in the document simultaneously
It is not suitable for the detection of the lactic acid bacteria in yellow rice wine source.However, the lactic acid bacteria with the primer detection in the present invention in yellow rice wine
It is all to be suitable for produce citrulling, and the present invention is also detected to other lactic acid bacterias of source (such as Yoghourt), is as a result also
Produce what citrulling ability was coincide with bacterial strain, the detection method validity for further illustrating the present invention is high, and applicability is wide.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with this skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore the protection model of the present invention
Enclosing should be by being defined that claims are defined.
Claims (10)
1. a kind of method of quick detection lactic acid bacteria arginine deiminase approach, it is characterised in that methods described is with lactic acid
Bacterium genomic DNA be template, enter performing PCR with primer pair A and primer pair B respectively, if it is possible to obtain respectively 200～300bp,
The PCR primer of 450bp～550bp, then the lactic acid bacteria has arginine deiminase approach, the otherwise lactic acid bacteria do not have essence
The de- imines enzymatic pathway of propylhomoserin；
Wherein primer pair A is classified as SEQ ID NO.1 by nucleotides sequence and nucleotides sequence is classified as the nucleotide fragments of SEQ ID NO.2
Composition, or two nucleotides for carrying out being obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.1 and SEQ ID NO.2
Fragment is constituted；Primer pair B is classified as SEQ ID NO.3 by nucleotides sequence and nucleotides sequence is classified as the nucleosides of SEQ ID NO.4
Acid fragment is constituted, or carry out being obtained after reverse complemental simultaneously by the sequence of SEQ ID NO.3 and SEQ ID NO.4 two
Nucleotide fragments are constituted.
2. method according to claim 1, it is characterised in that the lactic acid bacteria include Lactococcus lactis, lactobacillus curvatus,
Lactobacillus fermenti, Lactobacillus plantarum, lactobacillus, Lactobacillus brevis, Wei Si Salmonellas, Lactobacillus delbrueckii, Lactobacillus casei, bar-shaped newborn bar
Bacterium, lemon leukonid, leuconostoc pseudomesenteroides, Leuconostoc mesenteroides.
3. method according to claim 1, it is characterised in that the 200～300bp specifically refers to 250bp；The use
450bp～550bp specifically refers to 500bp.
4. method according to claim 1, it is characterised in that the reaction system of the PCR is：DNA profiling<500ng, on
Trip primer and downstream primer are each 0.2～1 μM, and the μ L of 10 × Ex Taq Buffer 5, the μ L of dNTP 4, sterile purified water is mended to 50 μ L.
5. method according to claim 1, it is characterised in that the reaction condition of the PCR is：Denaturation, 94 DEG C,
4min；Denaturation, 94 DEG C, 35s；Annealing, 50 DEG C, 35s extends, 72 DEG C, 40s；Denaturation, annealing, extension carry out 30 circulations, after
Renew and stretch, 72 DEG C, 10min, last 10 DEG C are maintained up to taking-up.
6. method according to claim 1, it is characterised in that the detection of the PCR primer is to use Ago-Gel electricity
Swimming detection amplified production.
7. it is a kind of to combine primer pair, it is characterised in that the combination primer pair includes primer pair A and primer pair B；Wherein primer pair
A is classified as SEQ ID NO.1 by nucleotides sequence and nucleotides sequence is classified as the nucleotide fragments of SEQ ID NO.2 and constitutes, or by
The sequence of SEQ ID NO.1 and SEQ ID NO.2 carries out the two nucleotide fragments composition obtained after reverse complemental simultaneously；It is described
Primer pair B is classified as SEQ ID NO.3 by nucleotides sequence and nucleotides sequence is classified as the nucleotide fragments of SEQ ID NO.4 and constitutes, or
Person is while carrying out the two nucleotide fragments groups obtained after reverse complemental by the sequence of SEQ ID NO.3 and SEQ ID NO.4
Into.
8. the application of primer pair lactic acid bacteria context of detection in food is combined described in claim 7.
9. application according to claim 8, it is characterised in that the food includes fermented food or beverage.
10. application according to claim 8, it is characterised in that the food is soy sauce, ferment sausage, yellow rice wine etc..
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584137A (en) * | 2021-08-09 | 2021-11-02 | 河南农业大学 | Degenerate primer for rapidly screening citrulline degrading bacteria and application thereof |
| CN115948316A (en) * | 2022-12-13 | 2023-04-11 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
-
2017
- 2017-03-06 CN CN201710127917.1A patent/CN106636448A/en active Pending
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| MARIA FERNANDEZ 等: "Amino Acid Catabolic Pathways of Lactic Acid Bacteria", 《CRITICAL REVIEWS IN MICROBIOLOGY》 * |
| 李晓敏 等: "黄酒发酵液中产瓜氨酸乳酸菌的分离鉴定与评价", 《酿酒科技》 * |
| 焦志华 等: "发酵酒精饮品中氨基甲酸乙酯前体物的代谢调控机制研究进展", 《食品科学》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113584137A (en) * | 2021-08-09 | 2021-11-02 | 河南农业大学 | Degenerate primer for rapidly screening citrulline degrading bacteria and application thereof |
| CN113584137B (en) * | 2021-08-09 | 2024-05-17 | 河南农业大学 | Degenerate primer for rapidly screening citrulline degrading bacteria and application thereof |
| CN115948316A (en) * | 2022-12-13 | 2023-04-11 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
| CN115948316B (en) * | 2022-12-13 | 2024-03-22 | 四川大学 | Method for improving acid resistance of lactic acid bacteria |
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Application publication date: 20170510 |