CN109725048A - A kind of mass spectrometry method of Rapid identification lactic acid bacteria - Google Patents
A kind of mass spectrometry method of Rapid identification lactic acid bacteria Download PDFInfo
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Abstract
The invention discloses a kind of mass spectrometry methods of Rapid identification lactic acid bacteria, including the substance assistant laser desorpted ion flight mass spectrum processing to lactic acid bacteria, treatment process includes: microbial cell breakage, microprotein extracts, the optimization of Mass Spectrometry Conditions and database analysis, this invention removes higher costs existing for the detection in the prior art to lactic acid bacteria, reliable unstable problem, this method is realized with mass spectrographic method Rapid identification lactic acid bacteria, and the analysis data of effective protein fingerprint pattern data and otherness and general character are obtained on the basis of qualification result, practical gate not only is opened to the Rapid identification of lactic acid bacteria, also theoretical guarantee is provided for the protein analysis of lactic acid bacteria and application.
Description
Technical field
The present invention relates to chemical methodes to identify microorganism, the mass spectrometry method of specifically a kind of Rapid identification lactic acid bacteria.
Background technique
MALDI-TOF MS (matrix desorption ionization flight time mass spectrum Matrix-Assisted Laser
Desorption/ Ionization Time of Flight Mass Spectrometry) a kind of soft ionization organic mass spectrometry, leads to
Extra pulse laser releases analyte from the solid state substrate containing intensive ultraviolet absorbing material, to generate singularly charged
Molecule or ion, generating amplification electric current on the detector can be used as the function of time for measuring abundance of ions and its quality and electricity
The ratio (karyoplasmic ratio m/z) of lotus.Microorganism nucleoprotein system is mainly controlled by inherent cause, media components and incubation time
Deng external because smaller on its element influence, with certain specificity [2], inspection can be passed through by the characteristic spectral peak of microorganism
Rope characteristic properties spectral peak database or compared with the mass spectra peak of known microorganisms, reaches and is used for quickly detecting, identifies to microorganism
With the purpose of parting.But from the point of view of science of heredity, though nucleic acid determines the expression of albumen, the abundance of protein is by translation efficiency
It is influenced with modification etc. after other albumen, therefore, microprotein level obtains analysis of the information compared with nucleic acid level and can get more
It is more.
Lactic acid bacteria (lactic acid bacteria, LAB) is that one kind can generate a large amount of creams with fermentable carbohydrate
The common name of the bacterium of the general name without gemma, gram-positive bacterium of acid.The pH range of lactic acid bacteria Adaptable growth is very wide, but
Some improve probiotic lactobacillus as gastrointestinal tract, can tolerate the highly acidity of gastric acid, go directly enteron aisle [4].The existing identification of lactic acid bacteria
Method is biochemical method, molecular method identification.The physical and chemical inspection method of lactic acid bacteria is based on " the micro- life of national food safety standard
Object lactic acid bacteria is examined " GB4789.35 and " identification of national food safety standard food microbiological examination Bifidobacterium "
GB4789.34 lactic acid bacteria biochemical reaction index specified in is identified, is needed in identification using single colonie Grain stain mirror
Procuratorial organ's formula.Such identification mode, there are biggish unstability for qualification result, and the result that can be identified is less.Phenotypic characteristic
Identification method is to be based on different strain in morphosis, biochemical reason with the lactic acid dientification of bacteria mode for being used for a long time history
Change the otherness on reaction and metabolite, intuitively rapidly clear lactic acid bacteria adheres to type separately.Such identification method is due to identification
Cost is relatively low, and application range is relatively wide in food industry, application time is longer, and majority is merely able to identify lactic acid bacteria when identification
It is affiliated, insufficient to specific Identification of Species, the reliability of identification is poor, and only passes through certain repetitive identified work ability
The specific type of enough clear lactic acid bacterias.
The multifarious identification method of lactobacillus protein matter is SDS-PAGE identification method, can be by entirely thin to lactic acid bacteria
Complicated operation for born of the same parents' protein analysis, the type of precise Identification lactic acid bacteria, but the identification method, and the efficiency of identification is lower,
It is difficult to a wide range of promotion and implementation.
The method advantage of identified for genes lactic acid bacteria is the influence that result not will receive lactic acid bacteria culture environment, is effectively promoted
The accuracy of identification and scientific, meanwhile, can be shown in the way of different identified for genes different classification standard and
It is horizontal.The multiple PCR technique being derived on the basis of round pcr, Real-time quantitative PCR, RFLP technology are lactic acid bacteria mirror
One of fixed effective means.RFLP identification method resolution ratio with higher, but there is also poor repeatability, identifying species are not deep enough
The defect entered.In lactic acid bacteria gene identification method, 16SrDNA and 16SrRNA sequencing methods identify non-perfect fungus
Effective means, the higher a kind of mode of accuracy in same or current lactic acid bacteria identification method.It is identified first in qualification process
Personnel will implement 16SrDNA and 16SrRNA specific amplification to lactic acid bacteria to be checked, then measure the nucleotides sequence of lactic acid bacteria
Column, on this basis, the gene order of acquisition are compared with specific lactic acid bacteria sequence, and then clearly identify lactic acid bacteria
Type.Such identification method has more significant advantage in lactic acid dientification of bacteria accuracy and accuracy, can be by lactic acid
The dientification of bacteria is divided into the level of kind.But appraisal cost needed for this identification method is higher, and qualification process is longer, is passing through
Ji property with have on efficiency it is to be hoisted.DGGE technology and TGGE technology have more significant in terms of micropopulation identification research
Advantage, can be applied in the identification of specific group's mushroom, by dropping into row specific amplification to specified germ, obtain thin
The relevant information that flora is fallen, and then reflect the truth of entire fermentation process, Molecular Identification is all based in microorganism in gene
Horizontal consistency, but there is still a need for the supports of more molecules experiment for its specificity.
The MALDI-TOF MS having been reported that the identification of microorganism based on the stability of microprotein and specificity and
Its protein fingerprint pattern is obtained, but is all that direct ionization or acid are handled to the processing of microorganism in existing report, cream
Sour bacterium has stronger acid-resistant property again, so existing it is not yet reported that more complete MALDI-TOF MS is to lactic acid bacteria detection side
Method, in addition, research of the MALDI-TOF MS as mass spectrography to microorganism, can not only obtain the information of microbe species distribution,
The protein information of microorganism specificity can be also obtained, this is of great advantage to the research and development of lactic acid bacteria.
In view of the above-mentioned problems, there is presently provided a kind of mass spectrometry method of Rapid identification lactic acid bacteria.
Summary of the invention
The purpose of the present invention is to provide a kind of mass spectrometry methods of Rapid identification lactic acid bacteria, to solve in above-mentioned background technique
The problem of proposition.
To achieve the above object, the invention provides the following technical scheme:
A kind of mass spectrometry method of Rapid identification lactic acid bacteria, including the substance assistant laser desorpted ion flight matter to lactic acid bacteria
Spectrum processing, treatment process includes: microbial cell breakage, microprotein extracts, the optimization of Mass Spectrometry Conditions and database divide
Analysis；
Microbial cell is broken: six 1.5ml sterile centrifugation tubes of picking are respectively put into appropriate bacterium amount, and 0.75mL is 2. added
75% ethanol water, liquid nitrogen frozen 30s, 37 DEG C melt 1min again, 3. 125 μ L, 75% ethanol water is added in 100HZ ultrasound 5min
Quartz sand 1-3g is added in solution simultaneously, after concussion uniformly, carries out liquid nitrogen grinding, and after abrasive material is transferred to centrifuge tube, 0.70mL is added
75% ethanol water,
6000-10000r/min is centrifuged 2min after above 3 kinds of processing, and it is spare to abandon supernatant；
Microprotein extracts:, c: being added 75 μ L, 75% aqueous formic acid, shakes ultrasound 10min, and 75 μ L second are added
Nitrile, uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant, d: 75 μ L, 50% aqueous formic acid is added, concussion is super
75 μ L acetonitriles are added in sound 10min, and uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant.
The optimization of Mass Spectrometry Conditions and database analysis: Mass Spectrometry Conditions: fixed-focus 337nm, the linear formpiston formula of detector,
MALDI ion source, laser beam energy frequency 500-900Hz, capture range 1000~30000 (m/z), 8739 large intestine of calibration object ATCC
Bacillus, threshold value peak accumulate type baseline mode, threshold deviation 0.020mV, and threshold value is corresponding 1.200 ×；
As a further solution of the present invention: the mass spectra peak diagram data that separate microorganism obtains imports MALDI-TOF/
SARAMIS microbial identification system is identified peak identification, using in SARAMIS Premium software
Superpectrum screens the mark peak of lactic acid bacteria of the same race, finds the shared characteristic peak of microorganism, carries out super spectrogram
Building, and characteristic peak is weighted, constructs the super spectrum library of identified specific kind of lactic acid bacteria, the identification for lactic acid bacteria.
As a further solution of the present invention: the mass spectrometry method of the Rapid identification lactic acid bacteria, which is characterized in that ethyl alcohol
Aqueous solution and acetonitrile, the order of addition of aqueous formic acid and additive amount are the key that influence Protein Extraction control direction.
As a further solution of the present invention: the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1,
It is characterized in that, laser beam energy frequency 750hz in EI-MS process.
As a further solution of the present invention: mass spectrum mark peak weight has the algorithm of the identification of lactic acid bacteria after being adjusted
Weighting adjustment, mark peak karyoplasmic ratio respectively (m/z) (3363.1,3512.2.3841.0,4409.0,5730.4,6544.4,
6727.5,6966.7,7025.0,7682.9).
As a further solution of the present invention: liquid nitrogen grinding is added for microbial cell breakage process and that 2g quartz sand is added is auxiliary
Assistant research fellow's mill.
As a further solution of the present invention: the concentration that aqueous formic acid is added in protein extraction procedure is 50%,
And shake ultrasonic 10min.
As a further solution of the present invention: clasmatosis and microprotein sequence of extraction grind for quartz sand auxiliary liquid nitrogen
Mill --- --- abandon supernatant --- centrifugation of aqueous formic acid ultrasound --- acetonitrile and extract 75% methanol aqueous solution by ultrasound centrifugation.
As a further solution of the present invention: Mass Spectrometry Conditions are optimized in laser beam energy frequency 750hz and database analysis
The weight at corresponding mark peak increase mark peak karyoplasmic ratio be respectively (m/z) (3363.1,3512.2.3841.0,4409.0,
5730.4,6544.4,6727.5,6966.7,7025.0,7682.9).
Compared with prior art, the beneficial effects of the present invention are: this invention removes in the prior art to the inspection of lactic acid bacteria
Survey existing higher cost, reliable unstable problem, this method is realized with mass spectrographic method Rapid identification lactic acid bacteria, and
The analysis data that effective protein fingerprint pattern data and otherness and general character are obtained on the basis of qualification result, not only to cream
The Rapid identification of sour bacterium opens practical gate, also provides theoretical guarantee for the protein analysis of lactic acid bacteria and application.
Detailed description of the invention
The lower lactic acid bacteria mass spectrum appearance fingerprint image of Fig. 1 method of production processing.
The lower lactic acid bacteria mass spectrum appearance fingerprint image of Fig. 2 conventional method processing.
Fig. 3 mass spectrum calibrates map Escherichia coli ATCC8739.
Fig. 4 lactic acid bacteria weighting mark peak schematic diagram.
Difference analysis dendrogram between the processing of Fig. 5 distinct methods.
Fig. 6 lactic acid bacteria DNA method clustering figure
Specific embodiment
In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", "upper", "lower",
The orientation or positional relationship of the instructions such as "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outside" is
It is based on the orientation or positional relationship shown in the drawings, is merely for convenience of description of the present invention and simplification of the description, rather than instruction or dark
Show that signified device or element must have a particular orientation, be constructed and operated in a specific orientation, therefore should not be understood as pair
Limitation of the invention.In addition, term " first ", " second " etc. are used for description purposes only, it is not understood to indicate or imply phase
To importance or implicitly indicate the quantity of indicated technical characteristic.The feature for defining " first ", " second " etc. as a result, can
To explicitly or implicitly include one or more of the features.In the description of the present invention, unless otherwise indicated, " multiple "
It is meant that two or more.
In the description of the present invention, it should be noted that unless otherwise clearly defined and limited, term " installation ", " phase
Even ", " connection " shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or be integrally connected；It can
To be mechanical connection, it is also possible to be electrically connected；It can be directly connected, can also can be indirectly connected through an intermediary
Connection inside two elements.For the ordinary skill in the art, above-mentioned term can be understood by concrete condition
Concrete meaning in the present invention.
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
Please refer to Fig. 1-6, in the embodiment of the present invention, a kind of mass spectrometry method of Rapid identification lactic acid bacteria, including to lactic acid bacteria
Substance assistant laser desorpted ion flight mass spectrum (MALDI-TOF MS) processing.
Microbial cell breakage method: in the appropriate bacterium amount of picking and 1.5ml sterile centrifugation tube, 1. 0.75mL 75% is added in
Ethanol water, liquid nitrogen frozen 30s, 37 DEG C melt 1min again, and 1 time repeatedly,.2. 75% ethanol water of 0.75mL, liquid nitrogen is added
30s is freezed, 37 DEG C melt 1min, 100HZ ultrasound 5min again.3. 125 μ L, 75% ethanol water is added while quartz sand is added
2g after concussion uniformly, carries out liquid nitrogen grinding, after abrasive material is transferred to centrifuge tube, 0.70 mL, 75% ethanol water is added and 4. adds
Enter 75% ethanol water of 0.75mL, 5. 75% formic acid water of 0.5mL 5%SDS aqueous solution and 0.25mL is added in ultrasonic 15min
6. 75% ethanol water of 0.70mL, 2 μ 75% aqueous formic acid of L, 3 μ L of lysozyme, concussion is added in solution, ultrasonic 15min
With rear 37 DEG C of water-bath 10min.6000-10000 r/min is centrifuged 2min after above 6 kinds of processing, and it is spare to abandon supernatant.
Microprotein extracting method: a: the not centrifuged 1 direct loading of μ L of stoste is taken.B: 75 μ L acetonitriles, shake is added
Centrifugation is swung, 1 μ L loading of supernatant is taken.C: being added 75 μ L, 75% aqueous formic acid, shakes ultrasound 10min, and 75 μ L acetonitriles, shake is added
It swings uniformly, 8000r/min is centrifuged 2min, takes 1 μ L loading of supernatant.D: 75 μ L, 50% aqueous formic acid, concussion ultrasound is added
75 μ L acetonitriles are added in 10min, and uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant.E: 75 μ L 15% are added
Aqueous formic acid shakes ultrasound 10min, and 75 μ L, 50% acetonitrile solution is added, and uniformly, 8000r/min is centrifuged 2min for concussion,
Take 1 μ L loading of supernatant.F: being added 125 μ L, 75% ethanol water, concussion, and 8000r/min is centrifuged 5min and abandons supernatant, is added 75
50% aqueous formic acid of μ L shakes ultrasound 10min, and 75 μ L acetonitriles are added, and uniformly, 8000r/min is centrifuged 2min for concussion, takes
Clear 1 μ L loading.
The optimization of Mass Spectrometry Conditions and database analysis: Mass Spectrometry Conditions: fixed-focus 337nm, the linear formpiston formula of detector,
MALDI ion source, laser beam energy frequency 758Hz, capture range 2000~20000 (m/z), 8739 large intestine bar of calibration object ATCC
Bacterium.Threshold value peak accumulation type (Threshold type) baseline mode, threshold deviation (threshold offset) 0.020mV,
Threshold value corresponding (threshold respond) 1.200 ×；
The mass spectra peak diagram data that separate microorganism obtains imports MALDI-TOF/SARAMIS microbial identification system and is marked
Know peak identification, is sieved using with mark peak of the superpectrum in SARAMIS Premium software to lactic acid bacteria of the same race
Choosing finds the shared characteristic peak of microorganism, carries out the building of super spectrogram, and be weighted to characteristic peak, construct identified spy
The super spectrum library of fixed kind of lactic acid bacteria, the identification for lactic acid bacteria.
The test combinations mass spectrogram that 2.+c is handled is shown in that Fig. 1,1 mass spectrogram of simple process method are shown in Fig. 2, Escherichia coli
ATCC8739 mass spectrogram is shown in Fig. 3, this experimental design is the fixed experiment of many factor designed with handling principle of Mass Spectrometry Conditions, two sections
30 kinds of processing methods are combined in processing altogether, carry out scanning of the mass spectrum to the Lactobacillus delbrueckii of 10 plants of separate sources under every kind of processing method,
Each sample is repeated 3 times, and is given a mark to appearance number and mark peak abundance, and marking algorithm is shown in that formula (1) calculates for weighted average
Method, score value is higher, and expression appearance effect is better, sees formula (2) between progress variance analysis internal group, the bigger expression of numerical value
Internal data is more discrete between group, and method combination score and variance analysis analysis the results are shown in Table 1, as can be seen from Table 1, appearance effect
Optimal processing concentrates on matrix [1.: 3., c::f] region, and internal variance difference is obvious between group, comprehensively considers between score and group
Data discrete degree, that is, appearance stability, 2.+c is the processing method of most stability and high efficiency.To under combined treatment sample identify peak and
It responds higher mark peak data and carries out clustering respectively, algorithm is shown in formula (3), (4), the clustering structure of distinct methods
Part Fig. 5, it can be seen that no matter always protein extracting method and method of cell disruption can all influence appearance effect, but on the whole,
Peak amount and the higher peak amount of response (peak appearance number and be the mark peak number that product peak area > maximum accumulates peak area 10%) albumen
The correlation of extracting method is stronger because arrangement of the sequence in correlation analysis tree of population characteristic valuve arrangement Protein Extraction method compared with
Obvious but clasmatosis does not have succession, this shows that clasmatosis is to influence the main cause of peak dose-effect fruit.From the level-one branch of Fig. 2
From the point of view of, Protein Extraction method has several groups in the number of level-one branch, but accounting is few, it is seen that appearance effect also needs several
Combined factors are considered.
For method handling principle, clasmatosis principle direction, optimization process region microbial disruption all uses liquid
1. number nitrogen, melting Method And Principle again using freezing is become larger using liquid icing volume, destroys eucaryotic cell structure, 2. -3. a number processing exist
1. ultrasonic vibration is used on the basis of and the method for quartzite sand grind is further processed, and 4. processing is only to be ultrasonically treated, and is being obtained
In result with 1. -3. number have a certain distance, 5. the purpose of SDS, which is added, in number processing but is drawn simultaneously to allow clasmatosis
Enter metal ion, has certain inhibition so the effective less score in mark peak finally obtained is lower to appearance, 6. processing uses
Lysozyme is to use for reference dissolution of the lysozyme to thick peptide glycan film in the DNA of lactic acid bacteria is extracted, and clasmatosis is helped, in mass spectrum
Appearance amount increases in the process, but the abundance at opposite mark peak reduces, and the introducing of foreign protein reduces the standard of microbial identification
True property.From the region c:f is all to be added to formic acid in treatment process and carry out ultrasound, but first in matrix from the perspective of Protein Extraction
The concentration of acid has a different, and significant difference is not present through mathematical method inspection is overall in experimental result, only 5. number processing at f
Poor score increases under reason, is because SDS will affect mass spectrum appearance rate, f processing has handled mostly step cleaning compared to other
Process, so definitive variation occurs in score, on the whole formic acid concn will not cause experimental result significant difference, but not add
Formic acid can be such that score reduces.
F is mark peak total number, and b is product peak area > maximum product peak area 10% mark peak number, and xi is mark peak score
Column vector, s are variance, and ai and aj are respectively to identify peak total number or mark peak appearance number and are product peak area > maximum product peak
Vector composed by m index of i-th and j-th sample of the mark peak number of area 10%, S is covariance matrix.
1 different disposal of table combines shot chart and standard deviation
Ultimate method optimization: optimal pre-treating method has been fitted using the method for mathematical analysis are as follows: bacterium is taken to be added in right amount
0.75 mL, 75% ethanol water, liquid nitrogen frozen 30s, 37 DEG C melt 1min, 100HZ ultrasound 5min again, and 75 μ L, 75% first is added
Aqueous acid shakes ultrasound 10min, and 75 μ L acetonitriles are added, and uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant.
Best Mass Spectrometry Conditions are as follows: MALDI ion source, laser beam energy frequency 758Hz, capture range 2000~20000 (m/z), calibration object
8739 Escherichia coli of ATCC.Threshold value peak accumulates type (Threshold type) baseline mode, threshold deviation (threshold
Offset) 0.020mV, threshold value corresponding (threshold respond) 1.200 ×.
Comparative analysis of 2 mass spectrum of embodiment to lactic acid dientification of bacteria classifying method and 16SrDNA method
The separation and the identification of 16SrDNA of lactic acid bacteria
Separation method: taking 25mL yak yoghourt to be placed in sterile homogenizing bag, and being added sufficiently to vibrate in 225g physiological saline makes
It is mixed, and is diluted, and continues gradient dilution to 10-3、10-4、10-5、10-6, 0.25mL sample liquid is drawn in culture dish, is toppled over
Bacterium, 28 DEG C of Anaerobic culturel 48h are mixed in MRS culture medium.
The identification of 16SrDNA: reaction system: 25 μ L PCR reaction systems, wherein 12.5 μ L MasterMix, primer 2 7f
With each 1 μ L of 1492r, 1 μ L of template DNA, 9.5 ddH2O μ L.PCR reaction condition: 95 DEG C of 5 min of initial denaturation；94 DEG C of amplification program
30s, 60 DEG C of 30s, 72 DEG C of 30s carry out 38 circulations altogether；72 DEG C of extension 7min.
Sequencing analysis: PCR product is carried out bidirectional sequencing and is spliced using DNAMAN software, and sequencing result is submitted
NCBI blast is compared, and identification correlated results is obtained, using MAGA7.0 software maximum parsimony method (Maximum parsimony)
Carry out sequence analysis building development tree.
The processing method of mass spectrum lactic acid bacteria are as follows: pre-treating method are as follows: take bacterium that 75% ethyl alcohol of 0.75mL is added in right amount water-soluble
Liquid, liquid nitrogen frozen 30s, 37 DEG C melt 1min, 100HZ ultrasound 5min again, and 75 μ L, 75% aqueous formic acid, concussion ultrasound is added
75 μ L acetonitriles are added in 10min, and uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant.Best Mass Spectrometry Conditions are as follows:
MALDI ion source, laser beam energy frequency 758Hz, capture range 2000~20000 (m/z), 8739 Escherichia coli of calibration object ATCC.
Threshold value peak accumulates type (Threshold type) baseline mode, threshold deviation (threshold offset) 0.020mV, threshold value
Accordingly (threshold respond) 1.200 ×.
Lactobacillus delbrueckii is identified using optimal processing method, 10 plants of microorganisms all obtain effective identification knot
Fruit, mass spectrogram are shown in Fig. 6, are identified peak to 10 plants of Lactobacillus delbrueckiis of separation and are fitted and carry out clustering, can see
It is out the same species of microorganism of Lactobacillus delbrueckii to qualification result, in the case where 16SrDNA does not have discrepant comparison substantially, in processing of the same race
Under the conditions of, the mutual difference of mass spectrum protein group peak figure is still fairly obvious.Experiment analysis results show 10 plants of separate sources
Lactobacillus delbrueckii be although it is of the same race, but obvious in protein level difference, can be used for special-shaped analysis.
It being compared through NCBI BLAST, 10 strains of lactic acid bacteria separated in 10 yak milks are Lactobacillus delbrueckii, with
Lactobacillus delbrueckii coverage rate and qualification result in GENEBANK are all 100%, to 10 plants of Lactobacillus delbrueckiis using maximum quasi- right
Method carries out the clustering of gene level, and experimental result is shown in Fig. 6, clustering figure be can be seen that in gene stability region
In 16SrDNA, available kind of horizontal identification of Lactobacillus delbrueckii of different fermentations cream separation, but interspecific difference can not be embodied substantially
It is different.
It is obvious to a person skilled in the art that invention is not limited to the details of the above exemplary embodiments, Er Qie
In the case where without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, the present embodiments are to be considered as illustrative and not restrictive, and the scope of the present invention is by appended power
Benefit requires rather than above description limits, it is intended that all by what is fallen within the meaning and scope of the equivalent elements of the claims
Variation is included within the present invention.Any reference signs in the claims should not be construed as limiting the involved claims
In addition, it should be understood that although this specification is described in terms of embodiments, but not each embodiment is only wrapped
Containing an independent technical solution, this description of the specification is merely for the sake of clarity, and those skilled in the art should
It considers the specification as a whole, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
The other embodiments being understood that.
Claims (6)
1. a kind of mass spectrometry method of Rapid identification lactic acid bacteria, which is characterized in that including to the substance assistant laser desorpted of lactic acid bacteria
The processing of ion flight mass spectrum, treatment process include: microbial cell breakage, microprotein extraction, the optimization of Mass Spectrometry Conditions
With database analysis；
Microbial cell is broken: six 1.5ml sterile centrifugation tubes of picking are respectively put into appropriate bacterium amount, and 1. 0.75mL75% second is added in
Alcohol solution, liquid nitrogen frozen 30s, 37 DEG C melt 1min again, 1 time repeatedly, 75% ethanol water of 0.75mL are 2. added, liquid nitrogen is cold
Freeze 30s, 37 DEG C melt 1min, 100HZ ultrasound 5min again, and 125 μ L, 75% ethanol water is 3. added while quartz sand 1- is added
3g after concussion uniformly, carries out liquid nitrogen grinding, after abrasive material is transferred to centrifuge tube, addition 75% ethanol water of 0.70mL, and above 3
6000-10000r/min is centrifuged 2min after kind processing, and it is spare to abandon supernatant；
Microprotein extracts: c: 75 μ L, 75% aqueous formic acid is added, shakes ultrasound 10min, 75 μ L acetonitriles, shake is added
It swings uniformly, 8000r/min is centrifuged 2min, takes 1 μ L loading of supernatant, d: 75 μ L, 50% aqueous formic acid, concussion ultrasound is added
75 μ L acetonitriles are added in 10min, and uniformly, 8000r/min is centrifuged 2min for concussion, takes 1 μ L loading of supernatant, e: 75 μ L, 15% first is added
Aqueous acid shakes ultrasound 10min, and 75 μ L, 50% acetonitrile solution is added, and uniformly, 8000r/min is centrifuged 2min for concussion, takes
1 μ L loading of supernatant, the optimization of Mass Spectrometry Conditions and database analysis: including setting Mass Spectrometry Conditions and database analysis；
Mass Spectrometry Conditions: fixed-focus 337nm, the linear formpiston formula of detector, MALDI ion source, laser beam energy frequency 750Hz are collected
Range 1000~30000 (m/z), 8739 Escherichia coli of calibration object ATCC, threshold value peak accumulate type baseline mode, threshold deviation
0.020mV, threshold value is corresponding 1.200 ×；
Database analysis: the mass spectra peak diagram data that separate microorganism obtains imports MALDI-TOF/SARAMIS microbial identification system
System is identified peak identification, utilizes the mark with the superpectrum in SARAMIS Premium software to lactic acid bacteria of the same race
Peak is screened, and mathematical method fitting mark peak carries out the building of super spectrogram, and be weighted to characteristic peak, constructs through reflecting
The super spectrum library of fixed specific kind of lactic acid bacteria, the identification for lactic acid bacteria.
2. the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1, which is characterized in that microbial cell breakage mistake
It is liquid nitrogen grinding that journey, which has quartz sand in liquid nitrogen grinding or addition,.
3. the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1, it is characterised in that the concentration of formic acid does not influence
The extraction of protein, but ultrasonic procedure influences the extraction of protein.
4. the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1, which is characterized in that ethanol water and second
Nitrile, the order of addition of aqueous formic acid and additive amount are the key that influence Protein Extraction control direction.
5. the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1, which is characterized in that laser beam in EI-MS process
It can frequency 750hz.
6. the mass spectrometry method of Rapid identification lactic acid bacteria according to claim 1, which is characterized in that mass spectrum identify peak weight into
There is weighting to adjust the algorithm of the identification of lactic acid bacteria after row adjustment.
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