CN105018607B - A method of based on Oil content and Protein content in DNA Quality Identification milk - Google Patents
A method of based on Oil content and Protein content in DNA Quality Identification milk Download PDFInfo
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Abstract
The invention discloses a kind of methods of Oil content and Protein content in Quality Identification milk based on DNA; this method identifies the content of fat and protein in milk using the quality of cow genome group DNA in milk; in a certain range, it establishes in milk the equation of linear regression of the content of cow genome group DNA in the content of protein and milk or establishes the equation of linear regression of the content of DNA in content fatty in milk and milk to judge fat or protein content in milk；The present invention establishes the relevance of cow genome group DNA mass and lactoprotein, milk fat content in milk in milk for the first time; a kind of new method is provided for the discriminating of lactoprotein, butter oil nutritional quality in milk; this method is easy to operate, expense is low, has a extensive future, and has great importance for the nutritional quality detection of milk.
Description
Technical field
It is specific main to rely in milk cow genome group DNA mass to identify fresh ox the invention belongs to field of food science
Oil content and Protein content situation of change in milk.DNA content quality in milk under the conditions of the proposed vertical refrigeration (1~5 DEG C) of this method
With the correlativity of milk Main Nutrient Quality (butter oil, lactoprotein), be conducive to as Dairy Processing enterprise and consumers in general
High-quality feedstocks milk is provided.
Background technology
Protein and fat content are the mostly important two indices of evaluation milk nutritional quality in raw milk, while
The standard often purchased as raw material milk.The detection method of two indices mostly uses physico-chemical process.
There are commonly 4 kinds of classical ways for protein content determination：Kjeldahl's method, biuret method, Folin- phenol reagent process
(Lowry methods), ultraviolet absorption method and Coomassie Brilliant Blue.The shortcomings that Kjeldahl's method is that time-consuming, and sensitivity is low, in sample
Nitrogenous compound can influence the measurement of protein content；Go out protein content to Accurate Determining, can first determine total nitrogen content, then
The albumen precipitation in sample solution is removed with trichloroacetic acid, then measures the non-protein nitrogen content of solution, finally from total nitrogenous
Non-protein nitrogen content is deducted in amount can relatively accurately determine the real protein content of sample.Biuret method measurement range
For 1~10mg protein, the substance of this measurement is interfered mainly to have：Ammonium sulfate, Tris buffer solutions and certain amino acid etc.；This method
The shortcomings that be poor sensitivity, be usually used in need quickly but do not need to protein determination with high accuracy.Folin- phenol reagent process
Developed on the basis of biuret method by Lowry, sensitiveer than biuret method, detectable minimum albumen quality is up to 5 μ g；
The disadvantage is that it is time-consuming longer, the operating time is accurately controlled, operations must accurately control the time, and standard curve is nor stringent
Form of straight lines, and specificity is poor, and interfering substance is more；To the ion that biuret reaction is interfered, equally it is easy to interfere
Lowry reacts, and to the influence bigger of Lowry reactions.The characteristics of ultraviolet absorption method is the accuracy for measuring protein content
Poor, specificity is poor, and interfering substance is more, if can absorb the substance of ultraviolet light in sample containing purine, pyrimidine and nucleic acid etc., can go out
Existing larger interference；Though can correct, but there are certain errors；When measuring protein content with calibration curve method,
The protein big with tyrosine in standard protein and tryptophane difference to those, there is certain error.Coomassie brilliant blue
Method is the highest protein determination of current sensitivity, thus is being widely used；Its main feature is that high sensitivity, minimum
Protein detection amount is up to 1 μ g；Quick, simplicity is measured, a kind of reagent need to be only added, the measurement for completing a sample generally only needs
5 minutes or so；Interfering substance is few, such as most of factor of Lowry methods is interfered not interfere this measuring method.
The method of fat test is more, have soxhlet extraction methods, acid-hydrolysis method, her Buddhist nun Huo Fushi alkaline process, it is Gothic in-sieve is purple
Method, lid Bo Shi methods, Ba Bukekeshi methods etc..Soxhlet extraction methods and acid-hydrolysis method are chiefly used in detection fatty in food, and sour water
It is free and the total amount of bound fat that solution, which measures,.- Luo Zifa, lid Bo Shi methods, Ba Bukekeshi methods and Yi Nihuo in Gothic
Fu Shi alkaline process is chiefly used in the traditional detection of breast and dairy fat.The quantitative detection of content of fatty acid and type uses in dairy products
Gas chromatography, but sample pretreatment process is complicated；Must all saponification and esterification be carried out to fat, recycle gas phase color first
Spectrometry measures the content of wherein aliphatic acid, and time-consuming for such method, consumptive material is more；High performance liquid chromatography can not only detect aliphatic acid
Content, while the position distribution situation of various aliphatic acid wherein, this method accuracy and favorable reproducibility can also be measured, but
Preparation of samples process is complicated, and time-consuming.Han Ruili etc. utilizes high performance liquid chromatography-evaporation photodetector and gas chromatography-mass spectrum
Method is used in conjunction and measures sn-2 aliphatic acid compositions of milk triglycerides, which eliminates thin-layer chromatography time-consuming and laborious during tradition measures
Separating step, precision is high, result is reliable.Woods sees equality and uses gas chromatograph-mass spectrometer (GC-MS) qualitative and quantitative analysis Zhanjiang
Lake-light butterfat acid forms and content, and this method is efficiently easy, accurately and reliably, saves the time, is suitble to the inspection of big scalar sample
It surveys.Wang Lijie etc. is diffused albumen in spectral technology and the offset minimum binary side's Return Law detection and analysis milk using near-infrared
Matter content, this method has many advantages, such as quick, at low cost and energy nondestructive measurement Multiple components simultaneously, but needs a large amount of milk
Model is established, and needs the numerical value of content of milk protein in milk.
Invention content
For the defects in the prior art and insufficient, the purpose of the present invention is to provide one kind can passing through ox base in milk
Because of the method for Oil content and Protein content in group DNA Quality Identification raw milks, this method passes through cow genome group DNA in milk
Separation and Extraction measures DNA content and purity, DNA sizes is measured using agarose gel electrophoresis method, with ox internal control primer and spy
Specific primer carries out PCR amplification, the quality of extract DNA in comprehensive analysis and judgement milk, according to breast egg in DNA content and milk
In vain, the relation equation of butter oil determines the content of lactoprotein and butter oil in milk.
In order to realize that above-mentioned task, the present invention are achieved by the following technical programs：
A method of based on Oil content and Protein content in DNA Quality Identification milk, this method includes carrying out in milk
The measurement of cow genome group DNA mass by fat content in the quality reaction milk of cow genome group DNA in milk and passes through milk
The quality reaction Protein Content in Milk of middle cow genome group DNA；
Fat content is negatively correlated in the quality of cow genome group DNA and milk in the milk, cow genome group in milk
The quality of DNA is proportionate with Protein Content in Milk；
Cow genome group DNA mass includes cow genome group in cow genome group DNA content, milk in milk in the milk
In DNA purity, milk in cow genome group DNA agarose gel electrophoresis band quality and milk cow genome group DNA PCR amplification energy
Power.
Specifically, cow genome group DNA mass in milk is represented using cow genome group DNA content in milk, by milk
The content back of cow genome group DNA answers fat content and the content back by cow genome group DNA in milk in milk to answer egg in milk
White matter content；
Fat content is negatively correlated in the content of cow genome group DNA and milk in milk, and cow genome group DNA's contains in milk
Amount is proportionate with Protein Content in Milk.
More specifically, this method further includes establishing the line of Protein Content in Milk and cow genome group DNA content in milk
Property regression equation and establish the equation of linear regression of cow genome group DNA content in fat content and milk in milk.
Compared with prior art, caused to have the technical effect that：
(1) relevance of cow genome group DNA mass and lactoprotein, milk fat content in milk in milk is established for the first time,
A kind of new method is provided for the discriminating of lactoprotein, butter oil nutritional quality in milk；
(2) present invention is easy to operate, expense is low, has a extensive future, and has for the nutritional quality detection of milk important
Meaning.
Description of the drawings
Fig. 1 is the cow genome group DNA purity testing results extracted in milk sample；
Fig. 2 is the cow genome group DNA content measurement result extracted in milk sample；
Fig. 3 is cow genome group DNA gel electrophoresis result in milk sample, M：DL2000marker；1,2,3,4,5,6,7,8,9 points
It Biao Shi not cow genome group DNA bands in the 0.1st, 1,2,3,4,5,6,7,15 day milk；
Fig. 4 is the PCR amplification of ox internal control primer as a result, M：DL2000marker；1, it 2,3,4,5,6,7,8,9 indicates respectively
Cow genome group DNA is the PCR product band of template amplification internal control primer in 0.1st, 1,2,3,4,5,6,7,15 day milk；
Fig. 5 is the PCR amplification of bovine primer as a result, M：DL2000marker；1,2,3,4,5,6,7,8,9 difference table
Show that cow genome group DNA is the PCR product band of template amplification specific primer in the 0.1st, 1,2,3,4,5,6,7,15 day milk；
Fig. 6 is milk content of milk protein measurement result；
Fig. 7 is milk milk fat content measurement result；
Fig. 8 is the correlation results of content of milk protein and cow genome group DNA content in milk；
Fig. 9 is the correlation results of milk fat content and cow genome group DNA content in milk；
The specific embodiment provided below in conjunction with specific experiment and inventor is for a more detailed description to the present invention.
Specific implementation mode
The study found that somatic number (SCC) has substantial connection in Protein Content in Milk, fat content and milk.
United States Department of Agriculture provides that SCC is necessarily less than 750,000 in every milliliter of raw milk, is otherwise not allowed for processing pasteurization milk etc.
Commercially available dairy products；European Union just provides that raw material milk, which is necessarily less than 400,000/mL, just permits being sold to processing station-service from June, 1992
In producing dairy products；Standard is also SCC< as defined in New Zealand and Australia;400000/mL.Meanwhile DNA in milk in milk
Content is mainly derived from the body cell in milk, and the body cell quantity in milk commonly reaches 10~300,000/mL and can carry
Cow genome group DNA in milk is taken out, this laboratory has been set up the extraction of cow genome group DNA in more stable milk early period
Method.
Cow genome group DNA mass includes ox base in cow genome group DNA content, milk in milk in milk of the present invention
Because the PCR of cow genome group DNA in cow genome group DNA agarose gel electrophoresis band quality in group DNA purity, milk and milk expands
Energization power；
Cow genome group DNA agarose gel electrophoresis band quality refers to the brightness of agarose gel electrophoresis band in milk
And size；
The PCR amplification ability of cow genome group DNA refers to using the cow genome group DNA in milk as template in milk, forms PCR bodies
System carries out PCR amplification, then judges amplification by the brightness of PCR product band and size.
The present invention measures the correlation of Oil content and Protein content and cow genome group DNA mass in milk by exploration, wishes
It hopes and relies on Oil content and Protein content situation of change in DNA quality discrimination raw milks, research through a large number of experiments is found not
Variation with cow genome group DNA content in milk under holding conditions and its purity, agarose gel electrophoresis and PCR amplification result is advised
Restrain it is almost the same, therefore, select the DNA content representation DNA quality with data come judge itself and in milk fat and protein
Between relationship.
Refrigeration of the present invention refers to being stored under the conditions of temperature is 2 DEG C；It is of the present invention at room temperature storage refer to
It is stored at 20 DEG C.
Embodiment one：
1, laboratory sample source
5 adult dairy cattles of the milk sample collection in the man of Xian District, Shanxi Province suburban area milk cattle cultivating family, each sample
The amount of taking is 15mL, is fitted into the centrifuge tube of 15mL.It after acquisition, is immediately placed in ice chest and takes back, be placed in stored refrigerated in refrigerator
15 days, for use.
2, test reagent
The reagents such as ethyl alcohol (more than 95% volume fraction), natrium carbonicum calcinatum, sodium chloride, isoamyl alcohol are analytical reagents or molten
Liquid, 95% ethyl alcohol, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate, Triton-x 100, Tris-Cl, EDTA-Na, dodecane
Base sodium sulphate, concentrated hydrochloric acid, protease, EDTA, Na2- EDTA, glacial acetic acid are purchased from the gloomy rich biological Co., Ltd in Xi'an.
The configuration of phosphate buffer (PBS)：NaCl 8g, KCl 0.2g, Na are weighed respectively2HPO41.44g
KH2PO4Various substances are dissolved in 800mL distilled water by 0.24g, adjust pH to 7.4, distilled water is added to be settled to 1L；
The configuration of OP emulsifiers：90% Triton-x 10020mL, 95% ethyl alcohol 125mL, 0.9g/L NaCl solution
855mL；
The configuration of DNA Extraction buffers：NaCl 1M/L, Tris-Cl 0.5M/L, EDTA-Na 0.5M/L, pH are adjusted to 7.5
~8.0；
The configuration of 20%SDS lysates：The lauryl sodium sulfate (SDS) of 20g is dissolved in 80mL distilled waters in 68 DEG C
It dissolves by heating, is adjusted to PH7.2 with dense HCl, is settled to 100mL, 4 DEG C save backup；
The configuration of Proteinase K：20mg protease is dissolved in the sterile tri-distilled waters of 1mL, -20 DEG C spare；
The configuration of TE buffer solutions：1M/L Tris-Cl 2mL and 0.5M/L the EDTA 0.4mL that pH is 8.0 are weighed respectively,
Its two kinds of solution are uniformly mixed, distilled water is added to be settled to 200mL, adjust pH to 8.0；
The configuration of electrophoretic buffer (1 × TAE)：Weigh 24.2g Tris-Cl, the Na of 10mL 0.5M/L2- EDTA,
The glacial acetic acid of 5.71mL is fully settled to 1000mL after dissolving, and pH is adjusted to 8.0~8.4.
3, key instrument
It is milk analyzer (Bulgarian Lactoscan), ultrahigh speed refrigerated centrifuge (3k30, Sigma Co., USA), low
Fast large capacity centrifuge (DL-4C, Anting Scientific Instrument Factory, Shanghai), high-speed refrigerated centrifuge (TGL-16G, Town in Shanghai pavilion science
Instrument plant), precise micro pipettor (German Eppendorf), whirlpool mixed instrument (XW-80A, its woods Bell's instrument system of Haimen City
Make Co., Ltd), micro-wave oven, electrophoresis apparatus (DYY-4C, Beijing Liuyi Instrument Factory), full automatic gel imaging analysis instrument (JS-
680B, Shanghai Peiqing Science Co., Ltd), grads PCR instrument (BLO-RAD), Palm type centrifugal machine (LX-100), ultraviolet spectrometry light
Spend meter, controllable temperature water-bath, thermostatic drying chamber, a ten thousandth assay balance, JHH-4 digital displays thermostat water bath (the mutual good public affairs in Shanghai
Department), TU1810 ultraviolet-uisible spectrophotometers (Beijing Pu Xi companies), beaker, pipette, the glasswares such as test tube.
4, test method
(1) DNA content extracts in milk
By fresh milk sample, first refrigeration is thawed, and subsequent room temperature is slowly thawed, excellent in ultrahigh speed low temperature (4 DEG C) centrifuge
Change and centrifuge 7500rpm, 30min under the rotating speed time, the butterfat on centrifuge tube upper layer is scraped off with small spoon, with suction pipe by the newborn egg of middle layer
Remove in vain, leaves one layer of precipitation of bottommost.600 μ L PBS are added to centrifugation bottom of the tube, bottom precipitation is beaten into suspension simultaneously
It is transferred in the centrifuge tube of 1.5mL, 12000rpm low-temperature centrifugation 10min, discards supernatant liquid, retain bottom precipitation.Again the bottom of to
Portion's precipitation plus 60 μ L of emulsifier, 540 PBS μ L are vibrated to precipitation with oscillator and are suspended completely, and 40 DEG C of waters bath with thermostatic control handle 10min
The butterfat around body cell, 12000rpm are sloughed, low-temperature centrifugation 10min abandons supernatant, adds 500 μ L of PBS to suspend and precipitates, in
12000rpm low-temperature centrifugations 10min makes body cell precipitation enrichment, abandons supernatant.The DNA extractions of 350 μ L are added in being precipitated to body cell
Buffer solution, the SDS of 50 μ L, the Proteinase K of 10 μ L, water-bath, which digests, at 56 DEG C stays overnight.
In order to accurately and efficiently extract DNA, this research extracts DNA using SDS phynol methods.First, add into digestion product
Isometric Tris saturation phenol solutions, centrifugation obtain supernatant；Supernatant is transferred in another 1.5mL centrifuge tube, is added
The mixture of isometric phenol, chloroform and isoamyl alcohol, phenol, chloroform and isoamyl alcohol are that 25 ︰, 24 ︰ 1 are prepared by volume, are run back and forth
, precipitation is made to dissolve, 12000rpm, centrifugation obtains supernatant；Supernatant is transferred to the centrifuge tube of another 1.5mL, is added
(- 20 DEG C) precipitations of ice absolute ethyl alcohol, gently shake, -20 DEG C, stand, 12000rpm centrifuges 10min, discards upper layer ethyl alcohol.Bottom
Precipitation is washed with ice ethyl alcohol (- 20 DEG C), 12000rpm, low-temperature centrifugation 10min, and careful to remove upper layer ethyl alcohol, quickly volatilize ethyl alcohol,
The TE dissolving DNAs of 25 μ L are added, -20 DEG C save backup.
(2) DNA quality testings
With the content and purity of UV spectrophotometer measuring DNA.5 μ L of DNA extracting solutions are taken, dilute 300 with TE buffer solutions
After times, its absorption value at 260nm and 280nm is detected on ultraviolet specrophotometer.Then the calculation formula of DNA content is：
DNA (ng/ μ L)=A260× extension rate × 50
By calculating it in A260And A280The ratio of absorption value obtain the purity of DNA, calculation formula is：A260/A280。
Each sample repeats to survey three times.If ratio is less than 1.8, protein content is higher, if ratio is more than 2.0, rna content is higher.
(3) agarose electrophoretic analysis
Take 3 μ LDNA samples, 1 μ L sample-loading buffers, the electrophoresis 30min under 1% agar gel, 100V burning voltages.Electrophoresis knot
It is observed under gel image analyser ultraviolet lamp after beam.
(4) the PCR detections of genomic DNA
The primer (Primer 1) for the ox reference gene that first group of amplification length is 212bp, sense primer：5'CAT CTG
TCT TTC CCT GCC GC 3', downstream primer：5'CTA CAG CCT TCC TCA TCT CCC CT 3'.According in NCBI
Ox B2M (NM_007308) gene order devises the specific primer in second group of amplification coding area through the analysis of 5 softwares of Primer
(Primer2), sense primer：5 ' GGC TTT CCC AGC ATC ACT AAC 3 ', downstream primer：5'TCA CAG CAC
CAC CAA ACT TAT CT 3 ', expand the segment of 729bp, are used as the evaluation index of thawing mode optimization.
The 10 μ LPCR reaction systems for amplification coding area segment：1 μ L, 2 × Taq Master of DNA content in milk
Buffer(5mmol/L Mg2+) 3.3 1 μ L of μ L, NTP (each 2.5mmol/L), sense primer (10pmol/ μ L) 0.3 μ L, downstream is drawn
Object (10pmol/ μ L) 0.3 μ L, Taq DNA polymerase (5U/ μ L) 0.1 μ L, ddH2O is mended to 10 μ L.
PCR amplification condition：The PCR reaction conditions of primer 1 are 95 DEG C, pre-degeneration 5min；94 DEG C, it is denaturalized 30s, 60 DEG C, is moved back
Fiery 30s, 72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 10min.The PCR reaction conditions of primer 2 are 95 DEG C, pre-degeneration 5min；
94 DEG C, it is denaturalized 30s, 62 DEG C, anneal 30s, 72 DEG C of extension 30s, 30 cycles；72 DEG C of extension 10min.Pcr amplification product is through 1%
Agarose gel electrophoresis detects.
(4) lactoprotein, butter oil detection in milk
It is detected using milk analyzer.
5, test result
Using the content and purity of UV spectrophotometer measuring cow genome group DNA, the result is shown in Figure 1.When obtained difference
Between the DNA purity variation ranges extracted in section milk sample be 1.69~1.99, average value 1.92, purity is relatively high；Preceding 4 days milk
The Reinheitszahl of cow genome group DNA is above the Reinheitszahl of cow genome group DNA in the 1.90, the 6th~15 day milk sample and is below in sample
1.80, overall purity is preferable.
DNA content value result is shown in Fig. 2.With the increase of number of days, cow genome group DNA content is in one on the whole in milk
Slow downward trend, ranging from 30~50ng/ μ L, 27 milk samples be averaged cow genome group DNA content reach 42ng/ μ L, DNA
Content and the relationship of time are：
Y=48.38e-0.0386X, R2=0.9782
Agarose gel electrophoresis analysis result is shown in Fig. 3.Electrophoretic band brightness gradually weakens with the passage of storage time,
The brightness change unobvious of preceding 7 days bands then have apparent brightness to compare on the 1st day compared with the 15th day, this phenomenon and DNA
Content curve graph result is consistent, and banding pattern is clearly consistent, substantially without miscellaneous band, almost without hangover and diffusing phenomenon.
Use cow genome group DNA for template, amplification length is the primer (Primer1) and length of the ox reference gene of 212bp
Degree is the specific primer (primer2) of 729bp, as a result sees Fig. 4 and Fig. 5 respectively.DNA in the milk that milk sample is carried in 15 days
Content can amplify ox internal control primer and the genetic fragment of specific primer；Ox in the milk extracted in preceding 7 days milk samples
The PCR band brightness changes of Genome DNA content are little, the PCR bands brightness of the 15th day sample compared with other band brightness,
Apparent dark, the content of cow genome group DNA significantly reduces in this 15th day milk sample of explanation.This and Gel electrophoresis results and DNA
Content results are consistent.
Content of milk protein measurement result is shown in Fig. 6, it is found that content of milk protein is generally on a declining curve, content range is
3.22%~3.26%, content of milk protein is fluctuated within the 2nd day to the 7th day, is changed with milk fat content almost the same.Lactoprotein
Content and the relationship of time are
Y=0.0001X2- 0.0041X+3.2523, R2=0.8765
Milk fat content measurement result is shown in Fig. 7, finds to increase with storage number of days, milk fat content is generally in rise
Gesture, content range are 4.00%~4.12%, and fat content is fluctuated within the 2nd day to the 7th day.The relationship of dairy fat content and time
For
Y=-0.0003X2+ 0.0094X+4.039, R2=0.9943
It is found according to result above, cow genome group DNA content and its agarose gel electrophoresis in milk under refrigerated condition,
The changing rule of PCR amplification result is almost the same, therefore, selects the cow genome group DNA content representation DNA quality with data,
The correlativity with lactoprotein, butter oil in milk is established, sees Fig. 8 and Fig. 9 respectively.
It is analyzed by application DPS processing softwares, content of milk protein is in extremely notable positive with cow genome group DNA content in milk
It closes (P=0.0053), polynomial equation is：
Y=4 × 10-5X2- 0.002X+3.2443, R2=0.9635
Y indicates content of milk protein, %；X indicates cow genome group DNA content in milk, ng/ μ L；
It was found that milk fat content is in significantly negatively correlated (P=0.0241), multinomial side with cow genome group DNA content in milk
Cheng Wei：
Y=-2 × 10-5X2+ 4.1983, R2=0.9921
Y indicates milk fat content, %；X indicates cow genome group DNA content in milk, ng/ μ L.
Claims (2)
1. a kind of method of Oil content and Protein content in Quality Identification milk based on DNA, which is characterized in that this method include into
The measurement of cow genome group DNA mass in row milk represents cow genome group DNA in milk using cow genome group DNA content in milk
Quality is answered fat content in milk by the content back of cow genome group DNA in milk and is contained by cow genome group DNA in milk
Quantitative response Protein Content in Milk；
Fat content is negatively correlated in the content of cow genome group DNA and milk in the milk, cow genome group DNA in milk
Content is proportionate with Protein Content in Milk；
This method identification is Oil content and Protein content in 2 DEG C of milk.
2. the method as described in claim 1 based on Oil content and Protein content in DNA Quality Identification milk, feature exist
Further include establishing Protein Content in Milk and the equation of linear regression of cow genome group DNA content in milk and building in, this method
The equation of linear regression of fat content and cow genome group DNA content in milk in vertical milk.
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