CN103822980A - Rapid identification method for quality of Jinhua ham - Google Patents
Rapid identification method for quality of Jinhua ham Download PDFInfo
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Abstract
The invention belongs to the field of food science and technology, and relates to a rapid identification method for the quality of a Jinhua ham. The rapid identification method comprises the following steps: (1) extracting flavor components in the Jinhua ham, wherein the flavor components in the Jinhua ham are extracted by adopting a solid phase micro-extraction technology; (2) performing GC-MS (gas chromatography-mass spectrometry) analysis on the Jinhua ham to obtain GC-MS total ion flow chromatogram of the flavor components, and identifying unknown compounds; (3) establishing a finger-print map; (4) identifying rapidly. According to the rapid identification method for the quality of the Jinhua ham, the aim of identifying rapidly, effectively and intuitively is fulfilled by analyzing the characteristics of the finger-print map, performing similarity identification and performing auxiliary identification on the basis of a PCA (principle component analysis) map; the requirement of industrial production is met.
Description
Technical field
The invention belongs to Food Science and technical field, particularly the Quick method of the Jinhua ham quality in different fermentations stage.
Background technology
Jinhua ham is the distinctive traditional meat pickled and cured meat of China, has peculiar flavour, is deeply subject to for many years liking of domestic and international consumer, enjoys high reputation.In Jinhua ham quality and ranking, fragrance is always a considerable quality index.By national standard, Jinhua ham is divided into select quality, primes and seconds.
Traditional " three label methods " is to be differentiated by the mode of inserting bamboo let on ham by experienced ham technician, is divided into upper, middle and lower three label according to the difference of sign inserting position, and good sign (head is signed) is near knee joint; Middle label (two sign) are at the inclined to one side leg dorsal part of bones of the body juxtra-articular; Lower label (three sign) are between vertebra and marrow bone.While playing label, bamboo let vertically must be inserted to ham from ham flesh noodles and beat label position, the degree of depth is 1/3 to 1/2 of das Beinfleisch face thickness.Superfine ham requires three label all will have good ham characteristic chicken flavor; One-level leg also requires three label perfume (or spice), but outward appearance is not as superfine leg; Secondary leg requires two label perfume and any in three label to sign all frees from extraneous odour.In " three label methods ", the flavor substance of ham mainly results from the curing process of self.In curing process, there is complicated physics, chemistry and biological variation in meat inside, as the lower degradation of the scattering and disappearing of the diffusion of curing agent, moisture, water activity.Meanwhile, the hydrolysis of protein, fat makes ham accumulate abundant amino acid and fatty acid, has formed certain flavour and local flavor.These materials further react, and as amino acid whose Strecker degraded, fatty acid oxidation, Maillard reaction etc., have formed a large amount of flavor substances, give the flavor characteristics of ham uniqueness.But at present,, traditional " three label methods " undue artificial experience of dependence, cannot meet the judgement of the ham of mass industrialized production being carried out to degree of ripeness and degree best in quality.
Summary of the invention
For prior art deficiency, the object of this invention is to provide one and by gas-matter coupling method (GC-MS), Jinhua ham odor characteristics fingerprint peaks is gathered, set up Jinhua ham standard local flavor finger-print.And by the method for fingerprint pattern technology and principal component analysis (PCA), the quality of ham is judged.
For achieving the above object, the invention provides a kind of Quick method of Jinhua ham quality, comprise the following steps:
(1) extraction of Jinhua ham flavor composition, adopts solid phase micro-extraction technique extraction Jinhua ham flavor composition.
(2) GC-MS of Flavor of Jin-hua Ham composition is analyzed, obtain the GC-MS total ion current figure of flavor components, identify unknown compound; Preferably, by adopting NIST library searching, the comparison of linear retention index and three kinds of modes of n-compound evaluation to carry out the evaluation of unknown compound; Adopt NIST library searching, when positive and negative matching degree >=800 of the compound retrieving are for best.
(3) foundation of finger-print:
1) preparation of finger-print
Utilize GC-MS respectively to ferment 9,12,15 and the Jinhua ham sample of 18 months measure, record total ion current figure.
2) determining of total peak
The total peak that proof strength is larger from the total ion current figure of step 1) is as the characteristic fingerprint peak of finger-print, and wherein to account for 80% of total aroma compound peak area be characteristic fingerprint peak to peak area, and preferred characteristic fingerprint peak is 10; With standard finger-print finger-print in contrast, and the relative peak area of calculated characteristics fingerprint peaks.
Utilize finger-print to calculate similarity, utilize respectively Cosin method and correlation coefficient process, the characteristic fingerprint peak relative peak area that the Data Source that calculates similarity is each sample.
Cosin method computing formula is,
Wherein: i representative sample number, k representative feature variable, r represents mean vector,
represent the average of i all characteristic variables of sample.
Correlation coefficient process computing formula is,
Wherein: i representative sample number, k representative feature variable, r represents mean vector, x
ikrepresent k characteristic variable of i sample.
(4) Quick
Get Jinhua ham sample, to step (3) operation, obtain sample finger-print by step (1), adopt similarity evaluation software, take standard finger-print as benchmark, sample finger-print is analyzed, and calculated its similarity.
The processing of experimental data is by SPSS Statistics17.0, and Microsoft Excel and similarity evaluation 2004A version data processing software complete jointly.
Preferably, also comprise and utilize principal component analysis (PCA) (PCA) method to carry out assistant analysis to finger-print, analysis result is understood more with visual, multiple variablees (relative peak area at the characteristic fingerprint peak of each sample) are converted into a few generalized variable (being major component) by principal component analysis (PCA), wherein each major component is the linear combination of original variable, uncorrelated mutually between each major component, thereby these major components can reflect most information of beginning variable, and contained information non-overlapping copies, and finally scheme to show intuitively the difference between variable by PCA.
Preferably, described step (3) also comprises step: the methodological study of finger-print, is specially:
1) reappearance experiment
Get 5~10 parts, the Jinhua ham sample of same lot number, prepare parallel sample, sample introduction, calculates the relative retention time at each peak and the relative standard deviation of relative peak area.
2) stability experiment
Get same sample, place respectively after 0,1,2,3,4,5,6,7,8 hour sample introduction and measure, more each total peak relative retention time and relative peak area, calculate the relative standard deviation at each peak.
3) Precision Experiment
Get same sample, continuous sample introduction 5~10 times, more each total peak relative retention time and relative peak area, calculate the relative standard deviation at each peak.
Preferably, in described step (2), the GC-MS analysis condition of Flavor of Jin-hua Ham composition is:
1) GC conditions is set: injector temperature is 250 ℃, carrier gas is He, and flow rate of carrier gas is 1.2ml/min, split ratio 5:1, temperature programme: 40 ℃ of initial temperatures, retention time 3min, then rise to 200 ℃ with 5 ℃/min, then with 10 ℃/min to 230 ℃ of temperature, keep 3min.
2) mass spectrum condition is set: 230 ℃ of ion source temperatures, voltage 70eV, ionization mode is EI
+; Filament emission current 35 μ A, full scan mass range: 45~650m/z, sweep time, 200ms, detected voltage 350V.
Beneficial effect of the present invention: by the foundation of finger-print, in the time carrying out Quick method without hand picking characteristic flavor on basis fingerprint peaks, without characteristic flavor on basis compound is identified, by the signature analysis to overall finger-print, the judgement of carrying out similarity differentiation and assisting according to PCA figure, thereby reach fast, efficiently, differentiate intuitively object, meet the requirement of suitability for industrialized production.
Accompanying drawing explanation
Fig. 19 months Flavor of Jin-hua Ham finger-prints that ferment;
Fig. 2 12 months Flavor of Jin-hua Ham finger-prints that ferment;
Fig. 3 15 months Flavor of Jin-hua Ham finger-prints that ferment;
Fig. 4 18 months Flavor of Jin-hua Ham finger-prints that ferment;
Fig. 5 total peak of 9 months Jinhua ham finger-prints relative peak area value of fermenting;
Fig. 6 total peak of 12 months Jinhua ham finger-prints relative peak area value of fermenting;
Fig. 7 total peak of 15 months Jinhua ham finger-prints relative peak area value of fermenting;
Fig. 8 total peak of 18 months Jinhua ham finger-prints relative peak area value of fermenting;
Fig. 99 months Jinhua ham fingerprint similarities that ferment;
Figure 10 12 months Jinhua ham fingerprint similarities that ferment;
Figure 11 15 months Jinhua ham fingerprint similarities that ferment;
Figure 12 18 months Jinhua ham fingerprint similarities that ferment;
The Jinhua ham principal component analysis (PCA) figure in Figure 13 different fermentations month;
The principal component analysis (PCA) figure of Jinhua ham quality is differentiated in Figure 14 express delivery;
Figure 15 differentiates the principal component analysis (PCA) figure of Jinhua ham unknown sample.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Foundation and the checking of embodiment 1 finger-print
In order to strengthen food quality control, guarantee the steady quality of product in production line, controlled.According to the variation of food smell active substance in different production procedures and in conjunction with gas-phase fingerprint pattern technology, food quality is differentiated.
1, material
(1) sample
Jinhua ham sample is taken from the drafting unit of standard GB/T/T19088-2008 " geography symbol product Jinhua ham ", Jinzi Ham Co., Ltd.Ham is made by traditional handicraft, selects wherein High-quality ham and also therefrom chooses the material that position, top (biceps muscle of thigh place) is analyzed as local flavor.In yeast phase ham finger-print process of establishing, sample is chosen respectively the Jinhua ham in 9,12,15 and 18 month of fermenting.In Jinhua ham finger-print process of establishing, the Jinhua ham of choosing 18 month of fermenting is ripe Jinhua ham.
(2) processing of ham sample
By the muscle parts of locating above ham (the about 0.5cm that is cut into small pieces
3), and freeze-80 ℃ of refrigerator and cooled, seal and keep in Dark Place, stand-by.
(3) reagent and instrument
Canonical reference compound is all purchased from Sigma company of the U.S., chromatographically pure.
Gas chromatography-triple level Four bar mass spectrometers (GC – MS QQQ), gas chromatograph-mass spectrometer device model: 7890A-7000B, purchased from Agilent company of the U.S.; Chromatographic column: DB-Wax(30m × 0.25mm × 0.25 μ m), purchased from J & W company of the U.S.; Hand sampling handle is purchased from Supelco company of the U.S.; SPME extraction syringe needle (DVB/CAR on PDMS) is purchased from Supelco company of the U.S.; Head space bottle (40ml) is purchased from QEC company of the U.S..
2, method
(1) the GC-MS analysis condition of Flavor of Jin-hua Ham composition is:
1) GC conditions is set: injector temperature is 250 ℃, carrier gas is He, and flow rate of carrier gas is 1.2ml/min, split ratio 5:1, temperature programme: 40 ℃ of initial temperatures, retention time 3min, then rise to 200 ℃ with 5 ℃/min, then with 10 ℃/min to 230 ℃ of temperature, keep 3min;
2) mass spectrum condition is set: 230 ℃ of ion source temperatures, voltage 70eV, ionization mode is EI
+; Filament emission current 35 μ A, full scan mass range: 45~650m/z, sweep time, 200ms, detected voltage 350V.
(2) extraction of flavor components
Solid phase micro-extraction technique (SPME): solid-phase micro-extracting device is made up of SPME handle, SPME extraction syringe needle and head space bottle, in sample pretreatment process, the sample that adds respectively 5g to shred in 40ml head space bottle, and bathe balance 20min at 45 ℃ of Water Unders.After water-bath balance, insert solid-phase microextraction syringe needle, head space absorption 40min.Respectively the Flavor of Jin-hua Ham composition of extractive fermentation 9,12,15 and 18 months, will extract after absorption in pin insertion gas chromatograph injection port and resolve 3min under 250 ℃ of conditions, analyze by GC-MS, obtain the total ion current figure of aroma compound.
(3) GC-MS analyzes
Adopt NIST2.0 library searching, linear retention index comparison (RI value) and n-compound to identify that three kinds of modes complete jointly to the evaluation of unknown compound.Wherein: NIST2.0 library searching is that the smell unknown compound in mass spectrogram is retrieved in NIST spectrum storehouse, requires positive and negative matching degree>=800 of compound that retrieve; Linear retention index (RI value) Comparison Method is with C
6~C
25n-alkane as standard and adopt the heating schedule identical with testing sample, calculate the RI value of unknown compound, and compare with standard RI, thereby material is carried out to method qualitatively; It is that the compound standard items that Preliminary Identification goes out are carried out to sample introduction with the same heating schedule of sample that n-compound is identified, finally, according to both RI values, the compound result of determination in ± 10 is true.
To ferment respectively 9,12,15 and 18 months Jinhua ham carry out GC-MS analysis, in result, identify altogether 48 kinds of aroma compounds.Wherein 13 kinds of aldehyde materials, as: hexanal, enanthaldehyde and 3-methyl butyraldehyde etc.; 9 kinds of alcohols materials, as: n-amyl alcohol, n-hexyl alcohol and 1-OCOL etc.; 8 kinds of acids, as: 3 Methylbutanoic acid, acetic acid and caproic acid etc.; 5 kinds of Esters, as: nonyl lactone, γ-decalactone and gamma-undecalactone etc.; 4 kinds of letones, as: 3-octene-2-ketone, (anti-, anti-)-3,5-octadiene-2-ketone and 1-penten-3-one etc.; 5 kinds of benzene homologues, as: benzaldehyde, phenylacetaldehyde and phenylethyl alcohol etc.; 2 kinds of furans, as: 2-amyl furan and 2-ethyl furan; 1 kind of pyrazine class material, as: 2,6-dimethyl pyrazine.
The processing of experimental data is completed jointly by SPSS Statistics17.0, Microsoft Excel and similarity evaluation 2004A version data processing software.(4) Jinhua ham fingerprint is learned and is investigated
Main normative reference: " technical requirement (provisional) of traditional Chinese medicine finger-print research ", " geography symbol product Jinhua ham " and " People's Republic of China's inspection and quarantining for import/export industry standard ".
The experiment of table 1 finger-print relative retention time reappearance
The experiment of table 2 finger-print relative peak area reappearance
Table 3 finger-print relative peak area stability and Precision Experiment
Table 4 finger-print relative retention time stability and Precision Experiment
Reappearance test: the reappearance of mainly investigating experimental technique.Get 7 parts, the ham sample of same lot number, prepare test sample and detect according to the preparation and determination methods method of test sample, investigate the relative retention time of chromatographic peak, the consistance of peak area ratio.
Stability test: the stability of mainly investigating ham sample.Get same ham sample, according to the method described above sample is prepared and respectively in the each integer of 0~8h time sample detection.Investigate the relative retention time of chromatographic peak, the consistance of peak area ratio.
Precision test: the precision of mainly investigating instrument.Get same ham sample, be prepared according to above-mentioned pre-treating method and sample preparation methods, and continuous sample introduction 9 times, the relative retention time of chromatographic peak, the consistance of peak area ratio investigated.
Conclusion: shown in table 1, table 2, table 3 and table 4, the RSD of relative retention time and relative peak area all, below 10, illustrates that instrument state and sample-pretreating method are respond well, meets the requirement of setting up finger-print.
(5) foundation of Jinhua ham finger-print
Utilize GC-MS respectively to ferment 9,12,15 and the Jinhua ham sample of 18 months measure according to said method, from GC-MS total ion current figure, choose 10 the total peaks (peak area account for total aroma compound peak area 80%) that are accredited as aroma compound as the characteristic fingerprint peak of finger-print, standard finger-print as shown in Figure 1, Figure 2, Figure 3 and Figure 4, is numbered every group of each sample characteristic fingerprint peaks in figure.With standard finger-print collection of illustrative plates in contrast, and calculate the characteristic fingerprint peak relative peak area of every group of 1~No. 10 sample, the corresponding fingerprint peaks absolute peak of fingerprint peaks relative peak area=sample characteristic fingerprint peaks absolute peak area/standard finger-print area.What wherein, the coupling at characteristic fingerprint peak adopted is the Auto-matching function in " similarity evaluation 2004A version " software.Fermentation 9,12,15 and the Jinhua ham sample relative peak area of 18 months respectively as shown in Fig. 5, Fig. 6, Fig. 7 and Fig. 8,1~10 representative sample Article Number, STD represents standard finger-print.
Utilize finger-print data to calculate similarity.Utilize respectively Cosin method and correlation coefficient process, calculate fermentation 9,12,15 and 1~No. 10 Jinhua ham sample fingerprint similarity of 18 months, can find out that from Fig. 9, Figure 10, Figure 11 and Figure 12 similar fingerprint similarity all more than 0.95, meets the requirement of setting up finger-print.
Use again principal component analytical method, as shown in figure 13, S0~S9, S10~S19, S20~S29, S30~S39 represents respectively fermentation 9,12,15 and the Jinhua ham sample of 18 months, can find out ham sample cluster preferably, reach the effect of distinguishing difference month product.
Sample is chosen respectively and has been fermented 6,9,12,15,18 and 21 months and the rotten Jinhua ham that ferments, and the ham wherein having fermented 18 months is defined as ripe Jinhua ham.Other steps are with embodiment 1.
GC-MS data analysis: after solid-phase micro-extracting device absorption Flavor of Jin-hua Ham, to extract in pin insertion gas chromatograph injection port and resolve 3min under 250 ℃ of conditions, obtain analyzing data, jointly complete data processing by SPSS Statistics17.0, Microsoft Excel and similarity evaluation 2004A version data processing software.The TXT chromatographic data of derivation is directly imported to " similarity evaluation 2004A version " data processing software, carry out full spectrogram coupling, contrast collection of illustrative plates generation method is average method, and time window width is 0.1, is the fermentation ripe Jinhua ham of 18 months with reference to spectrogram.Finally by the finger-print of fermentation 6 months, 21 months and rotten ham and the standard finger-print in 9,12,15 and 18 month of fermentation are carried out to software analysis, calculating similarity is as shown in table 5, wherein S-6 represents the Jinhua ham fermenting 6 months, the rotten ham of B-1 representative fermentation, its similarity difference is very large, can distinguish preferably; And be accompanied by the intensification of attenuation degree, the similarity of finger-print presents the trend increasing progressively successively, illustrate that the variation tendency of ham fermenting local flavor is consistent.
Table 5 Jinhua ham sample detection fingerprint similarity
In conjunction with principal component analysis (PCA), as shown in figure 14, can find out fermentation 6,9 and Dec ham sample comparatively close, fermentation is 15,18 comparatively close with 21 months ham samples, and all obvious with the fermentation ham difference that goes bad, thus the quality of differentiation Jinhua ham.
Testing sample: V1 is 9 months fermented sample standard diagrams, V2 is 12 months fermented sample standard diagrams, and V3 is 15 months fermented sample standard diagrams, and V4 is 18 months fermented sample standard diagrams (fermenting-ripening sample), V5~V10 is unknown sample, and discriminating step is with embodiment 2.
Table 6 Jinhua ham unknown sample detects fingerprint similarity
Differentiate result: referring to table 6, V6 similarity is similar to V1, can differentiate for fermentation initial production, V8 similarity is similar to V4, can differentiate for fermentation product in latter stage.In conjunction with principal component analysis (PCA), as shown in figure 15, V9 and V10 are rotten ham again, V5 insufficient fermentation 9 months, and V7 ferments between 9 months to 12 months.
Although, above use general explanation, embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. a Quick method for Jinhua ham quality, is characterized in that, comprises the following steps:
(1) extraction of Jinhua ham flavor composition, adopts solid phase micro-extraction technique extraction Jinhua ham flavor composition;
(2) GC-MS of Flavor of Jin-hua Ham composition is analyzed, obtain the total ion current figure of aroma compound, identify unknown compound;
(3) foundation of finger-print:
1) preparation of finger-print
Utilize GC-MS respectively to ferment 9,12,15 and the Jinhua ham sample of 18 months measure, record total ion current figure;
2) determining of total peak
The total peak that proof strength is larger from the total ion current figure of step 1) is as the characteristic fingerprint peak of finger-print, with standard finger-print finger-print in contrast, and the relative peak area of calculated characteristics fingerprint peaks;
(4) Quick
Get Jinhua ham sample, to step (3) operation, obtain sample finger-print by step (1), adopt similarity evaluation software, take standard finger-print as benchmark, sample finger-print is analyzed, and calculated its similarity.
2. Quick method according to claim 1, is characterized in that, described step (3) also comprises step: the methodological study of finger-print, is specially:
1) reappearance experiment
Get 5~10 parts, the Jinhua ham sample of same lot number, prepare parallel sample, sample introduction, calculates the relative retention time at each peak and the relative standard deviation of relative peak area;
2) stability experiment
Get same sample, place respectively after 0,1,2,3,4,5,6,7,8 hour sample introduction and measure, more each total peak relative retention time and relative peak area, calculate the relative standard deviation at each peak;
3) Precision Experiment
Get same sample, continuous sample introduction 5~10 times, more each total peak relative retention time and relative peak area, calculate the relative standard deviation at each peak.
3. Quick method according to claim 1 and 2, is characterized in that, in described step (2), the GC-MS analysis condition of Flavor of Jin-hua Ham composition is:
1) GC conditions is set: injector temperature is 250 ℃, carrier gas is He, and flow rate of carrier gas is 1.2ml/min, split ratio 5:1, temperature programme: 40 ℃ of initial temperatures, retention time 3min, then rise to 200 ℃ with 5 ℃/min, then with 10 ℃/min to 230 ℃ of temperature, keep 3min;
2) mass spectrum condition is set: 230 ℃ of ion source temperatures, voltage 70eV, ionization mode is EI
+; Filament emission current 35 μ A, full scan mass range: 45~650m/z, sweep time, 200ms, detected voltage 350V.
4. Quick method according to claim 3, is characterized in that, described step (2) is by adopting NIST library searching, the comparison of linear retention index and three kinds of modes of n-compound evaluation to carry out the evaluation of unknown compound.
5. Quick method according to claim 4, is characterized in that, adopts NIST library searching, positive and negative matching degree >=800 of compound that retrieve in described step (2).
6. Quick method according to claim 5, is characterized in that, in described step (3), the characteristic fingerprint peak of finger-print is 10.
7. Quick method according to claim 6, is characterized in that, in described step (4), also comprises and utilizes principal component analytical method to carry out assistant analysis to finger-print.
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