CN112179746B - Method for quantitatively evaluating quality of egg gelatin protective film - Google Patents
Method for quantitatively evaluating quality of egg gelatin protective film Download PDFInfo
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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Abstract
The invention discloses a method for quantitatively evaluating the quality of an egg gelatin protective film, and belongs to the technical field of Raman spectroscopy. The invention adopts malachite green dye solution as a novel dye for evaluating the quality of the protective film of the glue, and simultaneously adopts surface enhanced Raman as an evaluation method. Compared with the MST gelatin protective film blue dye liquor commonly used at present, the dyeing effect of the malachite green dye liquor is similar in appearance, meanwhile, as the main component of the gelatin protective film is glycoprotein, along with the deterioration of the quality of the gelatin protective film, the content of the glycoprotein is gradually reduced, and the malachite green dye liquor can be combined with protein to realize dyeing. Therefore, the malachite green dye liquor is used for dyeing, and then the quantitative evaluation is carried out on the malachite green combined on the protective film by the surface enhanced Raman, so that the quality of the protective film on the surface of the egg can be represented. In addition, the malachite green has lower price, is easy to purchase and can be popularized and used; compared with the traditional colorimetric method, the method for quantifying the spectral characteristic peak is more based on material basis and has more evaluation accuracy.
Description
Technical Field
The invention relates to a method for quantitatively evaluating the quality of an egg gelatin protective film, and belongs to the technical field of Raman spectroscopy.
Background
The eggs are a high-nutrition food and can provide necessary nutrients such as protein, fat, minerals, vitamins and the like for human bodies. However, eggs are particularly prone to spoilage in the production, processing, marketing and circulation links, so that the economic benefits of producers are damaged, and the physical health of consumers is even harmed. According to the research, the surface of the eggshell is provided with a layer of glue protective film which is a liquid protein, generally covers or is embedded into pores on the surface of the eggshell, is the outermost protective film for maintaining the quality of the egg, can not only physically block pollutants, but also effectively inhibit cross-shell pollution of bacteria from the aspect of chemical components, is a natural antibacterial barrier, and is an important evaluation index for optimizing and breeding the chicken species.
The more complete the protective film is, the better the quality is, and the stronger the protective effect on the internal quality of the egg is. For the same batch of eggs, the quality of the protective film gradually deteriorates as the storage time is prolonged. The quality evaluation of the protective rubber film is mainly carried out by a dyeing method, at present, eggs are mostly dyed by MST protective rubber film blue dye liquor produced by British MS company, the quality of the protective rubber film is evaluated by dyeing depth, and the deeper dyeing represents that the quality of the protective rubber film is better. However, MST (MST) gelatin protective film blue dye solution is difficult to purchase, expensive, needs to be dyed for 24 hours and then is tested, and has long detection time (Bain, MM, Mcdade, K, et al. In addition, the quantitative evaluation method of the staining method mainly adopts a colorimetric method, and quantification is carried out according to L, a and b values before and after staining, which is relatively extensive.
Therefore, there is a need to develop a more accurate and faster evaluation method based on material basis.
Disclosure of Invention
In order to solve at least one problem, the invention provides a method for quantitatively evaluating the quality of an egg gelatin protective film based on the combination of malachite green dye and Raman spectrum, which can quantitatively detect the quality of the egg gelatin protective film according to the quantity of the dye on the egg gelatin protective film.
The invention aims to provide a method for quantitatively evaluating the quality of an egg gelatin protective film, which comprises the following steps:
(1) preparing a dye solution; dissolving malachite green in water to prepare dye liquor;
(2) dyeing experiment: immersing eggs into a dye solution, and dyeing the protective film;
(3) washing: washing the dyed eggs, and removing dye liquor which is not combined with the protective colloid film on the surfaces of the eggs;
(4) evaluation: and after washing, wiping the surface with absorbent paper, dripping gold glue on the surface of the eggshell to serve as a reinforced substrate, collecting the Raman spectrum of the surface, and evaluating the quality of the glue protective film according to the characteristic peak intensity of the dye solution in the spectrum.
In one embodiment of the present invention, the concentration of the dye liquor in step (1) is 0.1% to 1%, preferably 0.5%.
In one embodiment of the present invention, the dyeing time in step (2) is 5min to 15min, preferably 10 min.
In one embodiment of the present invention, the washing time in step (3) is 0.5-2min, preferably 1 min.
In one embodiment of the present invention, the rinsing in step (3) is rinsing under tap water.
In one embodiment of the present invention, the spectrum collection in step (4) is performed immediately after the eggs are dried.
In one embodiment of the present invention, the gold colloid in step (4) is prepared by the classic potassium citrate trisodium chloride reduction method, which can be prepared according to the reference (Sun Y, Xie Y F, et al, adsorption of 2, 6-di-t-butyl-p-hydroxyurea (BHT) on gold nanoparticles: analysis and interpretation of Surface-enhanced Raman scattering [ J ] Applied Surface Science, 2012, 261(1): 431-) 435.).
In one embodiment of the present invention, the addition amount of the gold colloid in the step (4) is 10 μ l/egg.
In one embodiment of the present invention, the detection distance of the Raman spectrum used in step (4) is 8 mm.
In one embodiment of the present invention, the raman spectrum in step (4) is collected under the following conditions: the laser wavelength is 532nm, the integration time is 1-5s, and the average times are 1-3.
In one embodiment of the present invention, the raman spectrum in step (4) is collected under the following conditions: laser wavelength 532nm, integration time 1s, average times 2.
In one embodiment of the present invention, the method for quantitatively evaluating the quality of the egg gelatin protective film comprises the following steps:
(1) collecting the original color of the surface of the egg: and measuring the values of L, a and b before dyeing by using a colorimeter.
(2) Preparing a dye solution: dissolving malachite green in water to prepare a dye solution with the concentration of 0.1-1 percent;
(3) dyeing experiment: soaking egg in dye solution, and dyeing the protective film for 5-15 min;
(4) washing: washing the dyed eggs with water flow for 0.5-2min to remove dye liquor which is not combined with the protective film;
(5) evaluation: after the washing, the surface is wiped dry by absorbent paper, the values of L, a and b after dyeing are measured by a color difference meter, and the color difference before and after dyeing is calculated according to a formula
Computing
Dropping gold glue on the surface of egg shell as reinforcing substrate, collecting surface Raman spectrum, and collecting 1613cm characteristic peak of dye liquor in spectrum-1Peak intensity evaluation of the protective film quality.
The second purpose of the invention is the application of the method in egg breeding.
The invention has the beneficial effects that:
(1) the method for quantitatively evaluating the protective film quality of the egg gelatin has higher correlation with the traditional colorimetric method.
(2) Compared with the traditional colorimetric method, the method provided by the invention is more based on material basis and has more evaluation accuracy by adopting the quantitative spectrum characteristic peak.
(3) The method for quantitatively evaluating the protective film quality of egg gelatin has short time, can carry out quality detection immediately after dyeing, and can carry out colorimetric detection only after 24 hours by using the currently common MST dye.
(4) The invention also provides a matched dye liquor for evaluating the quality of the egg gelatin protective film, and compared with the MST gelatin protective film blue dye liquor which is commonly used at present, the dyeing effect is similar in appearance; because the main component of the protective colloid membrane is glycoprotein, the content of the glycoprotein is gradually reduced along with the deterioration of the quality of the protective colloid membrane, and the malachite green dye solution can be combined with protein to realize dyeing, so that the quality of the protective colloid membrane on the surface of the egg can be represented through the dyeing result of the malachite green dye solution.
(5) The dye malachite green adopted by the method is low in price and easy to purchase, and can be popularized and used.
Drawings
Fig. 1 is a raman spectrum of a malachite green standard.
FIG. 2 is a Raman spectrum collected after staining of example 1.
FIG. 3 is a graph showing the effect of dyeing eggs in example 1.
Fig. 4 is a raman spectrum collected after staining in example 2.
FIG. 5 is a graph showing the effect of dyeing eggs in example 2.
FIG. 6 shows the result of staining the surface of eggs with malachite green after the removal of the protective film.
FIG. 7 is a graph showing the effect of removing the protective film after dyeing the eggs of example 2.
FIG. 8 is a graph of the effect of brown-shelled eggs on example 2 (three eggs were selected for testing, avoiding the problem that individual specificity does not account for generality).
FIG. 9 is a graph of the effect of example 2 on white-shelled eggs (three eggs were selected for testing, avoiding the problem that individual specificity does not account for generality).
FIG. 10 is a graph of the effect of staining MST stain with brown shell eggs (three eggs were selected for testing, avoiding the problem that individual specificity does not account for universality).
FIG. 11 is a graph of the effect of MST stain on white shell eggs (three eggs were selected for testing, avoiding the problem that individual specificity does not account for generality).
FIG. 12 is the surface enhanced Raman spectrum 1613cm after staining in example 2-1And (4) analyzing the correlation between the characteristic peak intensity and the result of the colorimetry.
FIG. 13 shows Raman spectra of eggs before, after, and after dyeing with addition of gold gel.
Fig. 14 is a raman spectrum collected after staining in example 3.
FIG. 15 is a graph showing the effect of dyeing eggs in example 3.
FIG. 16 is a graph showing the effect of malachite green staining on eggs.
FIG. 17 is a graph showing the effect of methyl blue staining on eggs.
FIG. 18 is a graph showing the effect of Coomassie blue staining of eggs.
Detailed Description
The following description of the preferred embodiments of the present invention is provided for the purpose of better illustrating the invention and is not intended to limit the invention thereto.
Because different dye liquor concentrations and dyeing time can influence the final dyeing result, the higher the concentration is, the longer the dyeing time is, and the higher the dyeing degree is relatively in a certain range, so that a certain same condition needs to be adopted in the process of comparing the qualities of different egg gelatin protective films in practical application. According to the existing experimental results, under the condition that the concentration of the dye liquor is 0.5 percent and the dyeing time is 10min, 1613cm-1The peak intensity is more than or equal to 35300, namely the quality of the egg gelatin protective film with the chromatic aberration more than or equal to 40 is excellent; 31800 is less than or equal to 1613cm-1The peak intensity is less than 35300, namely the color difference is more than or equal to 35 and less than 40, and the protective film of the egg gelatin has medium quality; 1613cm-1The peak intensity is less than 31800, namely the quality of the egg gelatin protective film with the color difference less than 35 is poor.
Example 1
A method for quantitatively evaluating the quality of egg gelatin protective film comprises the following steps:
(1) collecting the original color of the surface of the egg: measuring the values of L, a and b before dyeing by using a color difference meter;
(2) preparing a dye solution: dissolving the malachite green solid in water to prepare a malachite green dye solution with the concentration of 0.1 percent;
(3) dyeing experiment: soaking egg in malachite green dye solution, and dyeing the protective film for 15 min;
(4) washing: washing the dyed eggs with water for 1min to remove dye liquor on the surfaces of the eggs, which is not combined with the protective films;
(5) evaluation: after the washing, the surface is wiped dry by absorbent paper, the values of L, a and b after dyeing are measured by a color difference meter, and the color difference before and after dyeing is calculated according to a formula
Computing
Dripping 10 microliter of gold glue on the surface of an eggshell to serve as an enhanced substrate, collecting a surface enhanced Raman spectrum, and collecting 1613cm according to a characteristic peak of a dye solution in the spectrum-1Peak intensity evaluation of the protective film quality.
The raman spectrum of the malachite green standard is shown in fig. 1, and the raman spectrum collected in this example is shown in fig. 2. From a comparison of fig. 1 and 2, it can be seen that: after the eggs are dyed with malachite green and the golden glue is added, the characteristic peak of the malachite green is 1613cm-1Clearly visible here is a peak intensity of 25140.
The effect graph of the dyed eggs in this example is shown in fig. 3, and it can be seen from fig. 3 that: if a conventional colorimetric method is adopted, the color difference measured 0h after dyeing is 25.82.
Example 2
A method for quantitatively evaluating the quality of egg gelatin protective film comprises the following steps:
(1) collecting the original color of the surface of the egg: and measuring the values of L, a and b before dyeing by using a colorimeter.
(2) Preparing a dye solution: dissolving the malachite green solid in water to prepare a malachite green dye solution with the concentration of 0.5 percent;
(3) dyeing experiment: soaking egg in malachite green dye solution, and dyeing the protective film for 10 min;
(4) washing: washing the dyed eggs with water for 1min to remove dye liquor on the surfaces of the eggs, which is not combined with the protective films;
(5) evaluation: after the washing, the surface is wiped dry by absorbent paper, the values of L, a and b after dyeing are measured by a color difference meter, and the color difference before and after dyeing is calculated according to a formula
Computing
Dripping 10 microliter of gold glue on the surface of an eggshell to serve as an enhanced substrate, collecting a surface enhanced Raman spectrum, and collecting 1613cm according to a characteristic peak of a dye solution in the spectrum-1Peak intensity evaluation of the protective film quality.
The raman spectrum of the malachite green standard is shown in fig. 1, and the raman spectrum collected in this example is shown in fig. 4. From a comparison of fig. 1 and 4, it can be seen that: after the eggs are dyed with malachite green and the golden glue is added, the characteristic peak of the malachite green is 1613cm-1Clearly visible here, the peak intensity is 32282.
The effect graph of the dyed eggs in this example is shown in fig. 5, and it can be seen from fig. 5 that: if a conventional colorimetric method is adopted, the color difference measured 0h after dyeing is 36.70.
Fig. 6 shows the result of staining the surface of the egg with malachite green after the treatment of removing the protective film, which indicates that the egg shell cannot be stained after removing the protective film, and thus the malachite green staining solution has a certain specificity for the protective film and can reflect the information of the protective film.
Fig. 7 is a graph showing the effect of removing the protective film after dyeing the egg of the present embodiment, and it can be seen from the graph that: the glue cuticula on egg surface can be dyed with malachite green, and will glue the cuticula again after the dyeing and remove, can see: the dyed cuticle was peeled off the eggshell surface leaving an unstained eggshell surface, further demonstrating that malachite green can specifically dye cuticles without staining the eggshell.
Fig. 8 and 9 are graphs showing the effect of dyeing brown-shell eggs and white-shell eggs on the dye liquor of the present example (three eggs were selected for testing, and the problem that the specificity of an individual cannot explain the universality) and fig. 10 and 11 are graphs showing the effect of dyeing brown-shell eggs and white-shell eggs on the MST dye liquor. As can be seen from the figure: the dyeing effect of malachite green and the dyeing and finishing body effect of the MST dye are relatively equivalent in the embodiment.
FIG. 12 shows the surface-enhanced Raman spectrum 1613cm after staining in this example-1And (4) analyzing the correlation between the characteristic peak intensity and the result of the colorimetry. As can be seen from the figure: surface enhanced Raman spectroscopy 1613cm after dyeing-1The intensity of the characteristic peak has stronger correlation with the quantitative result of the current commonly used colorimetric method, which shows that the quantitative method has reliability and can evaluate the quality of the glue protective film through the intensity of the characteristic peak. In addition, because the characteristic peak intensity is based on the material of malachite green, the method is more accurate compared with a direct rough colorimetric method, if the conditions do not allow the colorimetric method to be adopted, the characteristic peak intensity can be predicted by substituting the result obtained by the colorimetric method into a standard curve, and a more accurate quantitative evaluation result is obtained.
Comparative example 1
The gold paste in example 2 was omitted and the raman spectrum was obtained, keeping the same as in example 2.
As shown in fig. 13, (a) is a raman line before dyeing, (b) is a raman line after dyeing, and (c) is a raman line after dyeing and dropping gold paste (ser after dyeing), from which: the Raman spectrum is directly detected after dyeing, the peak position of the Raman spectrum is almost unchanged from the Raman spectrum before dyeing, the information of the malachite green dye liquor can not be obtained, the characteristic peak of the malachite green is obviously shown after the gold glue is dripped, wherein the characteristic peak is 1613cm-1The most clear.
Example 3
A method for quantitatively evaluating the quality of egg gelatin protective film comprises the following steps:
(1) collecting the original color of the surface of the egg: and measuring the values of L, a and b before dyeing by using a colorimeter.
(2) Preparing a dye solution: dissolving the malachite green solid in water to prepare a malachite green dye solution with the concentration of 1 percent;
(3) dyeing experiment: soaking egg in malachite green dye solution, and dyeing the protective film for 5 min;
(4) washing: washing the dyed eggs with water for 1min to remove dye liquor on the surfaces of the eggs, which is not combined with the protective films;
(5) evaluation: after the washing, the surface is wiped dry by absorbent paper, the values of L, a and b after dyeing are measured by a color difference meter, and the color difference before and after dyeing is calculated according to a formula
Computing
Dripping 10 microliter of gold glue on the surface of an eggshell to serve as an enhanced substrate, collecting a surface enhanced Raman spectrum, and collecting 1613cm according to a characteristic peak of a dye solution in the spectrum-1Peak intensity evaluation of the protective film quality.
The raman spectrum of the malachite green standard is shown in fig. 1, and the raman spectrum collected in this example is shown in fig. 14. From a comparison of fig. 1 and 14, it can be seen that: after the eggs are dyed with malachite green and the golden glue is added, the characteristic peak of the malachite green is 1613cm-1Clearly visible here is a peak intensity of 37139.
The effect of removing the protective film from the dyed eggs in this embodiment is shown in fig. 15, and it can be seen from fig. 15 that: if a conventional colorimetric method is adopted, the color difference measured 0h after dyeing is 41.47.
Comparative example 2
A method for quantitatively evaluating the quality of egg gelatin protective film comprises the following steps:
(1) collecting the original color of the surface of the egg: and measuring the values of L, a and b before dyeing by using a colorimeter.
(2) Preparing a dye solution: dissolving dye in water to prepare dye solution with the concentration of 0.5 percent;
(3) removing the glue protective film: removing the glue protective film at the bottom of the egg;
(4) dyeing experiment: soaking the eggs in a dye solution, and dyeing the protective film for 10 min;
(5) washing: washing the dyed eggs under water flow for 1min, and removing dye liquor which is not combined with the glue protective film on the surfaces of the eggs to obtain the dyed eggs;
wherein, the dye in the step (2) is malachite green, methyl blue and Coomassie blue.
FIG. 16 is a graph showing the effect of malachite green staining on eggs; FIG. 17 is a graph showing the effect of methyl blue-dyed eggs; FIG. 18 is a graph showing the effect of Coomassie blue staining of eggs. As can be seen from the three figures: for the malachite green dye solution, the normal glue protective film part can be dyed normally, and the bottom part without the glue protective film can not be dyed almost, which shows that the malachite green dye solution has certain specific dyeing effect on the glue protective film and can be used for evaluating the quality of the glue protective film; for methyl blue dye liquor, a normal protective film part can be dyed normally, but the dyeing phenomenon also occurs at the bottom part without the protective film, which shows that the methyl blue dye liquor can also dye the surface of an eggshell while dyeing the protective film, and the protective film lacks specificity and cannot be used for evaluating the quality of the protective film; in the coomassie brilliant blue dyeing solution, the gum protective film is also dissolved while dyeing the gum protective film, and a definite gum protective film dyeing result cannot be obtained, so that the quality of the gum protective film cannot be evaluated.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. The method for quantitatively evaluating the quality of the egg gelatin protective film is characterized by comprising the following steps of:
(1) preparing a dye solution; dissolving malachite green in water to prepare dye liquor; wherein the concentration of the dye liquor is 0.1% -1%;
(2) dyeing experiment: immersing eggs into a dye solution, and dyeing the protective film; wherein the dyeing time is 5min-15 min;
(3) washing: washing the dyed eggs, and removing dye liquor which is not combined with the protective colloid film on the surfaces of the eggs;
(4) evaluation: after washing, wiping the surface with absorbent paper, dripping gold glue on the surface of eggshell as a reinforced substrate, collecting the Raman spectrum of the surface, and collecting 1613cm from the characteristic peak of dye liquor in the spectrum-1The peak intensity of the resin coating is evaluated.
2. The method as claimed in claim 1, wherein the concentration of the dye liquor in step (1) is 0.5%.
3. The method according to claim 1, wherein the dyeing time in step (2) is 10 min.
4. The method of claim 1, wherein the collection of spectra in step (4) is performed immediately after the eggs are dried.
5. The method of claim 1, wherein the Raman spectrum of step (4) is collected under the following conditions: the laser wavelength is 532nm, the integration time is 1-5s, and the average times are 1-3.
6. The method of claim 1, wherein the Raman spectrum used in step (4) has a detection range of 8 mm.
7. The method of claim 6, wherein the Raman spectrum of step (4) is collected under the following conditions: laser wavelength 532nm, integration time 1s, average times 2.
8. Use of the method of any one of claims 1 to 7 in egg breeding.
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