CN114018842A - Method for measuring decoloration effect of protein polypeptide solution - Google Patents
Method for measuring decoloration effect of protein polypeptide solution Download PDFInfo
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Abstract
Dissolving dried polypeptide samples before and after decolorization in ultrapure water or deionized water according to the same concentration ratio to obtain sample solutions, placing the sample solutions at room temperature for a certain time, scanning the sample solutions and the ultrapure water at intervals of 1-10nm at 380-780nm by using an enzyme-labeling instrument or an ultraviolet spectrophotometer respectively, and determining the absorbance of the sample solutions and the ultrapure water in the range; (2) making a wavelength-absorbance line graph; (3) and determining the decoloring effect according to the change trend of the line graph or calculating the decoloring rate and the change of a qualitative or quantitative index. The method for measuring the decolorizing effect of the protein polypeptide solution makes up the defects in the prior art, has more intuitive decolorizing effect observation, and is suitable for solutions which are difficult to distinguish visually and have small differences.
Description
Technical Field
The invention relates to the technical field of polypeptide decoloration, in particular to a method for measuring the decoloration effect of a protein polypeptide solution.
Background
A large number of researches show that the polypeptide has physiological activities of resisting tumor, resisting inflammation, inhibiting bacteria, resisting oxidation and the like, has functional characteristics closely related to human health, and is widely applied to the fields of medicines, cosmetics, foods and the like. The polypeptide has related standards of more polypeptides in China, but the requirements on the color of the polypeptide are lower. For example, GB31645-2018 requires that a 1% collagen peptide solution is white or light yellow; GB/T22729-2008, SB/T10634-2011, QB/T2879-2007 and QB/T4588-2013 require that the peptide powder is white and light yellow in color; GB/T22492-2008 requires that the color of the soybean peptide powder is white, light yellow or yellow; QB/T4707-2014 requires that the corn oligopeptide powder is light yellow to brown yellow; QB/T5298-2018 requires wheat oligopeptide powder to be white to light gray. Consumers tend to prefer lighter polypeptide products, which is particularly prominent in the cosmetic industry. In the preparation process of the polypeptide, the quality of the final product is affected by the carrying of the raw materials, the Maillard reaction and pigments generated under severe reaction conditions, so that the decolorization treatment is needed, and a reliable method for measuring the decolorization effect is particularly important. Most enterprises compare the whiteness of the dried powder by sensory analysis or color difference meter, and many of the whiter polypeptide powder still presents darker color after being dissolved in water. In contrast, direct determination of the color of a polypeptide solution is somewhat more reliable.
At present, the related research, Zhang Yi and the like are available in China for the method for measuring the decoloration effect of the polypeptide[1]Measuring the decolorizing effect of the soybean polypeptide by using the absorbance change of 360 nm; sale etc[2]Comparing the decolorizing effects of the conus chinensis peptide by adopting 410 nm; huqing apple and the like[3]Zhengzheng golden baby[4]Respectively adopt 458nm andmeasuring the polypeptide decolorization rate of sea cucumber at 540 nm. In addition, 365nm was also used in the related studies[5]、367nm[6]、370nm[7]、380nm[8]、 410nm[9]And 450nm[10]And (5) measuring the decoloring effect of the polypeptide and the enzymolysis liquid by the change of the absorbance. In the above studies, a single wavelength was selected for determining the decolorization ratio, and the selection is mostly based on the absorption peak of the sample solution under the full-wavelength scan of 200nm to 900nm, while some studies[10]Since the sample has no absorption peak in this wavelength range, a complementary color wavelength of the color of the sample solution is used for the measurement.
However, the above measurement method has the following problems and disadvantages: firstly, the color of the polypeptide solution cannot be simply de-identified by a single color word, and the complementary absorption wavelength is not single, thereby causing inaccuracy of the method for measuring the complementary color wavelength of the sample color; secondly, the colors carried by the raw materials are different, so that the colors of the enzymolysis liquid are different, and the measurement of the decoloring effect of the enzymolysis liquid is limited; and thirdly, the color of part of the polypeptide solution can become darker along with oxidation under the condition of room temperature, so when the decolorization effect of the polypeptide solution is measured, the consistency of the measurement time after the dry sample is dissolved is ensured as much as possible, or the color of the polypeptide solution reaches a stable state at room temperature. The prior art does not pay much attention to the problem; and fourthly, different sample solutions have different absorption effects on the full wavelength of 200nm to 900nm, and part of the sample solutions may not have the highest absorption peak under the full wavelength of 200nm to 900nm, so that the measurement is not necessarily correct by adopting a single absorption wavelength. And fifthly, researching enzymolysis of the same raw material (oyster) by adopting different proteases, and finding that oyster polypeptide solutions of different proteases under different enzymolysis time show different colors when pH, enzymolysis temperature, feed-liquid ratio and enzyme adding amount are controlled to be constant. Thereby causing measurement errors; sixthly, the color of the polypeptide solution is not necessarily accurate by visual observation, and the color which is possibly displayed when a part of sample solution is filled in a small container (such as a 10mL test tube) is lighter, but the color which is possibly displayed when the sample solution is filled in a large container (such as a 50mL colorimetric tube) is darker, and the color of the polypeptide solution with small difference is difficult to distinguish by visual observation; seventhly, a plurality of whiter polypeptide powder still presents darker color after being dissolved in water; eighth, the turbidity of the polypeptide solution also affects its quality, and for polypeptide solutions with lighter color and higher turbidity, the measurement of the wavelength of the complementary color is not necessarily accurate.
[1] Zhang Yi, Hua Lifei, Kongxiangzhen, etc. research on the decolorizing effect of active carbon on soybean polypeptide [ J ] grain and feed industry, 2010(08):31-33.
[2] Thaxon decolorization process and its antioxidant activity [ J ] food science 2020,41(6).
[3] Huqing apple, Kai-Mai dong, ginger red plum, and the like, a response surface method optimizes a sea cucumber polypeptide decolorization process [ J ] food research and development, 2017(16) 81-86.
[4] Zhengjinwa, Wangqiu Guangdong, which Yunyan, and the like, optimization research on a sea cucumber polypeptide decolorization and deodorization process [ J ], university of Dalian sea, 2013,28(003): 303-wall 306.
[5] Study on decolorization and antioxidant activity of green soybean paste polypeptide stock solution in Guanqing and Ruangqing [ J ] Chinese brewing, 2012 and 06: 96-100.
[6] Plum blossom, xuwei, shoanrong, etc. the response surface method optimizes the polypide decolorization process [ J ] of Huidouba [ food industry technology ], 2015(13): 194-.
[7] The Welian meeting, the Raney green, the response surface method optimizes the decolorization process of the polypeptide in the soy sauce [ J ] the food industry science and technology, 2013(10):235-238.
[8] Xiaohuai autumn, Li Yuzhen, Lin Qin records, and the like, the response surface optimization research of the cold-pressed peanut proteolytic liquid activated carbon decolorization process [ J ] Chinese oil, 2014,39(10):34-38.
[9] Wangshun, Wanhan, Zhou Guangdong, and the like, maize polypeptide decolorization process optimization research [ J ]. proceedings of Wuhan industry academy of academic, 2009,28(004):25-29.
[10] Huqing apple, Wei Jiang Teng, Mei dong, etc. the response surface method optimizes the decolorization process of the fish roe polypeptide enzymolysis liquid [ J ] food industry science and technology, 2016,37(21): 189-.
Disclosure of Invention
The invention provides a method for measuring the decolorizing effect of a protein polypeptide solution, which overcomes the defects in the prior art, is more intuitive in decolorizing effect observation, is suitable for solutions which are difficult to distinguish visually and have small difference, and solves the problems in the prior art.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for measuring the decoloring effect of a protein polypeptide solution comprises the following operation steps:
(1) dissolving the polypeptide dry sample before and after decolorization in ultrapure water or deionized water according to the same concentration ratio to obtain a sample solution, standing at room temperature for a certain time, scanning the sample solution and the ultrapure water at an interval of 1-10nm at 380-780nm by using an enzyme-labeling instrument or an ultraviolet spectrophotometer, and measuring the absorbance of the sample solution and the ultrapure water in the range;
(2) making a wavelength-absorbance line graph;
(3) determining the decolorizing effect according to the change trend of the line graph or calculating the decolorizing rate by the change of qualitative or quantitative indexes:
a qualitative index of the decoloring effect: when the broken line trend of the sample solution is closer to the broken line of the ultrapure water, the decoloration effect is more thorough, and when the broken line trend of the sample solution is more inclined to the upper part, the decoloration effect is poorer;
and b, calculating the decolorization rate according to the line graph, and determining the decolorization effect according to the numerical value of the decolorization rate.
Further, the decolorization ratio is calculated by the following formula:
wherein, taking scanning at intervals of 10nm as an example:
s1 is the area enclosed by the broken line of the sample solution before decolorization and the abscissa axis, x is 380nm and x is 780 nm;
s2 is the area enclosed by the broken line of the sample solution after decolorization and the abscissa axis, x is 380nm and x is 780 nm;
s3 is the area enclosed by ultrapure water or deionized water and the abscissa axis, x is 380nm and x is 780 nm;
in the above formula, 10 represents 10nm spaced in the spectrum from 380nm to 780nm, i.e. considered to constitute the height of the trapezoid in area.
Further, the method for measuring the decoloring effect of the protein polypeptide solution further comprises the step of drying the polypeptide solution before and after decoloring treatment in the same drying manner to prepare a polypeptide dry sample before the step (1).
Further, the drying mode is freeze drying or spray drying.
Further, scanning the sample solution and the ultrapure water in the step (1) at an interval of 10nm in the range of 380nm-780 nm; and (3) standing at room temperature for 30min in the step (2).
Further, a wavelength-absorbance line graph was prepared using Excel or Origin software.
Further, the same concentration proportion in the step (1) is 5-20%.
Further, the same concentration ratio is 10% -20%.
The invention has the beneficial effects that:
the method provided by the invention eliminates the defects of the existing determination method, and can be used for comparing the decolorizing effects of decolorized samples in the same line graph for researching the differences and effects of different decolorizing modes, and is more intuitive, especially for solutions which are difficult to distinguish visually and have small differences. The method can also obviously show the polypeptide solution with lighter color but higher turbidity, has outstanding advantages of practicability and accuracy, provides a new way and method for measuring the color of the existing polypeptide solution, and is expected to be used in the industry as a new standard.
Drawings
FIG. 1 is a schematic diagram of a formula for calculating a decolorization ratio according to the present invention;
FIG. 2 is a schematic illustration of the human visible wavelength range in accordance with the principles of the present invention;
FIG. 3 is a line graph of absorption wavelength-absorbance at 380-;
FIG. 4 is a plot of the wavelength versus absorbance of the scan for the solution of example 2 in which the six pigments were treated at different dilutions;
FIG. 5 is a plot of the wavelength vs. absorbance curves scanned by solutions obtained after treatment of six additional pigments at different dilutions for example 2;
FIG. 6 is a plot of the wavelength vs. absorbance curves scanned by the solution resulting from the treatment of five more pigments at different dilutions of example 2;
FIG. 7 is a graph showing the decoloring effect of activated carbon AC1 in example 3 at different addition amounts and different decoloring times;
FIG. 8 is a graph showing the decoloring effect of activated carbon AC2 in example 3 at different addition amounts and different decoloring times;
FIG. 9 is a graph showing the decoloring effect of activated carbon AC3 in example 3 at different addition amounts and different decoloring times;
FIG. 10 is a graph showing the decoloring effect of activated carbon AC4 in example 3 at different addition amounts and different decoloring times;
FIG. 11 is a graph showing the decoloring effect of activated carbon AC5 in example 3 at different addition amounts and different decoloring times;
FIG. 12 is a plot of wavelength vs. absorbance against decolorization for the preferred treatment protocol for activated carbon AC1-AC4 of example 3;
FIG. 13 is a graph showing the effect of decolorization by continuous suction filtration using 8 kinds of filter cloths after decolorization of 4 kinds of activated carbons AC1, AC2, AC3 and AC4 in example 4;
FIG. 14 is a graph showing the decolorization effect of the sample solution after being continuously ultrafiltered and decolorized by different ultrafiltration membranes based on the treatment with activated carbon AC2 and the suction filtration with FC6 filter cloth in example 5;
FIG. 15 is a line graph of the discoloration of different polypeptide products treated by the method of the present invention in example 6;
wherein, in fig. 3, (a) is a wavelength-absorbance line graph of an enzymolysis liquid after the animal protease enzymolysis treatment;
fig. 3 (b) is a graph of wavelength-absorbance curves of the enzymatic hydrolysate after the enzymatic hydrolysis treatment by lactalbumin; FIG. 3 (c) is a graph showing the wavelength-absorbance line of the enzymatic hydrolysate after the trypsin enzymatic treatment; FIG. 3(d) is a wavelength-absorbance line graph of an enzymatic hydrolysate after the flavourzyme enzymatic treatment; FIG. 3(e) is a wavelength-absorbance line graph of the enzymolysis solution after the composite protease enzymolysis treatment; FIG. 3 (f) is a wavelength-absorbance line graph of an enzymatic hydrolysate after an alkaline protease enzymatic treatment;
the corresponding nine colors in fig. 4 are in order: SCARLET (SCARLET), ROSE bengal (ROSE), VERMILION (VERMILION), CRIMSONRED (CRIMSONRED), purplish red (PURPLERED), and transparent iron red (trasparentridoxide); the corresponding six colors in fig. 5 are in order: orange (orange yellow), yellow earth (yellow ochre), transparent iron yellow (TRANSPARENTYLELLOWOXIDE), GAMBOGE (GAMBOGE), light Yellow (YELLOWPALE), lemon yellow (LEMONYELLOW); the corresponding five colors in fig. 6 are in turn: grass green (sapgren), phthalocyanine green (phtalocyaniangren), light green (GREENLIGHT), emerald green (VIRIDIAN), and dark Green (GREENDEEP);
the left line graphs in fig. 7-11 are line graphs prepared by scanning at the interval of 10nm of absorption wavelength of 380-780nm after decolorizing treatment with different addition amounts of activated carbon AC and different decolorizing times, and the solution in the right colorimetric dish is a photographed graph of the solution after decolorizing treatment with different addition amounts of AC and different decolorizing times at the same viewing angle.
Detailed Description
In order to clearly explain the technical features of the present invention, the following detailed description of the present invention is provided with reference to the accompanying drawings. The scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
The method of the invention conforms to the beer-Lambert law.
As shown in FIG. 1, 380nm-780nm is generally used as the visible light range of human, and in the electromagnetic spectrum, this range can stimulate the human eye to feel different to light with different wavelengths, resulting in color difference. Human color perception goes from red to violet corresponding to its wavelength going from long to short.
Objects viewed by humans appear differently in color because they absorb light of different ranges of wavelengths, while the remaining unabsorbed light is reflected and appears, also referred to as fill-in light. Complementary Colors (also called Complementary Colors) refer to any two Colors that appear white or gray after being mixed in a proper proportion, i.e., the two Colors are Complementary Colors.
Similarly, the same is true for colored solutions, and when light with different wavelengths of 380nm-780nm passes through the solution, the solutions with different colors can show different absorbance characteristics due to complementary absorption; and the higher the concentration of the same color solution, the lower the universal transmittance of all light. The light and shade of the color can be reflected by measuring the absorbance of the light by adopting a spectral scanning mode.
Most of polypeptide solutions have certain colors, so that color difference is caused by different pigments carried by preparation raw materials or difference in concentration, 380nm-780nm spectrum scanning can be better suitable for the polypeptide solutions, and the measurement of the decoloring effect of the polypeptide solutions is more visual.
Example 1
Cleaning fresh oysters, shelling, adding deionized water with the mass being 2 times that of the fresh oysters into a beater, crushing and mixing the oysters uniformly, taking out oyster liquid, adding animal protease (A), lactalbumin (R), trypsin (Y), flavourzyme (F), compound protease (M) and alkaline protease (J) into the oyster liquid according to 3000U/g, carrying out enzymolysis for 1, 3, 5, 7 and 9 hours respectively under the corresponding proper pH and temperature conditions, collecting enzymolysis liquid under different enzymolysis conditions, carrying out enzyme deactivation for 10min in a boiling water bath, and carrying out freeze drying.
Re-dissolving the dried powder in deionized water according to the proportion of 10 percent and 20 percent, standing for 30min at room temperature, taking a proper amount of sample liquid, scanning in an enzyme-labeling instrument at the interval of 380nm-780nm and 10nm, and measuring the absorbance of the sample liquid in the range.
The enzymatic conditions are shown in table 1 below.
TABLE 1 conditions of the enzymatic hydrolysis
The results of the color difference of the oyster enzymolysis liquid under different enzymolysis conditions are shown in fig. 3(a) - (f).
As can be seen from FIG. 3, the color depth difference of the oyster enzymolysis liquid under different enzymolysis conditions can be clearly seen by the method established in the embodiment, wherein the deionized water is colorless and transparent, and the broken line appearing in the 380nm-780nm absorption wavelength range is tightly attached to the abscissa axis. For oyster enzymolysis liquid, the absorbance of the oyster enzymolysis liquid at 380nm-780nm does not have the highest peak, the deeper the oyster enzymolysis liquid is, the higher the absorbance of the spectrum is, the higher the refraction is, and conversely, the lighter the color is, the lower the absorbance is, and the closer the broken line is to the broken line of the deionized water below. By this method we can see that the sample solution enzymolyzed with alkaline protease is relatively dark in color and that with trypsin is relatively light in color.
From the results, the enzymolysis liquid of the same raw material oyster shell can show different colors under different enzymolysis conditions, and the color of the enzymolysis liquid is different even if the same protease is used under different enzymolysis time. And in FIG. 3(a), the absorbance of A5-10% at 380 nm-550 nm is higher than that of A9-10%, while the absorbance of A5-10% at 550 nm-780nm is lower than that of A9-10%; in FIG. 3(e), a part of the enzymatic hydrolysate such as M3-10%, M5-10%, M7-10% and M5-20% has a distinct absorption peak at 670nm, but the absorption peak is not significant in the components such as M9-10%, M1-20% and M9-20%, and a similar phenomenon also appears in FIG. 3 (d). Therefore, when comparing the colors of the enzymatic hydrolysate or the polypeptide solution, or measuring the decoloring effect of the enzymatic hydrolysate, the selection of a single wavelength is not necessarily accurate.
Example 2
As the polypeptide solution samples have different colors, the experiment adopts watercolor pigment for measuring the basic color of the solution, and simultaneously verifies the feasibility of the establishment method and the explanation of the related principle. Since the gouache is easy to precipitate and the color is darker after the gouache is dissolved, the determination is influenced. The watercolor raw materials have certain transparency, are made of gum arabic (an adhesive), glycerol/sugar water (a wetting agent), phenol (a preservative) and the like, and have small influence on the absorbance of the solution at 380nm-780nm, so the watercolor pigment is selected for the experimental determination in the experiment.
Diluting 36 watercolor pigments with different colors in deionized water according to the times of 500, 1000, 2000, 4000, 8000 and 16000 respectively, standing at room temperature for 30min, scanning with an enzyme labeling instrument at the interval of 380nm-780nm and 10nm, and measuring the absorbance in the range; meanwhile, the sample solution is placed in a 50mL glass cuvette and is photographed under the same visual angle, so that the accuracy of the method for decoloring the black liquor is more visual.
The absorbance line graphs and the photographs of several colors measured in the above experiment are selected for summary, and the results are shown in fig. 4-6.
The present example determines the research method, and from the experimental results, the shade of the color and the concentration difference thereof will cause the light source of the sample solution in the wavelength range of 380nm to 780nm to have different absorption degrees. The higher the dilution factor of the solution, the lower its concentration and thus the higher the light transmittance, the closer to the measurement broken line of the deionized water below in the broken line graph. Meanwhile, the absorption peak range of the solution gradually increases as the color deepens.
From the line graphs and the photographed graphs of 36 colors of the experiment, the absorption range of yellow solutions such as light yellow, lemon yellow, gamboge, khaki, orange yellow, transparent iron yellow and the like is higher at 380nm-500 nm; red solutions such as scarlet, rose bengal, vermilion, crimson, and mauve have high absorption degree at 450-600 nm; furthermore, green color solutions such as light green, grass green, phthalocyanine green, emerald green, and dark green have a specific absorption peak in the 550nm-700nm range. Part of the color solution may be due to the fact that it may be formed by mixing several other colors, which appear to produce specific absorption peaks in different wavelength ranges.
This phenomenon is consistent with the complementary color absorption of light, with lighter colored solutions having a higher absorbance for lower wavelength range light and darker colored solutions having a higher absorbance for higher wavelength range light. However, the color of part of the solution may be made of a mixture of colors, which does not have this rule for the absorption wavelength of light. Meanwhile, the higher the concentration of the solution is, the stronger the absorption effect of the solution on light is. The solutions with different colors have different absorption effects at 380nm-780nm, which correspond to the decolorization effect of the photographed sample of the cuvette, so that the method can well show the effects of different pigments possibly carried by different raw material sources of the enzymatic hydrolysate.
Example 3
The method comprises the steps of taking sturgeon skin as a raw material of an experiment in the embodiment, adding deionized water in an amount which is 4 times the mass of the sturgeon skin, adding compound protease, carrying out enzymolysis for the same time at 50 ℃, inactivating enzyme, and centrifuging to obtain supernatant which is a sturgeon skin protein polypeptide solution.
5 kinds of active carbon commonly used in industry are selected to carry out decoloration treatment on the sturgeon skin protein polypeptide solution, the active carbon is added according to the proportion of 0.15 percent, 0.30 percent, 0.45 percent and 0.60 percent respectively, the solution is decolored for 30min, 60min, 90min and 120min at the temperature of 50 ℃, filter paper is adopted for suction filtration, and simultaneously food-grade diatomite with the same mass is added as a filter aid. Placing the sturgeon skin protein peptide solution obtained after suction filtration at room temperature for 30min, scanning the sample solution and ultrapure water at an interval of 380nm-780nm by 10nm by adopting an enzyme-labeling instrument or an ultraviolet spectrophotometer, and measuring the absorbance of the sample solution and the ultrapure water in the range; and calculating the decolorization ratio. Meanwhile, the solution is placed in a 50mL glass cuvette and photographed under the same visual angle; the decolorization effect of the sample can be observed more obviously by combining the results of the photo and the spectrum scanning, so that the accuracy of the method for detecting the decolorization effect is more intuitive.
The following table 2 is a statistics of decolorization rates of activated carbon AC1 with different addition amounts and different decolorization times; table 3 shows the statistics of the decolorization ratio for different addition amounts of AC2 and different decolorization times; table 4 shows the statistics of the decolorization ratio for different addition amounts of AC3 and different decolorization times; table 5 shows the statistics of the decolorization rate of AC4 at different addition levels and different decolorization times; table 6 shows the statistics of the decolorization rate for different addition amounts of AC5 and different decolorization times. The decolorization ratio is calculated by the method of the invention.
The calculation process of the decolorization rate of 30min decolorization treatment with the addition of 0.15% of AC1 is shown as follows:
wherein, the measurement results of the absorbance value in the range of 380-780nm are shown in the following table:
an area S1 enclosed by a broken line of the sample solution before the decoloration treatment of AC1 and an abscissa axis, x is 380nm and x is 780 nm;
an area S2 enclosed by a broken line of the sample solution after decolorization and an abscissa axis, x is 380nm and x is 780nm is treated by AC1 decolorization;
s3 is the area enclosed by the ultrapure water and the abscissa axis, x is 380nm and x is 780 nm;
the above numerical calculation is carried into a decoloration ratio formula,
the calculation of the bleaching ratio values for the other processing conditions is not described in detail.
TABLE 2 decolorization ratio of AC1
TABLE 3 decolorization ratio of AC2
TABLE 4 decolorization ratio of AC3
TABLE 5 decolorization ratio of AC4
TABLE 6 decolorization ratio of AC5
As shown in FIGS. 7 to 11, the wavelength-absorbance line graphs obtained after different addition amounts of the above-mentioned activated carbons and different decoloring treatment times are compared with corresponding photographs of the solutions. FIG. 12 is a plot of wavelength versus absorbance for a preferred activated carbon treatment protocol.
Through the method researched and established by the embodiment, the characteristic that the decoloring effect of the sturgeon skin protein peptide solution is more obvious basically as the addition amount of the activated carbon is higher and the decoloring time is longer can be clearly seen. When a part of activated carbon such as AC1 and AC4 was added at 0.6%, the decolorization effect was slightly decreased, which may be related to a case where a part of fine activated carbon remained in the solution and could not be trapped by the filter paper, thereby darkening the color of the sample. The 5 th active carbon (AC5) has poor decolorization effect, and the method established by the research of the embodiment can clearly see that the absorbance of the polypeptide solution at 380nm-780nm is still very close to that of the polypeptide solution (Control) before decolorization after the decolorization treatment by AC5, particularly the addition of AC5 is more obvious when the addition is 0.15%. For sample solutions with very similar colors, the sample solutions are difficult to distinguish visually, and the decolorization effect is obtained in a qualitative and quantitative mode through the change trend of a line graph and the numerical value of the decolorization rate by combining the results of spectral scanning, so that the color shades of the sample solutions can be well distinguished. The research of the invention shows that the way of decolorization rate and the decolorization effect can also correspond well.
The results of the spectral scan under the optimal decolorization conditions for the superior 4 activated carbons AC1, AC2, AC3, and AC4 are summarized in fig. 12. Among them, the decoloring effect with AC2 was the best among the 4 kinds of activated carbon. On the basis of this, the decoloring treatment and the observation and verification of the decoloring effect in the subsequent embodiments are continued.
Example 4
The method is carried out on the basis of the embodiment 3, sturgeon skin is used as a raw material, 4 times of deionized water by mass is added, compound protease is added for enzymolysis at 50 ℃ for the same time, and then enzyme deactivation and centrifugation are carried out to obtain supernatant which is sturgeon skin protein polypeptide solution.
The better four types of activated carbon in the experiment of example 3 and the corresponding optimal decolorizing conditions are selected, and are AC1 (added amount of activated carbon is 0.45%, decolorizing time is 2h), AC2 (added amount of activated carbon is 0.6%, decolorizing time is 2h), AC3 (added amount of activated carbon is 0.6%, decolorizing time is 2h) and AC4 (added amount of activated carbon is 0.45%, decolorizing time is 2 h). The method comprises the steps of (1) simulating production, loading a plate-and-frame vacuum filter device, respectively replacing filter paper with 8 kinds of filter cloth (respectively marked as FC1, FC2, FC3, FC4, FC5, FC6, FC7 and FC8) commonly used in industrial production for vacuum filtration, placing sturgeon skin protein peptide solution obtained after vacuum filtration at room temperature for 30min, scanning the sample solution and ultrapure water at an interval of 10nm between 380nm and 780nm by using a microplate reader or an ultraviolet spectrophotometer, measuring absorbance of the sample solution and the ultrapure water in the range, and calculating decolorization rate; meanwhile, the solution is placed in a 50mL glass colorimetric dish, and a picture is taken under the same visual angle; the decolorization effect of the sample can be obviously observed by combining the results of the photo and the spectral scanning.
Table 7 shows the results of the decolorization by suction filtration of 8 filter cloths. The decolorization ratio was calculated in the same manner and in the same manner as in example 3, and is not shown here.
TABLE 7 decolorization rate after suction filtration treatment of eight kinds of filter cloth
As shown in fig. 13, in the results of the spectral scan on the left side, the uppermost broken line is the sample solution (Control) that has not been subjected to the decoloring treatment, and the lowermost broken line is deionized water; in the photograph on the right, the leftmost sample solution (Control) which had not been subjected to the decoloring treatment was darker.
In contrast to the decolorization effect exhibited by photographs taken on photographs, the other 2 activated carbons were similar and difficult to distinguish by eye, particularly from each other, except that the colors of AC1 and AC4 were relatively dark. The defect that the decoloring effect of the protein solution is difficult to distinguish by directly observing the conventional container is also overcome. And the suction filtration and decoloration effects of the AC2 (the addition amount of the activated carbon is 0.6 percent, and the decoloration is 2 hours) on 8 kinds of filter cloth can be obviously seen by combining a line graph of spectral scanning analysis, the suction filtration and decoloration effects are optimal in 4 kinds of activated carbon, all the line graphs are closer to the line graph of the deionized water below, and the decoloration effect of the suction filtration by adopting FC6 is the best, the decoloration rate is the highest, and the color is the lightest. Therefore, the experimental results further illustrate the feasibility of the method established in the present study.
Example 5
The optimum process optimized in the experiment of example 3 and the experiment of example 4 was used for decolorization, and AC2 (0.6% of added activated carbon, 2 hours for decolorization) was selected and filtered with FC6 to obtain a filtrate. Sequentially carrying out ultrafiltration decolorization on the filtrate by using a 50nm ceramic membrane, a 100kDa ultrafiltration membrane, a 20kDa ultrafiltration membrane, a 10kDa ultrafiltration membrane and a 3kDa ultrafiltration membrane, collecting corresponding component polypeptide solution, and freeze-drying. Dissolving the dried dry sample in deionized water according to the concentration of 10%, standing at room temperature for 30min, scanning the sample solution and ultrapure water at the interval of 380nm-780nm by using an enzyme labeling instrument or an ultraviolet spectrophotometer at the interval of 10nm, and measuring the absorbance of the sample solution and ultrapure water in the range; meanwhile, the solution is placed in a 50mL glass cuvette and is photographed under the same visual angle; the decolorization effect of the sample can be observed more obviously by combining the results of the photo and the spectral scanning, the filtrate obtained by filtering through the filter cloth is highlighted, and the line graph and the decolorization rate calculation result are adopted before and after the ceramic membrane treatment is continuously adopted for decolorization, so that the decolorization effect judgment has better intuition and accuracy.
TABLE 8 is
TABLE 8 decolorization ratio of ultrafiltration membrane treatment
As can be seen from the results in FIG. 14, the method established in this example clearly shows that ultrafiltration has a good decolorization effect on the polypeptide solution. After the activated carbon treatment, most of pigments, suspended turbid substances and the like in the solution are removed. The polypeptide solution can be better decolorized by adopting a 50nm ceramic membrane and a 100kDa ultrafiltration membrane, while 20kDa ultrafiltration has no better decolorization effect and the effect is even worse than that of the 100kDa ultrafiltration membrane. The polypeptide solution after ultrafiltration treatment by using the 3kDa ultrafiltration membrane is quite approximate to colorless and transparent, and the point can be verified visually.
Therefore, the change trend of the line graph or the numerical value of the decoloring rate shows that the method established by the invention can intuitively and definitely present the specific decoloring effect after different decoloring treatment modes are treated.
Example 6
3 kinds of polypeptide products (P1: Japanese imported pig skin collagen peptide; P2: Lusirolo French imported cod collagen peptide; P3: Japanese imported cod skin collagen peptide) on the market are dissolved in deionized water according to the concentration of 10% and 20%, the ultrasonic treatment is carried out until the solid sample is completely dissolved, after the solid sample is placed at room temperature for 30min, a microplate reader or an ultraviolet spectrophotometer is adopted, the sample solution and the ultrapure water are scanned at the interval of 380nm-780nm by 10nm, and the absorbance of the sample solution in the range is measured. And drawing a line graph.
As can be seen from the combination of FIG. 15, the method developed by the present invention is well applicable to the polypeptide products on the market, and the results show that the concentration difference of the sample can have a great influence on the absorbance or color of the polypeptide solution. Therefore, when the polypeptide solution decolorization effect is compared, the sample concentration should be controlled as close as possible, that is, the sample is dissolved in deionized water or ultra-pure water at the same concentration ratio required by the method of the present invention, so as to better compare the decolorization effect.
The above-described embodiments should not be construed as limiting the scope of the invention, and any alternative modifications or alterations to the embodiments of the present invention will be apparent to those skilled in the art.
The present invention is not described in detail, but is known to those skilled in the art.
Claims (6)
1. A method for measuring the decoloring effect of a protein polypeptide solution is characterized by comprising the following operation steps:
(1) dissolving the polypeptide dry sample before and after decolorization in ultrapure water or deionized water according to the same concentration ratio to obtain a sample solution, standing at room temperature for a certain time, scanning the sample solution and the ultrapure water at an interval of 1-10nm between 380nm and 780nm by using an enzyme-linked immunosorbent assay (ELIAS) or ultraviolet spectrophotometer, and measuring the absorbance of the sample solution and the ultrapure water in the range;
(2) making a wavelength-absorbance line graph;
(3) determining the decoloring effect according to the change trend of the line graph or calculating the decoloring rate and the change of qualitative or quantitative indexes:
a qualitative index of the decoloring effect: when the broken line trend of the sample solution is closer to the broken line of the ultrapure water, the decoloration effect is more thorough, and when the broken line trend of the sample solution is more inclined to the upper part, the decoloration effect is poorer;
and b, calculating the decolorization rate according to the line graph, and determining the decolorization effect according to the numerical value of the decolorization rate.
2. The method for measuring the decoloring effect of a protein polypeptide solution according to claim 1,
the decolorization ratio is calculated by adopting the following formula:
wherein, taking scanning at intervals of 10nm as an example:
s1 is the area enclosed by the broken line of the sample solution before decolorization and the abscissa axis, x is 380nm and x is 780 nm;
s2 is the area enclosed by the broken line of the sample solution after decolorization and the abscissa axis, x is 380nm and x is 780 nm;
s3 is the area enclosed by ultrapure water or deionized water and the abscissa axis, x is 380nm and x is 780 nm;
in the above formula, 10 represents 10nm spaced in the spectrum from 380nm to 780nm, i.e., viewed as the height of the trapezoid in the composition area.
3. The method according to claim 1, further comprising a step of drying the polypeptide solution before and after the decolorization treatment in the same drying manner to obtain a dried polypeptide sample before step (1).
4. The method for measuring the decoloring effect of a protein polypeptide solution according to claim 1, wherein the sample solution and the ultrapure water of the step (1) are scanned at an interval of 10nm in a range of 380nm to 780 nm; and (3) standing at room temperature for 30min in the step (2).
5. The method for measuring the decoloring effect of a protein polypeptide solution according to claim 1, wherein in the step (1), the concentration ratio is 5% to 20%.
6. The method for measuring the decoloring effect of a protein polypeptide solution according to claim 5, wherein the same concentration ratio is 10% to 20%.
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