CN114062620A - Evaluation method for stable storage of milk hypnotize peptides - Google Patents

Evaluation method for stable storage of milk hypnotize peptides Download PDF

Info

Publication number
CN114062620A
CN114062620A CN202111299332.0A CN202111299332A CN114062620A CN 114062620 A CN114062620 A CN 114062620A CN 202111299332 A CN202111299332 A CN 202111299332A CN 114062620 A CN114062620 A CN 114062620A
Authority
CN
China
Prior art keywords
hypnotize
milk
peptide
sample
cow milk
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111299332.0A
Other languages
Chinese (zh)
Inventor
曹庸
韦颖欣
李俊
刘星雨
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
South China Agricultural University
Original Assignee
South China Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by South China Agricultural University filed Critical South China Agricultural University
Priority to CN202111299332.0A priority Critical patent/CN114062620A/en
Publication of CN114062620A publication Critical patent/CN114062620A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/04—Dairy products
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/04—Preparation or injection of sample to be analysed
    • G01N30/06—Preparation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/30—Control of physical parameters of the fluid carrier of temperature
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/62—Detectors specially adapted therefor
    • G01N30/74—Optical detectors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N5/00—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid
    • G01N5/04—Analysing materials by weighing, e.g. weighing small particles separated from a gas or liquid by removing a component, e.g. by evaporation, and weighing the remainder
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/26—Conditioning of the fluid carrier; Flow patterns
    • G01N30/28—Control of physical parameters of the fluid carrier
    • G01N30/32—Control of physical parameters of the fluid carrier of pressure or speed
    • G01N2030/324—Control of physical parameters of the fluid carrier of pressure or speed speed, flow rate

Landscapes

  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Medicinal Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention belongs to the technical field of milk source active ingredient extraction, and particularly relates to an evaluation method for stable storage of milk hypnotize peptides, which comprises the following steps: (1) measuring the ash content of the cow milk hypnotize peptides under different storage conditions; (2) measuring the drying weight loss amount of the milk hypnotize peptides under different storage conditions; (3) measuring the pH value of the cow milk hypnotize peptides under different storage conditions; (4) and (3) measuring the content of the cow milk hypnotize dormancy peptide under different storage conditions. The optimal storage condition of the cow milk hypnotic peptide is evaluated through a series of tests, and guidance is provided for the storage mode of the cow milk hypnotic peptide.

Description

Evaluation method for stable storage of milk hypnotize peptides
Technical Field
The invention belongs to the technical field of milk source active ingredient extraction, and particularly relates to an evaluation method for stable storage of milk hypnotize peptides.
Background
Sleep is an extremely important ring in our lives, and calculated according to the total length of life, more than one third of time is allocated to sleep, so that the health of a human body has a direct relation with the quality of sleep. The sleep is also a global focus of attention in the field of health, a great deal of research considers that the occurrence or development of a plurality of chronic diseases is closely related to the sleep, sleep disorders such as insufficient sleep time and insomnia can improve the occurrence probability of various chronic diseases such as diabetes, hypertension, obesity, cardiovascular and cerebrovascular diseases and the like, the sleep time is related to the occurrence rate of breast cancer, colorectal cancer and the like, and the insomnia problem exists in a large proportion of patients such as lung cancer, breast cancer, head and neck tumors and the like according to statistics. The insomnia is a common sleep disorder, and refers to the feeling that the body feels unpleasant to the sleeping duration and the sleeping quality and influences the links of learning, working, living and the like in the daytime under proper opportunities and environments, and can be classified into primary and secondary. If the body is in a state of insufficient sleep and poor quality for a long time, adverse results such as memory decline, slow response and the like can be caused, furthermore, people have depressed emotions and mania, physical and mental diseases can be caused, even death can be caused in serious cases, and other social security accidents can be caused.
In daily life, people often choose to drink milk before sleeping in order to help sleep. Cow milk casein is a precursor of a plurality of potential bioactive substances, and small peptides with multiple biological functions, such as cow milk sleep-promoting peptides with sleep-promoting efficacy, can be obtained by adopting protease enzymolysis. The cow milk hypnotize dormancy peptide not only has strong functional activity, low price and good safety, but also is easy to carry out industrialized production. However, the active peptide undergoes quality change during storage due to various external factors such as temperature, time, etc., resulting in a decrease in the content of active ingredients, a decrease in activity, or a decrease in efficacy.
Disclosure of Invention
In order to solve the problem of stable storage of the milk hypnotizing peptide, the invention provides an evaluation method, the stored milk hypnotizing peptide is not obviously changed after the ash content, the pH value and the peptide content of different nodes are measured, the milk hypnotizing peptide has good stability, and the optimal storage condition is proper-temperature sealing and drying.
In order to achieve the purpose, the invention is realized by the following technical scheme:
an evaluation method for stable storage of milk hypnotize peptides comprises the following steps:
(1) determination of ash content of cow milk hypnotize dormancy peptide under different storage conditions
Figure 17790DEST_PATH_IMAGE001
The cow milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d are repeatedly tested in groups, each group: weighing the crucible as m1, adding a proper amount of cow milk hypnotize dormancy peptide sample into the crucible, and weighing the crucible as m 2;
Figure 582764DEST_PATH_IMAGE002
adding water into the cow milk hypnotize peptide sample in the crucible for dispersion, heating to fully carbonize the sample to be smokeless, then placing the sample in a muffle furnace, firing at 525-;
Figure 890773DEST_PATH_IMAGE003
respectively calculating the ash content of the sample under the condition of single storage time and storage temperature, and further comparing the content change under each time and temperature condition;
the ash content of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 755961DEST_PATH_IMAGE004
。
(2) determination of cow milk hypnotize peptide drying weight loss under different storage conditions
Figure 363660DEST_PATH_IMAGE001
Dividing milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d into a plurality of groups for repeated tests, wherein each group comprises: weighing the culture dish as m1 (with a cover), putting a proper amount of cow milk hypnotize dormancy peptide sample into the culture dish, covering, shaking on the side and spreading, then weighing again as m 2.
Figure 806142DEST_PATH_IMAGE002
And putting the culture dish filled with the cow milk hypnotizing peptide sample into an oven and a dryer with the temperature of 101-.
Figure 524700DEST_PATH_IMAGE003
And respectively calculating the drying weight loss under the condition of single storage time and storage temperature, and comparing the drying weight loss change under each time and temperature condition.
The weight loss on drying of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 244394DEST_PATH_IMAGE004
。
(3) determination of pH value of cow milk hypnotize dormancy peptide under different storage conditions
The cow milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d are repeatedly prepared into a solution of 20mg/mL by ultrapure water in groups, the pH value is measured, recorded and the influence of conditions on the pH value is evaluated.
(4) Determination of cow milk hypnotize peptide content under different storage conditions
Figure 413207DEST_PATH_IMAGE001
Establishing a cow milk hypnotizing peptide liquid chromatography method: preparing a standard stock solution (with the concentration of 200 mug/mL) by using a standard substance, measuring the standard stock solution, adjusting the elution gradient, and performing high performance liquid chromatography according to set liquid phase conditions, wherein the chromatographic conditions are as follows: welch C18 column, mobile phase: ultrapure water pumped by a pump, acetonitrile pumped by a pump B, the flow rate of 1 mL/min, the sample injection amount of 20 mu L, the column temperature of 28 ℃, the detection wavelengths of 214 nm and 280 nm, and the elution conditions (acetonitrile concentration): 10% -20% (0.01-10 min), 20% -30% (10-40 min), 30% -90% (40-48 min), 90% (48-53 min); and preparing standard mixed solutions with different concentrations by using the standard stock solutions to perform linear equation, linear range and quantitative limit tests.
Figure 218352DEST_PATH_IMAGE002
And measuring the content of each monomer of the milk hypnotize sleeping peptide by high performance liquid chromatography
Diluting milk hypnotize peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90 days to proper concentration according to water for pre-test, and adjusting the chromatographic conditions after elution gradient: welch C18 column, mobile phase: ultrapure water pumped by a pump, acetonitrile pumped by a pump B, the flow rate of 1 mL/min, the sample injection amount of 20 mu L, the column temperature of 28 ℃, the detection wavelengths of 214 nm and 280 nm, and the elution conditions (acetonitrile concentration): 10-20% (0.01-10 min), 20-30% (10-40 min) and 30-90% (40-48 min).
The invention has the following positive and beneficial effects:
according to the invention, the optimal storage condition of the cow milk hypnotic peptide is evaluated through a series of tests, the physicochemical index of the cow milk hypnotic peptide and the content of active peptide are not obviously changed, and guidance is provided for the storage mode of the cow milk hypnotic peptide.
Drawings
FIG. 1 is a graph of the ash content of cow milk for promoting sleep at different storage times;
FIG. 2 shows the loss on drying of milk hypnotize peptides at different storage times;
FIG. 3 is the pH values of cow milk hypnotize peptides at different storage times;
FIG. 4 is a liquid chromatogram of a bovine milk sleep-promoting peptide standard;
FIG. 5 is a liquid chromatogram of a milk hypnotize sleep peptide sample;
FIG. 6 is a liquid phase chromatogram of cow milk hypnotize peptide stored at 4 deg.C;
FIG. 7 is a liquid phase map of cow milk hypnotize peptide stored at 25 deg.C;
FIG. 8 shows the content changes of cow milk hypnotize dormancy peptide monomers 1 and 2 under different storage time;
FIG. 9 shows the changes of cow milk hypnotize dormancy peptide monomer 3 content under different storage time;
FIG. 10 shows the change of cow milk hypnotize dormancy peptide monomer 4 content under different storage time;
figure 114 ℃ milk hypnotize peptide monomer content change;
FIG. 1225 deg.C milk hypnotize dormancy peptide monomer content change.
Detailed Description
In order to better understand the present invention, the following embodiments are provided to illustrate the technical solution of the present invention. The drugs and reagents used in the following examples are commercially available, the bovine casein hydrolysate is a bovine milk polypeptide solution obtained by hydrolyzing bovine casein with protease, and is obtained from Guangzhou green extract biotechnology limited, and the milk source is bovine milk.
Example 1
The steady-state storage method of the cow milk hypnotize dormancy peptide is evaluated by the following steps:
(1) preparation of cow milk hypnotize peptide sample
Preparing the cow milk casein zymolyte into an aqueous solution with the content of 20wt%, adding a proper volume of hydrochloric acid to adjust the pH value of the aqueous solution to 4, standing for 10 min, centrifuging for 10 min at the rotating speed of 4000 rpm/min to respectively obtain a supernatant and a precipitate, and removing the precipitate to obtain the supernatant for later use. Adding 80% ethanol water solution into the supernatant, precipitating with ethanol for more than 5 h, centrifuging at 4000 rpm/min for 10 min to obtain supernatant and precipitate, respectively, and removing the precipitate to obtain supernatant. Concentrating the supernatant at 60-65 deg.C under reduced pressure until the solid matter is about 30wt%, spray drying the solid matter by spray drying technology with air inlet temperature of 180 deg.C, air outlet temperature of 90 deg.C, rotation speed of 12000 rpm/min, and feed speed of 3L/h, drying and concentrating to obtain cow milk hypnotize dormancy peptide for stability evaluation.
(2) Determination of ash content of cow milk hypnotize dormancy peptide under different storage time
Figure 6180DEST_PATH_IMAGE001
Taking 6 porcelain crucibles with proper sizes, numbering No. 1-6, cleaning, placing in a muffle furnace, burning at 550 +/-25 ℃ for 0.5 h, cooling to below 200 ℃, taking out, placing in a dryer for cooling to room temperature, precisely weighing, and burning repeatedly until the difference between the two weights is not more than 0.5 mg before and after the burning is constant weight, which is marked as m 1.
Figure 439435DEST_PATH_IMAGE002
And dividing the cow milk hypnotizing peptide samples stored for 0, 7, 15, 30, 60 and 90 days at 4 ℃ and 25 ℃ respectively into 3 groups for repeated tests, adding 2-3 g of cow milk hypnotizing peptide samples into each small crucible, precisely weighing, and recording as m 2.
Figure 654516DEST_PATH_IMAGE003
Adding a small amount of water into the milk hypnotize peptide sample to scatter (the liquid is not more than one third of the crucible), then placing the milk hypnotize peptide sample on an electric hot plate, heating the milk hypnotize peptide sample by small fire to fully carbonize the sample to be smokeless, then placing the milk hypnotize peptide sample in a muffle furnace, burning the milk hypnotize peptide sample at the temperature of (550 +/-25) DEG C for 4 hours, closing the muffle furnace, cooling the milk hypnotize peptide sample to 200 ℃, taking out the milk hypnotize peptide sample, placing the milk hypnotize peptide sample in a dryer to cool the milk hypnotize peptide sample to room temperature, and weighing the milk hypnotize peptide sample. Repeating the burning till the weight difference between the two times is not more than 0.5 mg, and recording as m 3. The weighing results are reported in table 1.
TABLE 1 ash content determination table under single storage time condition
Figure 681377DEST_PATH_IMAGE005
。
Figure 272896DEST_PATH_IMAGE006
And calculating the ash content of the sample under the condition of single storage time and storage temperature, and comparing the content change under each time and temperature condition, wherein the content change is shown in figure 1.
The ash content of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 560658DEST_PATH_IMAGE007
in the formula:
x-the content of ash in the sample%
m 1-crucible Mass, g
m 2-Mass of crucible and sample, g
m3 — mass of crucible and ash, g.
As can be seen from figure 1, the results of the ash content of cow milk hypnotize peptide stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90 days do not show obvious increase or decrease trend. The cow milk hypnotize dormancy peptide is stored for different time under different temperature conditions, the ash content change is not obvious, and the ash content is stable.
(3) Determination of cow milk hypnotize peptide drying weight loss amount under different storage time
Figure 946640DEST_PATH_IMAGE001
Preparing 6 culture dishes, numbering 1-6 respectively, cleaning, obliquely supporting the culture dish cover at the side of the bottle, drying for 1 h in a (101-.
Figure 726377DEST_PATH_IMAGE002
Dividing the cow milk hypnotizing peptide samples stored for 0, 7, 15, 30, 60 and 90 days at 4 ℃ and 25 ℃ respectively into 3 groups to perform repeated tests, putting about 2 g of cow milk hypnotizing peptide samples into a culture dish, covering the culture dish, shaking the culture dish on the side, laying the culture dish on the side, and weighing the culture dish as m 2.
Figure 980641DEST_PATH_IMAGE003
Placing the culture dish filled with the cow milk hypnotizing peptide sample into a (101-. The weighing results are reported in table 2.
TABLE 2 recording chart of the loss on drying measurement experiment under single storage time condition
Figure 263854DEST_PATH_IMAGE008
。
Figure 820738DEST_PATH_IMAGE006
And calculating the drying weight loss under the condition of single storage time and storage temperature, and comparing the change of the drying weight loss under each time and temperature condition, which is shown in figure 2.
The weight loss on drying of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 946826DEST_PATH_IMAGE004
in the formula:
x-loss on drying of the sample%
m 1-quality of the dish, g
m 2-Mass of Petri dish and sample, g
m 3-mass of the dish and dried sample, g.
As can be seen from fig. 2, the cow milk hypnotic peptides are stored at 4 ℃ and 25 ℃ respectively, when the storage time is 30 days, the drying weight loss is obviously increased, the drying weight loss is kept stable during the storage for 30-90 days without obvious increase, and compared with cow milk hypnotic peptides stored at 4 ℃ for 0-90 days, the drying weight loss of cow milk hypnotic peptides is slightly lower than that of cow milk hypnotic peptides stored at 25 ℃. According to the description, the cow milk hypnotizing peptide has the condition of water absorption and moisture absorption during the storage period, the cow milk hypnotizing peptide reaches a saturated state after 30 days, and the cow milk hypnotizing peptide is stored under the condition of drying and sealing.
(4) Determination of pH value of cow milk hypnotize dormancy peptide under different storage time
Preparing 3 groups of cow milk hypnotize peptide samples stored at 4 deg.C and 25 deg.C for 0, 7, 15, 30, 60, and 90d with ultrapure water into 20mg/mL solution, measuring pH of the sample with a precision pH meter, recording, and averaging to obtain figure 3.
According to the figure 3, during the storage time of 0-90 d, the pH value of the cow milk hypnotizing peptide is stable at 4 ℃ and 25 ℃, the pH value is stable between 5.00-5.50, the cow milk hypnotizing peptide is weakly acidic, and the number of acidic amino acids in the cow milk hypnotizing peptide molecule is more than that of basic amino acids due to the reason that the cow milk hypnotizing peptide or the cow milk hypnotizing peptide protein has obvious acidity (Martinez et al, 2006). Comparing that the pH of the cow milk hypnotic peptide stored at 4 ℃ and 25 ℃ is close, the pH value difference change degree of the cow milk hypnotic peptide at two storage temperatures in different time is slight, the storage temperature has little influence on the pH value of the cow milk hypnotic peptide, and the pH value is kept stable within 90 days of storage.
(5) Determination of cow milk hypnotize peptide content under different storage time
Figure 614567DEST_PATH_IMAGE001
The establishment of the cow milk hypnotizing peptide liquid phase method comprises the following steps: accurately weighing 1 mg of a proper amount of standard substance (the purity is more than 98 percent, and the peak sample connected for preparing the liquid phase) and ultrapure water to prepare a single standard stock solution with the concentration of 1 mg/mL, transferring 1 mL of the single standard stock solution, and using the ultrapure water to fix the volume to 5 mL to prepare a mixed standard stock solution with the concentration of 200 mu g/mL. The elution gradient was adjusted by measuring the standard stock solution, and high performance liquid chromatography analysis was performed under the set liquid phase conditions, the chromatographic conditions being shown in table 3.
TABLE 3 high Performance liquid chromatography conditions
Figure 17867DEST_PATH_IMAGE009
。
Sample solutions were prepared in the manner of table 4, and standard mixed solutions of different concentrations were prepared for linear equation, linear range, and quantitative limit tests.
TABLE 4 Linear test sample preparation
Figure 11231DEST_PATH_IMAGE010
。
And (3) optimizing the liquid chromatogram after the liquid phase analysis condition is optimized to obtain standard peaks of the milk hypnotize peptides, wherein the standard peaks are mainly distributed in 25-40 min, and the sum of the peak areas of the section is used as the quantitative detection of the milk hypnotize peptide content. The liquid chromatogram of the cow milk sleep promoting peptide standard product is shown in 4:
respectively detecting components of 4 monomers obtained by preparing a liquid phase, wherein the position of a peak 1 is closer to that of a peak 2, the monomers 1 and 2 are combined into a sample to be detected, the monomers 3 and 4 are independently calculated, the measured peak area is represented by (Y), the solution mass concentration is represented by (X), linear regression calculation is carried out to obtain a curve and a total content curve, the curve and the total content curve are shown in a table 5, the detection limit is calculated by using the concentration corresponding to 3 times of signal to noise ratio and is 6 mu g/kg, and the quantification limit of the method is calculated by using the concentration corresponding to 10 times of signal to noise ratio and is 20 mu g/kg.
As can be seen from the standard curves of the standard samples with different concentrations of each monomer in the cow milk hypnotizing peptide samples in the table 5, the linear relation of the standard curve of the cow milk hypnotizing peptide samples in the mass concentration range of 20-1000 mug/kg is higher than 0.99, the linear relation is good, the concentration range is proper, and the method can be used for measuring the content of the cow milk hypnotizing peptide samples under the experimental conditions.
TABLE 5 cow milk hypnotize peptide standard sample linear equation
Figure 359035DEST_PATH_IMAGE011
。
As can be seen from fig. 5, the crude milk hypnotizing peptide extract contains more polar and non-polar impurities, which affect the purity of hypnotizing peptide, the pH value is adjusted to 4, the supernatant obtained by centrifugation is precipitated by ethanol with the concentration of 80% for more than 5 h, the supernatant obtained by standing and centrifugation is subjected to reduced pressure concentration and spray drying to obtain a purified milk hypnotizing peptide sample, which is used as a sample for stability evaluation.
Figure 96047DEST_PATH_IMAGE002
And measuring the content of each monomer of the milk hypnotize sleeping peptide by high performance liquid chromatography
Respectively storing cow milk hypnotize peptide samples at 4 deg.C and 25 deg.C for 0, 7, 15, 30, 60, and 90 days, respectively, treating the solutions after pH value measurement with 0.22 μm filter membrane of pinhole filter, and analyzing with high performance liquid chromatograph. According to the preliminary test, the cow milk hypnotize dormancy peptide sample solution is diluted to a proper concentration before the sample loading, and the chromatographic conditions after the elution gradient is adjusted are shown in table 6.
TABLE 6 high Performance liquid chromatography conditions
Figure 353853DEST_PATH_IMAGE012
。
And (3) measuring the peptide content of 4 monomers of the hypnotized dormancy peptide stored at 4 ℃ and 25 ℃ for 90d, combining the monomer 1 and the monomer 2 into an active peptide section for measurement due to the approach of peak positions, and independently calculating the monomer 3 and the monomer 4. Measuring peptide content by high performance liquid chromatography, and respectively storing at 4 deg.C and 25 deg.C in liquid phase chromatogram of hypnotizing peptide from top to bottom for 0, 7, 15, 30, 60, and 90 days. The standard peak of the hypnotizing peptide is mainly distributed between 25-40 min, the monomer peak area in the period is collected and calculated to compare the content of each monomer peptide, and the peptide content is expressed by the proportion of each monomer in the total active ingredients.
FIGS. 8-10 show the change of the peptide content of 4 monomers of bovine milk sleeping peptide stored at 4 ℃ and 25 ℃ for 90 days, respectively. Comparing the proportion of each monomer in the total active ingredients of the milk hypnotize peptides stored at 4 ℃ and 25 ℃ within 90 days of storage time, the proportion of the monomers 1 and 2, the monomers 3 and the monomers 4 is not obviously changed at the two storage temperatures, and therefore, the content of the peptides of the monomers 1 and 2, the monomers 3 and the monomers 4 is not obviously changed and is basically kept stable when the monomers are stored at 4 ℃ and 25 ℃ for 90 days.
FIGS. 11-12 show the change of 90d content of each monomer of milk hypnotize peptide stored at 4 deg.C and 25 deg.C, expressed by the proportion of each monomer in total active ingredients of milk hypnotize peptide. Storage at 4 ℃ for 90 days gives the highest monomer 4 content, followed by monomers 1 and 2 and the least monomer 3. The highest monomer 4 content was followed by monomers 1 and 2 and the least monomer 3 during storage at 25 ℃ for 90 days. In summary, the monomer 4 content ratio is highest at storage temperatures of either 4 ℃ or 25 ℃, and each monomer content ratio ranges from high to low as monomer 4> monomer 1 and 2> monomer 3.
According to the experimental conditions, the content of the monomers 1 and 2, the monomer 3 and the monomer 4 is not obviously changed during the storage at 4 ℃ and 25 ℃ for 90 days, the cow milk hypnotize dormancy peptide has no special requirements on the storage temperature, and the cow milk hypnotize dormancy peptide keeps good stability during the storage time of 90 days.

Claims (7)

1. An evaluation method for stable storage of milk hypnotize peptides is characterized by comprising the following steps:
(1) measuring the ash content of the cow milk hypnotize peptides under different storage conditions;
(2) measuring the drying weight loss amount of the milk hypnotize peptides under different storage conditions;
(3) measuring the pH value of the cow milk hypnotize peptides under different storage conditions;
(4) and (3) measuring the content of the cow milk hypnotize dormancy peptide under different storage conditions.
2. The evaluation method according to claim 1, characterized in that: the step (1) specifically comprises the following operations:
Figure 51514DEST_PATH_IMAGE001
the cow milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d are repeatedly tested in groups, each group: weighing the crucible as m1, adding a proper amount of cow milk hypnotize dormancy peptide sample into the crucible, and weighing the crucible as m 2;
Figure 43741DEST_PATH_IMAGE002
adding water into the cow milk hypnotize peptide sample in the crucible for dispersion, heating to fully carbonize the sample to be smokeless, then placing the sample in a muffle furnace, firing at 525-;
Figure 739165DEST_PATH_IMAGE003
respectively calculating the ash content of the sample under the condition of single storage time and storage temperature, and further comparing the content change under each time and temperature condition;
the ash content of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 574266DEST_PATH_IMAGE005
。
3. the evaluation method according to claim 1, characterized in that: the step (2) specifically comprises the following operations:
Figure 52651DEST_PATH_IMAGE001
dividing milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d into a plurality of groups for repeated tests, wherein each group comprises: weighing the culture dish with a cover as m1, putting a proper amount of cow milk hypnotize dormancy peptide sample into the culture dish, covering, shaking on the side, spreading, and weighing again as m 2;
Figure 289598DEST_PATH_IMAGE002
placing the culture dish filled with the cow milk hypnotizing peptide sample into a drying oven and a dryer at 105 ℃ and at constant weight, and marking as m 3;
Figure 890343DEST_PATH_IMAGE003
respectively calculate the single storage timeThe drying weight loss under the condition of intermediate storage temperature is compared with the drying weight loss change under the condition of each time temperature;
the weight loss on drying of the sample under the condition of single storage time and storage temperature is calculated according to the formula:
Figure 88106DEST_PATH_IMAGE005
。
4. the evaluation method according to claim 1, characterized in that: the step (3) specifically comprises the following operations:
the cow milk hypnotize dormancy peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90d are repeatedly prepared into a solution of 20mg/mL by ultrapure water in groups, the pH value is measured, recorded and the influence of conditions on the pH value is evaluated.
5. The evaluation method according to claim 1, characterized in that: the step (4) specifically comprises the following operations:
Figure 763326DEST_PATH_IMAGE001
establishing a cow milk hypnotizing peptide liquid chromatography method: preparing a standard stock solution (with the concentration of 200 mug/mL) by using a standard substance, measuring the standard stock solution, adjusting the elution gradient, and performing high performance liquid chromatography according to set liquid phase conditions, wherein the chromatographic conditions are as follows: welch C18 column, mobile phase: ultrapure water pumped by a pump, acetonitrile pumped by a pump B, the flow rate of 1 mL/min, the sample injection amount of 20 mu L, the column temperature of 28 ℃, the detection wavelengths of 214 nm and 280 nm, and the elution conditions (acetonitrile concentration): 10% -20% (0.01-10 min), 20% -30% (10-40 min), 30% -90% (40-48 min), 90% (48-53 min); preparing standard mixed solutions with different concentrations by using the standard stock solutions to perform linear equation, linear range and quantitative limit tests;
Figure 464566DEST_PATH_IMAGE002
high performance liquid chromatographyContent of each monomer of milk calmette
Diluting milk hypnotize peptide samples stored at 4 ℃ and 25 ℃ for 0, 7, 15, 30, 60 and 90 days to proper concentration according to water for pre-test, and adjusting the chromatographic conditions after elution gradient: welch C18 column, mobile phase: ultrapure water pumped by a pump, acetonitrile pumped by a pump B, the flow rate of 1 mL/min, the sample injection amount of 20 mu L, the column temperature of 28 ℃, the detection wavelengths of 214 nm and 280 nm, and the elution conditions (acetonitrile concentration): 10-20% (0.01-10 min), 20-30% (10-40 min) and 30-90% (40-48 min).
6. The evaluation method according to claim 5, characterized in that: the standard peaks of the milk hypnotize peptides obtained by the liquid chromatogram after the liquid phase analysis are mainly distributed between 25-40 min, and the sum of the peak areas of the section is used as the quantitative detection of the milk hypnotize peptide content; respectively detecting components of 4 monomers obtained by preparing a liquid phase according to a map, wherein the position of a peak 1 is closer to that of a peak 2, the monomers 1 and 2 are combined into a sample to be detected, the monomer 3 and the monomer 4 are independently calculated, the measured peak area is represented by Y, the solution mass concentration is represented by X, linear regression calculation is carried out to obtain a curve and a total content curve, the detection limit is calculated by concentration corresponding to 3 times of signal to noise ratio and is 6 mug/kg, and the quantification limit of the method is calculated by concentration corresponding to 10 times of signal to noise ratio and is 20 mug/kg;
Figure 501792DEST_PATH_IMAGE006
。
7. the evaluation method according to claim 5, characterized in that: the preliminary test in the step (4) is as follows: preparing samples with different concentrations, carrying out liquid phase analysis, observing the display condition of a liquid phase diagram, and finally determining the appropriate concentration, namely the sample concentration when the liquid phase diagram is most regular.
CN202111299332.0A 2021-11-04 2021-11-04 Evaluation method for stable storage of milk hypnotize peptides Pending CN114062620A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111299332.0A CN114062620A (en) 2021-11-04 2021-11-04 Evaluation method for stable storage of milk hypnotize peptides

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202111299332.0A CN114062620A (en) 2021-11-04 2021-11-04 Evaluation method for stable storage of milk hypnotize peptides

Publications (1)

Publication Number Publication Date
CN114062620A true CN114062620A (en) 2022-02-18

Family

ID=80273723

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202111299332.0A Pending CN114062620A (en) 2021-11-04 2021-11-04 Evaluation method for stable storage of milk hypnotize peptides

Country Status (1)

Country Link
CN (1) CN114062620A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180368395A1 (en) * 2016-01-29 2018-12-27 Kaneka Corporation Peptide-containing composition and stabilizer, stabilizing method, and storage method for peptide
CN110024901A (en) * 2019-04-30 2019-07-19 集美大学 A kind of collagen peptide and its production method and process units and application
CN110596367A (en) * 2019-09-06 2019-12-20 广州科方生物技术股份有限公司 Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof
CN110595933A (en) * 2019-09-02 2019-12-20 大连工业大学 Method for rapidly detecting storage quality of aseptically packaged sea cucumber protein peptide powder based on DVS and GC-IMS technology
CN112409470A (en) * 2020-11-24 2021-02-26 中食都庆（山东）生物技术有限公司 Casein peptide with sleep improvement effect and preparation method and application thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20180368395A1 (en) * 2016-01-29 2018-12-27 Kaneka Corporation Peptide-containing composition and stabilizer, stabilizing method, and storage method for peptide
CN110024901A (en) * 2019-04-30 2019-07-19 集美大学 A kind of collagen peptide and its production method and process units and application
CN110595933A (en) * 2019-09-02 2019-12-20 大连工业大学 Method for rapidly detecting storage quality of aseptically packaged sea cucumber protein peptide powder based on DVS and GC-IMS technology
CN110596367A (en) * 2019-09-06 2019-12-20 广州科方生物技术股份有限公司 Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof
CN112409470A (en) * 2020-11-24 2021-02-26 中食都庆（山东）生物技术有限公司 Casein peptide with sleep improvement effect and preparation method and application thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
王允华 等: "谷氨酰胺结合肽组成及贮藏稳定性研究", 《粮食与油脂》 *
蔡木易: "食源性肽研究进展", 《北京工商大学学报》 *
陈媛媛: "牛乳源促睡眠肽的纯化鉴定、活性评价、机制及应用研究，", 《中国优秀硕士学位论文电子期刊》 *

Similar Documents

Publication Publication Date Title
Dodero et al. Biomolecular studies by circular dichroism
US20210109068A1 (en) Fingerprint detection method for antidepressant drug
CN107796892B (en) Fingerprint of Gentiana macrophylla capsule and its application in quality control and component analysis
CN108982700A (en) Research method of the PQQ to the inhibiting effect of AGEs
CN109633010A (en) The kit and method of glycosylated hemoglobin in a kind of measurement whole blood
Zou et al. PM2. 5 induces airway remodeling in chronic obstructive pulmonary diseases via the Wnt5a/β-catenin pathway
CN114062620A (en) Evaluation method for stable storage of milk hypnotize peptides
CN102590431B (en) Quality standard detection method for Chinese medicinal composition for treating cough
CN102269749B (en) Method for detecting adenosine content in hericium erinaceus tablet
CN106987566B (en) Type III polyketide synthase and its encoding gene and purposes
CN109232768A (en) One kind having the active carboxymethylpachymaran of inflammatory enteropathy and its preparation and application
CN114563496B (en) Quantitative fingerprint analysis method for components in ginger, ginger and pinellia tuber percolate
CN113447596B (en) Method for measuring 3 active ingredients in pharmaceutical preparation
CN101435812A (en) Puyuanhewei capsule quality standard detecting method
CN104155399A (en) Quality control method of four-time treated rhizoma cyperi decoction pieces
CN105506131A (en) Primer pair for detecting people AEG-1 gene expression quantity and relative expression quantity
CN111239284A (en) A quality analysis method of radix Isatidis or its product
CN113671066B (en) Quality control method of radix rosae multiflorae medicinal material
Qiao et al. Ephrin B3 exacerbates colitis and colitis-associated colorectal cancer
CN110609110A (en) Method for measuring content of tissue repair factor PA1 in periplaneta americana and related products thereof
CN108484694A (en) Natural products isostrictiniin and its preparation method and application
CN109061028A (en) Chinese medicine chastity lotus increases and exempts from oral solution quality determining method
CN108129550A (en) A kind of crystal form of reduced glutathione and preparation method thereof
CN116068096B (en) Specific marker related to stiff silkworm section silk gland ring, screening method and stiff silkworm quality detection method
CN113391003B (en) Method for simultaneously detecting content of active ingredients in pulse-activating decoction

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20220218