CN109112180A - Number of freezing and thawing measuring method during a kind of freezer storage - Google Patents
Number of freezing and thawing measuring method during a kind of freezer storage Download PDFInfo
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- CN109112180A CN109112180A CN201811050434.7A CN201811050434A CN109112180A CN 109112180 A CN109112180 A CN 109112180A CN 201811050434 A CN201811050434 A CN 201811050434A CN 109112180 A CN109112180 A CN 109112180A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96472—Aspartic endopeptidases (3.4.23)
- G01N2333/96475—Aspartic endopeptidases (3.4.23) with definite EC number
- G01N2333/9648—Chymosin, i.e. rennin (3.4.23.4)
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- Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Freezing, Cooling And Drying Of Foods (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Sampling And Sample Adjustment (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of discrimination methods of frozen product number of freezing and thawing, and in particular to number of freezing and thawing measuring method during a kind of freezer storage.The determination method of number of freezing and thawing: the enzyme activity or inactivation activation energy of rennin solns are measured;Step (1) rennin solns sealing is placed in frozen product to be measured and frozen product is together after freeze thawing, the enzyme activity or inactivation activation energy of the enzyme is measured again, the number of freeze thawing is judged by enzyme activity twice or the difference for inactivating activation energy.The present invention assesses number of freezing and thawing using indirect method, by changing with refrigerated products enzyme activity or thermal stability of the external standard enzyme preparation during multigelation of putting together, determines refrigerated products number of freezing and thawing.As a result more accurate, universality is strong, and application range is more extensive.
Description
Technical field
The present invention relates to a kind of discrimination methods of frozen product number of freezing and thawing, and in particular to freeze thawing time during a kind of freezer storage
Number measuring method.
Background technique
Low temperature can suppress growth of microorganism breeding, reduce enzymatic activity, Shelf-life.Therefore make all kinds of meat products, aquatic products
Product, and the most convenient to the storage of freshness demanding agricultural product, one of most efficient method.Refrigerating process can generate size
Different ice crystal causes mechanical damage to membrane structure and institutional framework, and causes albuminous degeneration and fat oxidation.It thawed
Journey also results in meat tenderness decline, nutrient loss, soluble protein content reduction, gel formation ability decline etc..
Since (low temperature, high when transport when as stored for cold chain unsound in transport, storage and process of consumption for refrigerated products
Temperature and the control of sales section temperature are improper), cause product to encounter biggish temperature fluctuation, the anti-of freeze-thaw occurs repeatedly
Multiple frozen-thaw process influences the product quality of frozen product very big.Need a kind of method that can indicate frozen product multigelation number.Mesh
Before, using direct method, by meat measurement, particularly preferred method can identify the meat that multigelation is crossed not yet.Document 3(base
In the Fresh Grade Breast number of freezing and thawing discrimination method of impedance operator and neural network) have studied fresh grade breast and different number of freezing and thawing
The quality of Fresh Grade Breast and the amplitude of impedance and phase angle variations situation, analytic process is considerably complicated, and only demonstrates jelly
The case where melting the Fresh Grade Breast within 3 times.To the identification effect of the number of freezing and thawing of other frozen products, the frozen product multiple to freeze thawing
Fruit does not know simultaneously.This method is indirect method, using milk-coagulating enzyme preparation as external standard product, can accurately identify freeze thawing time
Number.
Summary of the invention
The present invention assesses number of freezing and thawing using indirect method, to exist with refrigerated products external standard enzyme preparation of putting together
The linear change of thermal stability during multigelation is foundation, as a result more accurate;It can be applied as external standard indicator
In the judgement of all refrigerated products number of freezing and thawing, the universality of application is strong;Multigelation number within capable of being suitable for 10 times
Determine, it is more to be applicable in number of freezing and thawing;Have it is easy to operate it is preliminary identify it is optional with two methods of complicated Accurate Analysis, with compared with
Strong practicability.In addition, be food grade products as outer target milk-coagulating enzyme preparation, it is nontoxic, it is suitable for frozen food freeze thawing
The analysis of number.
A kind of determination method of number of freezing and thawing, using following steps:
(1) enzyme activity of rennin solns is measured;
(2) step (1) rennin solns sealing is placed in frozen product to be measured and frozen product is together after freeze thawing, measured again
The enzyme activity of the enzyme judges the number of freeze thawing by the difference of enzyme activity twice.
Preferably, renin used in rennin solns described in step (1) is pure enzyme powder;The rennin solns are matched
Method processed is with the potassium dihydrogen phosphate of 20mmol/L pH6.0-dipotassium hydrogen phosphate buffer dilution.
Preferably, freeze thawing described in step (2) is once rennin solns in -20 DEG C of refrigerator freezings 2 hours to completely cold
Freeze, then 20 DEG C of water-bath water-bath 70min melt to complete, and shaking gently mixing is a frozen-thaw process.
Preferably, the specific calculation method of the number of freezing and thawing are as follows:
(1) the curdled milk enzyme activity A without freeze thawing is measured0;
(2) the renin remnants vigor A after 2,4,6,8,10 freeze thawing is measured respectivelyn, according to renin remnants vigor and
The relationship of number of freezing and thawing establishes equation (I);
An=A0-nx
Wherein AnThe renin enzyme activity of n times is crossed for freeze thawing;N is number of freezing and thawing, and x is that enzyme activity caused by each freeze thawing loses
(3) renin remnants vigor of the detection Jing Guo n times freeze thawing calculates time of freeze thawing by renin remnants vis viva equation (I)
Number n.
A kind of determination method of above-mentioned number of freezing and thawing, using following steps:
1) the inactivation activation energy of renin is measured;
(2) sealing of renin described in step (1) is placed in frozen product to be measured and frozen product is together after freeze thawing, measurement should again
The inactivation activation energy of enzyme obtains the number of renin freeze thawing, judges the number of freezing and thawing of frozen product according to the variation of inactivation activation energy.
Preferably, the freeze thawing is to keep the temperature at absolute temperature 323K-331K;Soaking time is 0-300min.
Preferably, the number of freezing and thawing circular are as follows:
(1) measurement enzyme solution keeps the temperature the enzyme activity Ar after 0-300min at absolute temperature 323K-331K, with enzyme activity
Logarithm lnAr map to soaking time t, and establish equation (II)
Relative activity Ar=remnants vigor Al/ initial vigor A0×100%
lnAr=-kt (II)
Wherein, k is decay rate constants, and t is soaking time.
(2) according to the slope k and coefficient R of lnAr linear regression under enzyme solution different temperatures2, according to falling for absolute temperature
The relationship of number 1/T and slope k logarithm establishes equation (III):
(III)
(3) it is calculated separately to obtain inactivation activation energy of the enzyme solution after freeze thawing 2,4,6,8,10 times according to equation (III)a;And according to
Inactivate activation energyaInactivation activation energy equation (IV) is established with the relationship of number of freezing and thawing;
Ea’=Ea- n* Δ E(IV)
Wherein, Ea' be freeze thawing n times renin inactivation activation energy;EaFor the inactivation activation energy without freeze thawing, Δ E is to freeze every time
Melt the difference of renin inactivation activation energy.
(5) inactivation activation energy of the detection Jing Guo n times freeze thawinga, the number of freeze thawing is calculated by inactivation activation energy equation (IV)
n。
Beneficial effect
The present invention assesses number of freezing and thawing using indirect method, by putting external standard enzyme preparation together anti-with refrigerated products
Thermal stability variation in multiple frozen-thaw process, determines refrigerated products number of freezing and thawing.As a result more accurate, universality is strong, using model
It encloses more extensively.
Detailed description of the invention
Fig. 1 is the relationship of curdled milk enzyme activity and enzyme activity and number of freezing and thawing;
Fig. 2 is the enzyme activity that initial enzyme solution keeps the temperature different time at different temperatures;
Fig. 3 is the decay rate constants k of initial enzyme solution at different temperatures;
Fig. 4 is the calculating that initial enzyme solution inactivates activation energy;
Fig. 5 be freeze thawing 2,4,6,8,10 times renin at different temperatures, the result that enzyme activity changes with soaking time;
Fig. 6 is reciprocal mapping results of 2,4,6,8,10 lnk of Fig. 6 freeze thawing to absolute temperature;
Fig. 7 is the relationship that renin inactivates activation energy and number of freezing and thawing.
Specific embodiment
Invention is described in further details With reference to embodiment.
Embodiment 1: using curdled milk enzyme activity as the number of freezing and thawing of index preliminary judgement frozen product.
Rennin solns are packed into 50ml plastic centrifuge tube, freeze to complete within refrigerator freezing 2 hours in -20 DEG C;Then 20 DEG C
Water-bath water-bath 70min melts to complete, and shaking gently mixing is a frozen-thaw process.The result shows that renin freeze thawing is once lived
Power loss 7IU/ml or 4%(is shown in Fig. 1).
Certain frozen product, the initial enzyme activity A0 of external standard renin is 166.67IU/ml, after multigelation, curdled milk enzyme activity
It is reduced to 133.33IU/ml, enzyme activity has lost 20%, and according to Fig. 1, the every freeze thawing of renin is primary, and condenser water level probably loses
The 4% of the initial vigor of 6.67%(), therefore can be determined that, which may have passed through 5 freeze thawing in storing process.
Embodiment 2: the number of freezing and thawing of frozen product is determined using renin inactivation activation energy as index
When there are other factors that may generate interference (for example enzyme solution is diluted) to curdled milk enzyme activity, need to measure using this method
Number of freezing and thawing.It is measured after initial enzyme solution keeps the temperature at different temperatures respectively, ratio (the opposite enzyme of remaining vigor and initial enzyme activity
Vigor) as shown in Fig. 2, with the logarithm lnA of enzyme activityrTo soaking time mapping (see figure 3).
The slope k and related coefficient of lnAr linear regression under the initial enzyme solution different temperatures of table 1
It is converted into absolute temperature by degree Celsius, and with the 1/T reciprocal of the absolute temperature and logarithm lnk of slope k mapping (Fig. 4).
It can be calculated according to equation (III), the inactivation activation energy of initial enzyme solutiona0=8.314*(51770 ± 1852)=
430.42±15.40KJ/mol。
2,4,6,8,10 enzyme activities of freeze thawing are measured respectively with soaking time result of variations (figure according to the above method
5).
The result such as Fig. 6 of the logarithm lnk of the decay rate constants of different number of freezing and thawing to the 1/T reciprocal mapping of absolute temperature
It is shown.
We have been computed the renin inactivation activation energy for learning non-freeze thawinga0=430.42±15.40KJ/mol.By Fig. 6
Result bring equation (III) into, after being calculated freeze thawing 2-10 times, the inactivation activation energy of renin is respectively as follows: freeze thawing 2 times
Inactivate activation energya2=415.45±8.164KJ/mol;Freeze thawing 4 times inactivation activation energiesa4=403.06±42.58KJ/mol;
Freeze thawing 6 times inactivation activation energiesa6= 385.19±1.16KJ/mol;Freeze thawing 8 times inactivation activation energiesa8=374.96±
10.53KJ/mol;Freeze thawing 10 times inactivation activation energiesa10=369.64±21.65KJ/mol。
To inactivate activation energyaWith the mapping of number of freezing and thawing, as shown in Figure 7.It is in a linear relationship in a certain range, Ean=
Ea0-6.332n。
Certain sample, the inactivation activation energy of renin, then can determine that the frozen product by 5 freeze thawing after measured.
Certain frozen product, for external standard renin after multigelation, measurement renin keeps the temperature different time at different temperatures
The inactivation activation energy of the external standard renin is calculated according to equation (II) and equation (III) for remaining vigoran=394kJ/mol,
31.6kJ/mol is reduced than initial enzyme solution.The every freeze thawing of renin as the result is shown of Fig. 7 is primary, and inactivation activation energy reduces
6.32kJ/mol is calculated according to equation (IV) it is found that the frozen product have passed through 5 freeze thawing in storing process.
Claims (7)
1. a kind of determination method of number of freezing and thawing, which is characterized in that use following steps:
(1) enzyme activity of rennin solns is measured;
(2) step (1) rennin solns sealing is placed in frozen product to be measured and frozen product is together after freeze thawing, measured again
The enzyme activity of the enzyme judges the number of freeze thawing by the difference of enzyme activity twice.
2. determination method according to claim 1, which is characterized in that coagulated used in rennin solns described in step (1)
Galactenzyme is pure enzyme powder;The preparation method of the rennin solns is with the potassium dihydrogen phosphate of 20mmol/L pH6.0-phosphoric acid hydrogen two
The dilution of potassium buffer.
3. determination method according to claim 1, which is characterized in that freeze thawing described in step (2) is once that renin is molten
Liquid freezes for refrigerator freezing 2 hours in -20 DEG C to complete, and then 20 DEG C of water-bath water-bath 70min melt to complete, shake gently mixed
Even is a frozen-thaw process.
4. determination method according to claim 1-3, which is characterized in that the specific side of calculating of the number of freezing and thawing
Method are as follows:
(1) the curdled milk enzyme activity A without freeze thawing is measured0;
(2) the renin remnants vigor A after 2,4,6,8,10 freeze thawing is measured respectivelyn, according to renin remnants vigor and jelly
The relationship for melting number establishes equation (I);
An=A0- nx (I)
Wherein AnThe renin enzyme activity of n times is crossed for freeze thawing;N is number of freezing and thawing, and x is that enzyme activity caused by each freeze thawing loses;
(3) renin remnants vigor of the detection Jing Guo n times freeze thawing calculates time of freeze thawing by renin remnants vis viva equation (I)
Number n.
5. a kind of determination method of number of freezing and thawing, which is characterized in that use following steps:
(1) the inactivation activation energy of renin is measured;
(2) sealing of renin described in step (1) is placed in frozen product to be measured and frozen product is together after freeze thawing, measurement should again
The inactivation activation energy of enzyme obtains the number of renin freeze thawing, judges the number of freezing and thawing of frozen product according to the variation of inactivation activation energy.
6. determination method according to claim 5, which is characterized in that the freeze thawing is at absolute temperature 323K-331K
Heat preservation;Soaking time is 0-300min.
7. determination method according to claim 5 or 6, which is characterized in that the number of freezing and thawing circular are as follows:
(1) measurement enzyme solution keeps the temperature the enzyme activity Ar after 0-300min at absolute temperature 323K-331K, with enzyme activity
Logarithm lnAr map to soaking time t, and establish equation (II)
Relative activity Ar=remnants vigor Al/ initial vigor A0×100%
lnAr=-kt (II)
Wherein, k is decay rate constants, and t is soaking time;
(2) according to the slope k and coefficient R of lnAr linear regression under enzyme solution different temperatures2, according to the 1/T reciprocal of absolute temperature
Equation (III) is established with the relationship of slope k logarithm:
(III)
(3) it is calculated separately to obtain inactivation activation energy of the enzyme solution after freeze thawing 2,4,6,8,10 times according to equation (III)a;And according to mistake
Activation energy livingaInactivation activation energy equation (IV) is established with the relationship of number of freezing and thawing;
Ea’=Ea- n* Δ E(IV)
Wherein, Ea' be freeze thawing n times renin inactivation activation energy;EaFor the inactivation activation energy without freeze thawing, Δ E is each freeze thawing
The difference of renin inactivation activation energy;
(5) inactivation activation energy of the detection Jing Guo n times freeze thawinga, the frequency n of freeze thawing is calculated by inactivation activation energy equation (IV).
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Cited By (3)
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CN109932386A (en) * | 2019-04-03 | 2019-06-25 | 王信青 | A kind of monitoring method of refrigerating process |
CN110243260A (en) * | 2019-07-29 | 2019-09-17 | 中国电建集团中南勘测设计研究院有限公司 | A kind of ice layer thickness calculation method and system |
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CN110243260A (en) * | 2019-07-29 | 2019-09-17 | 中国电建集团中南勘测设计研究院有限公司 | A kind of ice layer thickness calculation method and system |
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CN111337532A (en) * | 2020-03-29 | 2020-06-26 | 上海伟星新材料科技有限公司 | Low-temperature performance test method for water-based emulsion |
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