CN107389722A - The method that ESR characterizes beef fat quality comparison during multigelation - Google Patents

Abstract

The invention discloses an electron spin resonance (ESR) method for characterizing the quality change of beef fat in the process of repeated freezing and thawing. Sweep field width, microwave power, modulation amplitude, receiver gain, time constant, conversion time, and frequency of x-band; The extracted fat is measured by spin resonance, and the measured objects include free radical intensity, free radical quantity or induction time; (3) analyze and process the obtained electron spin resonance spectrum, so as to realize the detection of beef fat in the process of repeated freezing and thawing. Symptoms of quality change. The method of the invention has the advantages of high detection sensitivity, less analysis solvent consumption, simple sample processing, etc., and can quickly and efficiently detect the quality of beef fat during repeated freezing and thawing processes.

Description

ESR method to characterize the change of beef fat quality during repeated freezing and thawing

technical field

The invention relates to a method for detecting changes in beef quality during repeated freezing and thawing, in particular to a method for characterizing lipid deterioration in beef quality during repeated freezing and thawing by electron spin resonance (ESR).

Background technique

Freezing storage is still the most widely used preservation method for meat and meat products in long-distance transportation. It can better maintain product quality and make products have a longer shelf life. Due to the poor cold chain transportation system in our country, the beef in our country is more likely to undergo repeated freezing-thawing (freezing-thawing) processes. Many studies have confirmed that repeated freezing and thawing accelerates the deterioration of meat texture, flavor, color, microbial activity and nutritional value. Most researchers believe that the main cause of meat deterioration caused by repeated freezing and thawing is the formation of ice crystals during freezing. The formed ice crystals lead to the rupture of the cell membrane, which releases the main catalyst for lipid oxidation in the cell and accelerates lipid oxidation. The free radicals produced in the process of fat deterioration are closely related to many chemical reactions, which are the signs of the oxidation process of food. The chain reaction triggered by free radicals is the essence of fat auto-oxidation, and free radicals can also affect the flavor of food.

At present, the main indicators for fat quality evaluation are peroxide value, TBARS thiobarbituric acid reaction products (Thiobarbituric Acid Reactive Substances, TBARS) and acid value. The main detection methods mainly include high performance liquid chromatography, chemiluminescence, and fluorescence detection, etc., but these methods require many reagents and consumables, cumbersome sample pretreatment, and long analysis time.

Contents of the invention

The main purpose of the present invention is to provide a kind of method that electron spin resonance (ESR) characterizes the beef fat quality change in repeated freezing and thawing process, and it has the characteristics such as high sensitivity, easy and simple operation, rapid analysis, thus overcomes the problem in the prior art. insufficient.

In order to realize the above-mentioned purpose of the invention, the present invention has adopted following technical scheme:

The embodiment of the present invention provides a method for ESR characterizing the quality change of beef fat in the process of repeated freezing and thawing, which includes:

(1) setting electron spin resonance working parameters, said working parameters include central magnetic field strength, sweep field width, microwave power, modulation amplitude, receiver gain, time constant, conversion time, x frequency band frequency;

(2) Carry out repeated freezing and thawing treatment to beef, then carry out fat extraction to beef after thawing, adopt electron spin resonance to measure the extracted fat afterwards, the object of measurement includes free radical intensity, free radical quantity or induction time;

(3) Analyzing and processing the obtained electron spin resonance spectrum, so as to realize the characterization of the quality change of beef fat during repeated freezing and thawing.

In some preferred embodiments, the central magnetic field strength of the step (1) is set to 3350.00G, the sweep field width is set to 100.00G, the microwave power is set to 20mW, the modulation amplitude is set to 1.000G, and the receiver gain is set to 30dB, the time constant is set to 0.01ms, the transition time is set to 40.96ms, and the x-band frequency is set to 9.42GHz. In some embodiments, in the step (2), in the process of repeatedly freezing and thawing the beef, the beef is frozen at -18 to -20°C for more than 24 hours and then thawed at 2 to 4°C to the center of the beef A freeze-thaw cycle was performed at a temperature of 2-4°C.

In some embodiments, the step (2) includes: crushing the thawed beef, mixing the extractant with the ground beef and shaking overnight, and then removing the extractant by rotary evaporation at 40-50°C to achieve fat extraction; the extractant includes petroleum ether, and its boiling point is 30-60°C.

In some preferred embodiments, the step (2) includes: mixing the free radical scavenger and the extracted fat in the ESR tube according to a volume ratio of 1:10, and starting timing from the ESR tube entering the ESR detection chamber, every 2 to 5 minutes as a time interval to record once, to obtain electron spin resonance detection spectrum.

Preferably, the step (2) includes: recording every 2 to 5 minutes as a time interval, and each fat sample is required to obtain 15 to 25 electron spin resonance detection spectra, that is, the time required for each fat sample to be measured The number of intervals is 15 to 25. In some embodiments, the information of the electron spin resonance detection spectrum includes the peak height value of the free radical, the total spin number of the free radical, the spin concentration and the induction time.

Further, the calculation method of the aforementioned total spin number and spin concentration of free radicals is to use the spin quantification function in the Xenon software to integrate the spectral lines, and obtain the total number of spins and the spin concentration through computer calculation. The calculation of the induction time is the time corresponding to the inflection point of the free radical from the slow growth interval to the fast growth interval.

In some embodiments, the step (3) includes: measuring the change of the peak intensity of the selected free radical peak in the electron spin resonance detection spectrum, so as to realize the characterization of the quality change of beef fat during repeated freezing and thawing.

Further, the selected free radical peak is the central peak of the multiplet. More specifically, if the number of peaks in the multiplet is odd, take the central peak. If the number of peaks in the multiplet is even, take the taller peaks on either side of the center.

In some embodiments, the step (3) includes: define the free radical intensity, induction time or free radical quantity with the size of the free radical peak peak value in the selected time, so as to achieve the effect of beef fat in the process of repeated freezing and thawing. Symptoms of quality change.

In some preferred embodiments, the temperature in the ESR chamber is 95-100° C. during the ESR detection process.

Preferably, the step (2) includes: mixing the toluene solution of the free radical scavenger and the extracted fat in the ESR tube, and then putting them into the ESR detection chamber for detection.

Preferably, the concentration of the toluene solution of the radical scavenger is 0.1wt%-0.7wt%.

Further, the free radical scavenger includes N-tert-butyl-phenylnitrone (PBN) or 5,5-dimethyl-1-pyrroline nitrogen oxide (DMPO), etc., but is not limited thereto.

Compared with the prior art, the present invention adopts electron spin resonance (ESR) to characterize the quality change of beef fat in the process of repeated freezing and thawing, and has the advantages of high detection sensitivity, less analysis solvent consumption, simple sample processing, etc. During the melting process, the quality of beef fat was tested.

Description of drawings

Figures 1a-1c respectively show the changes in fat peroxide value, TBARS and acid value in beef after different repeated freezing and thawing in Example 1 of the present invention.

Fig. 2 shows the changes of the ESR free radical signal spectrum of fat in beef after seven freeze-thaw cycles in Example 1 of the present invention, wherein it is recorded every 5 minutes until 50 minutes.

Fig. 3 shows the signal intensity of free radicals in the beef fat in different freeze-thaw cycles in Example 1 of the present invention when it is heated for 50 minutes. Fig. 4 shows the spectrum for measuring free radical intensity in Example 1 of the present invention.

Fig. 5 shows the measurement pattern of induction time in Example 1 of the present invention.

detailed description

As mentioned above, in view of the defects of the prior art, the inventor of this case was able to propose the technical solution of the present invention after long-term research and a lot of practice, which is mainly an electron spin resonance (ESR) characterization of repeated freezing The method for the change of beef fat quality during the melting process has the characteristics of sensitive method and simple sample processing.

In some more specific embodiments, the method includes the steps of:

(1) Determination of detection conditions:

Electron spin resonance (ESR) parameter setting;

(2) Sample testing

a. The beef is repeatedly frozen and thawed, and the thawed beef is subjected to fat extraction;

b. The extracted fat is carried out to measure free radical strength, quantity and induction time, etc.;

(3) Electron spin resonance (ESR) spectrum analysis

The obtained electron spin resonance (ESR) spectra were analyzed accordingly.

Further, in the foregoing step (1), the parameters of the electron spin resonance (ESR) can be set as follows: the center field (Center Field) is 3350.00G; the sweep field width (Sweep Width) is 100.00G; the microwave power ( MicrowavePower) is 20m W; modulation amplitude (Modulation Amplitude) is 1.000G; receiver gain (receiver gain) 30dB; time constant (time constant) 0.01ms; conversion time (conversion time) 40.96ms; x-band frequency (x-band frequency )9.42GHz.

Further, the aforementioned step (2) may include: repeatedly freezing and thawing the beef (frozen at -18 to -20°C for more than 24 hours and then thawed at 2 to 4°C until the central temperature of the beef is 2 to 4°C as a first freeze. Thaw cycle, specifically, for example, freezing at -20°C for more than 24 hours and then thawing at 4°C for more than 20 hours is a freeze-thaw cycle). Thawed beef is crushed, mixed with petroleum ether (boiling point 30-60° C.) and minced beef, placed in a shaker and shaken overnight, and then recovered with a vacuum rotary evaporator to obtain fat.

Further, the aforementioned step (2) may also include: mixing the toluene solution (concentration of about 0.1wt% to 0.7wt%) of the free radical scavenger with the fat volume ratio of 1:10, starting with the ESR tube entering the ESR cavity Timing, record once at a certain time interval (for example, record once every 2 to 5 minutes as a time interval, each fat sample requires 15 to 25 electron spin resonance detection spectra, that is, the time required for each fat sample to be measured The number of intervals is 15-25), and all the spectra are obtained, and the information includes the peak height value, the total spin number of free radicals, the spin concentration, and the induction time.

Further, the aforementioned step (3) may include:

a. Determination of free radical intensity with regard to peak selection and fixation.

b. Characterization method of free radical intensity Select the size of the peak height value, the length of induction time or the number of free radicals within a period of time.

The technical solutions of the present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings, but the present invention is not limited to the following embodiments.

In the following examples, the beef is first cut into pieces (4cm×4cm×4cm) and packaged for freezing. Freeze at -20°C for more than 24 hours and then thaw at 4°C for more than 20 hours as a freeze-thaw cycle.

In the following examples, the following detection conditions are adopted:

Electron Spin Resonance (ESR) parameter setting: Center Field is 3350.00G; Sweep Width is 100.00G; Microwave Power is 20m W; Modulation Amplitude is 1.000G ; Receiver gain (receiver gain) 30dB; time constant (time constant) 0.01ms; conversion time (conversion time) 40.96ms; x-band frequency (x-band frequency) 9.42GHz.

Example 1 In this example, raw beef was subjected to 7 freeze-thaw cycles (referred to as the freeze-thaw group), and the completely thawed beef was crushed, mixed with petroleum ether (boiling point 30-60°C) and ground beef, placed in Vibrate on a shaker overnight, and then use a vacuum rotary evaporator to recover petroleum ether at 40-50°C to obtain fat. Add 200 μL of extracted fat and 20 μL of PBN to the ESR tube, shake until well mixed. The strength, quantity and induction time of free radicals were measured, and the ESR spectrum of beef fat was obtained.

This embodiment also uses peroxide value, TBARS and acid value as evaluation indicators to verify the quality change of beef fat in the process of repeated freezing and thawing.

Determination of peroxide value is determined according to the method in GB5009.227-2016: 2g~3g of beef sample (accurate to 0.001g) is placed in a 250mL iodine measuring bottle, and 30mL of chloroform-glacial acetic acid solution (volume ratio 40:60) is added Mix the liquid and shake gently to dissolve the fat completely. Accurately add 1.00mL saturated potassium iodide solution, stopper the cap tightly, shake gently for 0.5min, and place in the dark for 3min. Take out and add 100mL of water, shake well and immediately use sodium thiosulfate standard solution (when the estimated value of peroxide value is 0.15g/100g and below, use 0.002mol/L standard solution, when the estimated value of peroxide value is greater than 0.15g/100g , with 0.01mol/L standard solution) to titrate the precipitated iodine, when titrated to pale yellow, add 1mL of 1% starch indicator, continue the titration and shake vigorously until the blue color of the solution disappears as the end point. Simultaneously conduct a blank test. The volume V ₀ of the 0.01mol/L sodium thiosulfate solution consumed in the blank test shall not exceed 0.1mL.

X ₁ (mmol/kg)=C×(VV ₀ )×0.1269×100/m

X ₁ —peroxide value, the unit is grams per hundred grams (g/100g)

V—the volume of sodium thiosulfate standard solution consumed by the sample, in milliliters (mL)

V0—the volume of sodium thiosulfate standard solution consumed by the blank test, in milliliters (mL)

c—the concentration of sodium thiosulfate standard solution, in moles per liter (mol/L)

0.1269—the quality of iodine equivalent to 1.00mL sodium thiosulfate standard titration solution [c(Na2S2O3)=1.000mol/L]

m—sample mass, in grams (g)

100—conversion factor.

TBARS determination is as follows: take 5g meat sample and mix with 10mL 7.5% TCA-EDTA (0.1%), homogenize at high speed, filter, take 5ml supernatant, add 5ml TBA (2.88g/L) solution. After 40 minutes in the boiling water bath, take it out and let it cool at room temperature. Then the above reaction solution was transferred to a clean colorimetric tube, and 5ml of chloroform was added to shake it. After standing for stratification, the supernatant was taken for ultraviolet detection, and the absorbance value at a wavelength of 532nm was obtained. A standard curve was drawn with 1,1,3,3-tetraethoxypropane.

The acid value is determined according to the method in GB5009.229-2016: take a clean 250mL Erlenmeyer flask, take a sample of about 10g, and the unit of its mass m is gram. Add 50mL-100mL of ether-isopropanol (volume ratio 1:1) mixed solution and 3-4 drops of phenolphthalein indicator, and shake fully to dissolve the sample. Then titrate with 0.1mol/L standard titration solution. When the sample solution initially appears reddish and there is no obvious fading within 15s, it is the end point of titration. Immediately stop the titration, record the number of milliliters of the standard titration solution consumed by this titration, and this value is V.

Take another clean 250mL Erlenmeyer flask, accurately add the same volume and type of organic solvent mixture and indicator used in the determination of the above sample, and shake to mix. Then titrate with 0.1mol/L standard titration solution. When the sample solution initially appears reddish and there is no obvious fading within 15s, it is the end point of titration. Stop the titration immediately, record the milliliters of the standard titration solution consumed by this titration, and this value is V ₀ .

X _AV ＝(VV ₀ )*c*56.11/m

X _AV —acid value, in milligrams per gram (mg/g)

V—the volume of the standard titration solution consumed by the sample determination, in milliliters (mL)

V ₀ —the volume of the standard titration solution consumed by the corresponding blank determination, in milliliters (mL)

c—the molar concentration of the standard titration solution, in moles per liter (mol/L)

56.1—the molar mass of potassium hydroxide in grams per mole (g/mol)

m—the weight of the fat sample, in grams (g).

If the acid value is ≤1mg/g, the calculation result shall be kept to 2 decimal places; if the acid value is 1mg/g<acid value≤100mg/g, the calculation result shall be kept to 1 decimal place; if the acid value is >100mg/g, the calculation result shall be kept to an integer.

In addition, each freeze-thaw cycle corresponds to the control group (that is, beef that has been frozen) as a comparison, and other experimental conditions are the same as those of the freeze-thaw group.

Please refer to Figure 1a-Figure 1c and Table 1. The test results show that with the increase of freeze-thaw times, the peroxide value, TBARS and acid value of the fat all increase and change. It shows that with the increase of freeze-thaw times, the quality of beef fat deteriorates. At the same time, please refer to Figure 2, Figure 3 and Table 1, as the number of freezing and thawing increases, the intensity of free radicals also increases. Correlation analysis between the fat quality evaluation index and the free radical intensity shows that electron self-selected resonance (ESR) can accurately characterize the changes in beef fat quality caused by repeated freezing and thawing.

Table 1 Correlation analysis between fat quality evaluation index and ESR

Embodiment 2: Same as Embodiment 1, but replace the free radical scavenger PBN with DMPO.

It should be understood that the above preferred embodiments are only used to illustrate the content of the present invention. In addition, the present invention also has other implementation modes, but those skilled in the art adopt equivalent replacements or equivalents due to the technical inspiration involved in the present invention. The technical solutions formed by effective deformation methods all fall within the protection scope of the present invention.

Claims (10)

1. A kind of method that ESR characterizes the beef fat quality variation in repeated freeze-thaw process, it is characterized in that comprising: (1) setting electron spin resonance working parameters, said working parameters include central magnetic field strength, sweep field width, microwave power, modulation amplitude, receiver gain, time constant, conversion time, x frequency band frequency; (2) Carry out repeated freezing and thawing treatment to beef, then carry out fat extraction to beef after thawing, adopt electron spin resonance to measure the extracted fat afterwards, the object of measurement includes free radical intensity, free radical quantity or induction time; (3) Analyzing and processing the obtained electron spin resonance spectrum, so as to realize the characterization of the quality change of beef fat during repeated freezing and thawing. 2. The method according to claim 1, characterized in that: the central magnetic field strength of the step (1) is set to 3350.00G, the sweep field width is set to 100.00G, the microwave power is set to 20mW, and the modulation amplitude is set to is 1.000G, the receiver gain is set to 30dB, the time constant is set to 0.01ms, the transition time is set to 40.96ms, and the x-band frequency is set to 9.42GHz. 3. The method according to claim 1, characterized in that: in the step (2), in the process of repeatedly freezing and thawing the beef, the beef is frozen at -18～-20°C for more than 24h and then placed in the Thawing at 2-4°C until the core temperature of the beef is 2-4°C is one freeze-thaw cycle. 4. The method according to claim 1 or 3, characterized in that, the step (2) comprises: the beef after thawing is crushed, then the extractant is mixed with the ground beef and shaken overnight, and then after 40 The extractant is removed by rotary evaporation at ~50°C to realize fat extraction; the extractant includes petroleum ether with a boiling point of 30-60°C. 5. The method according to claim 1, characterized in that the step (2) comprises: mixing the free radical scavenger and the extracted fat in the ESR tube according to the volume ratio of 1:20 to 1:10, and automatically The ESR tube enters the ESR detection cavity and starts timing, and records every 2-5 minutes as a time interval to obtain an electron spin resonance detection spectrum; preferably, the temperature in the ESR cavity is 95-100° C. during the ESR detection process. 6. The method according to claim 5, characterized in that the step (2) comprises: recording once every 2 to 5 minutes as a time interval, and the electron spin resonance detection spectrum required to be obtained for each fat sample is 15 to 5 minutes. 25, that is, the number of time intervals required for the determination of each fat sample is 15 to 25. 7. The method according to claim 5 or 6, characterized in that: the information of the electron spin resonance detection spectrum includes the peak height value of free radicals, the total number of free radical spins, spin concentration and induction time. 8. The method according to claim 1, characterized in that, said step (3) comprises: measuring the change in peak intensity of selected free radical peaks in said electron spin resonance detection spectrum, realizing that for repeated freeze-thaw processes Characterization of fat quality changes in medium beef. 9. method according to claim 1, is characterized in that, described step (3) comprises: be defined as free radical intensity, induction time or free radical quantity with the size of free radical peak height value in selected time, thereby Realize the characterization of beef fat quality changes during repeated freezing and thawing. 10. The method according to claim 5, characterized in that: said free radical scavenger comprises N-tert-butyl-phenylnitrone or 5,5-dimethyl-1-pyrroline nitrogen oxide; Preferably, the step (2) includes: mixing the toluene solution of the free radical scavenger with the extracted fat in the ESR tube, and then placing it into the ESR detection chamber for detection; preferably, the toluene solution of the free radical scavenger The concentration of the solution is 0.1wt%-0.7wt%.