Milk freezing protection status
The procedures and steps of cryopreservation are also important factors affecting the cryopreservation of samples. According to the method of cryopreservation, ice crystals are formed inside and outside the cells during the process of cryopreservation, which causes physical damage to the milk when the temperature drops. Some studies have shown that ice crystallization can only be formed by slowly cooling under the temperature range of -60 and 0℃. The slower the cooling, the larger the ice crystals. The ice crystals formed at about -25℃ are the most and have the largest impact on the liquid. Therefore, in the process of cryopreservation, try to avoid the harmful temperature zone or accelerate the speed of cryopreservation to reduce the impact of physical injury on the experimental results, in order to better preserve the liquid.The samples including the following groups were randomly selected and numbered: -20℃ ordinary frozen storage group, -20℃ vacuum protected frozen storage group, slow frozen instant protection group, quick frozen slow soluble protection group, quick frozen instant protection group, and freeze-thaw protection group.
For the -20℃ ordinary frozen storage group, fresh milk was divided into aliquots in high-pressure sterile EP tubes, marked, placed in a -20℃ freezer, and repeatedly frozen and thawed every 30 minutes. The freezing and thawing times were divided into groups in which the samples were repeatedly mixed, the sealing film was removed, and the samples were centrifuged at 2000 r/min for 5 minutes and equilibrated to room temperature. Each freeze and thaw time was performed in three groups of parallel samples for group identification.In the process of gradually cooling, the cooling process avoids the thermal shock caused by rapid freezing and melting, which causes physical damage to the components in the liquid. It is better to use 1℃ per minute. During the experiment, it was found that the milk could be cooled to -20℃ in a -20℃low-temperature refrigerator at 30 minutes, and this time was used to carry out the freezing cycle for subsequent test grouping.
For the -20℃ frozen storage group under vacuum protection, the test milk was placed in an EP tube after autoclaving, labeled, and subjected to food-grade vacuum standards used for vacuum storage and frozen storage. The compressor was evacuated for storage. Samples were placed in a freezer at -20℃, and freeze-thaw cycles were repeated every 30 minutes. The samples were grouped according to the number of freeze-thaw cycles. The samples were repeatedly mixed each time, the sealing film was removed, and samples were centrifuged at 2000 r/min for 5 minutes and equilibrated to room temperature. Each number of fusions was performed in three sets of parallel samples, which were identified by grouping.
For the slow-freezing and fast-dissolving protection group, fresh milk was placed in an EP tube after autoclaving, then incubated at 4℃, -20℃ and -80℃ in order, for 0.5 h at each temperature. The samples were then warmed in a 37℃ water bath immediately after freezing. Repeated freeze-thaw was performed according to this method, and the samples were repeatedly mixed each time. The sealing film was removed, and samples were centrifuged at 2000 r/min for 5 minutes and equilibrated to room temperature. Each freeze-thaw cycle was performed in three groups that included parallel samples for group identification.
For the quick-freezing and slow-dissolving protection group,
Four different conditions of freeze-thaw protection were investigated: quick-freezing, quick-freezing, slow-freezing, slow-freezing, and slow-freezing. Under different frozen storage conditions, ordinary cryopreservation used as a control group to compare with other groups in protein content changes, the protein carbonylation content index under various frozen storage conditions increased with the number of freeze-thaw cycles, and the protein carbonylation content also increased.milk was divided into aliquots in high-pressure sterile EP tubes, labeled, and immediately placed in liquid nitrogen and then stored in a -80℃ freezer. This was followed by storage at -20℃, 4℃ and 37℃ for 0.5 h at each temperature. The samples were repeatedly mixed each time, the sealing film was removed, and the samples were centrifuged at 2000r/min for 5min and equilibrated to room temperature. Three sets of parallel samples were examined.
For the quick-frozen and instant-protected group, fresh milk was divided into aliquots in plastic EP tubes after autoclaving, randomly marked, immediately frozen in liquid nitrogen and transferred to a -80℃ freezer. After freezing for 0.5 h, samples were transferred to a 37℃ water bath. The samples were repeatedly mixed each time, the sealing film was removed, and samples were centrifuged at 2000 r/min for 5 min and equilibrated to room temperature. Three sets of parallel samples were examined and identified in groups.
For the slow-freezing and slow-dissolving protection group, fresh milk was divided into aliquots in high-pressure sterile EP tubes, labeled and incubated at 37℃, 4℃, -20℃ and -80℃ sequentially for 0.5 h each. The samples were then incubated at -20℃, 4℃ and 37℃ for 0.5 h each. Freeze-thaw cycles were repeated five times according to this method. Three sets of parallel samples were examined for each number of freeze-thaw cycles for group identification.
Determination of protein carbonylation
The total protein TP detection kit was provided by Mindray. The testing was performed with a Mindray BS-800 automatic biochemical analyzer. The protein carbonyl content was determined according to the method of Levine (1994) with a protein carbonylation content determination kit provided by Beijing Solibao Technology Co., Ltd. in a 1.5 mL centrifuge tube, to which 0.1 mL of protein solution and 0.5 mL of 0.02 mol/L 2,4-dinitrophenylhydrazine in 2 mol/L HCl solution were added and reacted at 25℃ for 40 min. For blank samples, 0.5 mL of a 2 mol/L HCl solution without 2,4-dinitrophenylhydrazine was added. Then 0.5 mL of 20% trichloroacetic acid was added to the above reaction solution. Samples were centrifuged (11000 × g, 5 min, 4℃), the supernatant was discarded, and 1 mL of ethanol-acetic acid was used for protein precipitation. The ethyl acetate solution (volume ratio 1:1) was washed three times. After the solvent was evaporated, the protein was suspended in 1 mL of a 6 mol/L guanidine hydrochloride solution and incubated in a water bath at 37℃ for 30 minutes. The blank sample was used as a control, and the absorbance at 370 nm was measured. The content of the protein carbonyl derivative (nmol/mg protein) was calculated by using the molar absorption coefficient of 22000 M-1 cm-1. Three replicates of each sample were measured, and the median value was taken.
The formula for calculating the carbonyl content was:
carbonyl content (nmol/g myofin) = (A×n)/(ε × ρ)×109
In the formula, A represents the absorbance at the wavelength of 370 nm; n represents the dilution factor; ε represents the molar absorption coefficient of 22000/(L/(mol · cm)); and ρ represents the protein mass concentration (mg/mL).