Three-section variable-temperature liquid nitrogen quick-freezing fresh-keeping method for beef and application

Abstract

The invention relates to the technical field of food processing and storage and transportation preservation, in particular to a three-section variable-temperature liquid nitrogen quick-freezing preservation method for beef and application, and the method comprises the following steps: S1, performing pretreatment such as trimming, fascia removal and pre-cooling on beef; s2, performing three-stage variable-temperature liquid nitrogen quick freezing to enable the central temperature of the meat to reach-18 DEG C; and S3, after the sample is frozen and stored for 28 days, performing quality detection and analysis. The three-stage variable-temperature liquid nitrogen quick freezing provided by the invention can effectively reduce the freezing time and cooking loss of the beef, delay the spoilage of proteins and lipids, maintain a better muscle tissue form, and maintain the fresh appearance characteristic of the beef.

Description

Technical Field

[0001] The invention relates to the technical field of food processing and storage and preservation, and in particular to a three-stage variable temperature liquid nitrogen quick-freezing and preservation method for beef and its application. Background Art

[0002] Yaks live on the Qinghai-Tibet Plateau and mainly graze on natural grasslands. Their meat is delicious, has a unique flavor and extremely high nutritional value. They enjoy a high reputation throughout the country and have become a high-end meat resource with both regional characteristics and market potential.

[0003] Currently, yak beef is mainly sold in the form of jerky, canned meat and fresh meat. Fresh yak beef is rich in high-quality protein, fat, amino acids, etc., but it has a high water content and is prone to spoilage after slaughter, which seriously affects sales and food safety. Traditional freezing methods can extend the shelf life of yak beef, but they have the disadvantages of poor thermal conductivity and long freezing time, which leads to the formation of large ice crystals, seriously damaging the muscle tissue of yak beef, and causing a series of deteriorations such as color deterioration, decreased water holding capacity, protein denaturation and lipid oxidation, resulting in a decline in edible quality. Compared with traditional air freezing, immersion freezing, magnetic field freezing, ultrasonic freezing, high-pressure freezing, liquid nitrogen freezing and other technologies can increase the freezing rate and improve the freezing quality. Among them, liquid nitrogen quick freezing can achieve rapid passage through the maximum ice crystal formation zone, greatly improving the quality of frozen food. However, liquid nitrogen quick freezing is currently mostly used for seafood and fruits such as blueberries, crabs and shrimps, and there is little research on liquid nitrogen quick freezing in beef. The present invention studies the important role of three-stage liquid nitrogen quick freezing in maintaining the fresh quality of yak meat and extending its shelf life. The study of the effect of liquid nitrogen quick freezing on the quality characteristics of yak meat has great practical significance.

[0004] Generally speaking, the freezing process of meat can be divided into three stages: pre-cooling stage, phase change stage and deep freezing stage. Among them, the phase change stage, also known as the maximum ice crystal formation zone, is where about 70% to 80% of the water forms ice. The faster the speed through this stage, the smaller the ice crystals produced, and the less damage to the quality of the meat after thawing. Liquid nitrogen quick freezing technology is a technology that uses liquid nitrogen as a cooling medium to achieve rapid freezing. The boiling point of liquid nitrogen is -195.8℃, and it has an extremely low temperature under normal pressure. When it comes into contact with food, it does not directly contact the food itself, but transfers cold energy indirectly. Due to the extremely fast cooling speed, the water inside the food does not have time to form large ice crystals, but forms small and uniform ice crystals, which helps to avoid damaging the cell wall, reduce water loss and structural tissue damage, thereby preserving the original taste, color, texture and nutrients of the food to the greatest extent.

[0005] According to previous studies, liquid nitrogen quick freezing can inhibit ice crystal formation to a great extent and better maintain the integrity of tissue structure, but past studies have adopted a single temperature of -80°C through cooling and phase change stages, which on the one hand increases the consumption of liquid nitrogen and on the other hand limits the room for improving food quality. However, maintaining -80°C requires a continuous supply of liquid nitrogen, which increases economic costs. In order to solve these problems, the present invention aims to find a segmented beef freezing mode that can minimize quality deterioration after thawing and save liquid nitrogen. Summary of the invention

[0006] The purpose of the present invention is to provide a method and application of three-stage variable temperature liquid nitrogen quick freezing and preservation of beef, which effectively reduces liquid nitrogen consumption and can better maintain the edible quality of beef after thawing, significantly reduces cooking loss, delays the deterioration of protein and lipids, maintains a more complete muscle tissue morphology, and maintains the fresh appearance characteristics of beef.

[0007] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0008] A three-stage variable temperature liquid nitrogen quick freezing and fresh-keeping method for beef, comprising:

[0009] S1: pre-processing the beef such as trimming, removing fascia, and pre-cooling;

[0010] S2: Use three-stage variable temperature liquid nitrogen quick freezing to make the core temperature of the meat reach -18℃;

[0011] S3: After the samples were frozen and stored for 28 days, quality testing and analysis were performed.

[0012] Furthermore, the step S1 specifically includes:

[0013] S1.1: Select fresh beef longissimus dorsi muscle and remove the surface fascia;

[0014] S1.2: Cut the meat into chunks with a thickness of 2-3 cm and a weight of 150 g;

[0015] S1.3: Use sealed polyethylene bags and pre-cool and store in a refrigerator at 4°C to balance the initial temperature of the meat.

[0016] Furthermore, the three-stage variable temperature liquid nitrogen quick freezing in step S2 includes a precooling stage, a phase change stage, and a deep cooling stage; specifically includes:

[0017] S2.1: Set CF as the control group, and freeze the beef in a -18℃ refrigerator until the core temperature of the meat reaches -18℃;

[0018] S2.2: Use -80℃ liquid nitrogen treatment to quickly pass through the cooling stage. In the phase change stage, the liquid nitrogen temperature is set to three different treatments of -60℃, -80℃ or -100℃ for 5 minutes to quickly pass through the maximum ice crystal formation area. The -80℃ liquid nitrogen treatment in the phase change stage is defined as LNF1 group, the -100℃ liquid nitrogen treatment is defined as LNF2 group, and the -60℃ liquid nitrogen treatment is defined as LNF3 group;

[0019] S2.3: During the deep freezing stage, the liquid nitrogen temperature is set to -80°C until the core temperature of the meat reaches -18°C, so as to make full use of the cold energy in the first two stages and save liquid nitrogen consumption.

[0020] Furthermore, the step S3 specifically includes:

[0021] S3.1: Use an insertion thermometer placed in the center of the meat, and the temperature is -18°C as the end point of quick freezing; record the temperature curve;

[0022] S3.2: Transfer the samples of CF, LNF1, LNF2 and LNF3 to a -20℃ refrigerator for 28 days and then thaw at 4℃ for quality index determination;

[0023] S3.3: The experimental data were counted, processed, analyzed and plotted using Excel 2019, SPSS26.0 and Origin 2021.

[0024] Furthermore, the index determination in step S3 includes: pH value determination, thiobarbituric acid value TBARS determination, volatile basic total nitrogen TVB-N determination, L* / a* / b* colorimetry determination, total thiol content determination, moisture distribution determination, scanning electron microscopy observation, and HE staining observation.

[0025] On the other hand, the present invention provides the application of the above method in quick-freezing and fresh-keeping beef.

[0026] Beneficial effects of the present invention:

[0027] The highlight of the three-stage variable temperature liquid nitrogen quick freezing proposed by the present invention is its fine control of the temperature in the phase change stage. By adjusting the liquid nitrogen temperature to -60°C, -80°C or -100°C for 5 minutes, it quickly passes through the maximum ice crystal formation zone to form fine and uniform ice crystals. This fine and uniform ice crystal structure reduces the physical damage to muscle fibers and cell membranes, thereby maintaining the texture and original taste of the beef. The distribution of fine ice crystals can significantly reduce blood loss after thawing, which is directly related to the juiciness and intact appearance color of the beef after thawing, and maintains the high sensory quality of the meat.

[0028] The present invention reduces protein denaturation and lipid oxidation by quickly passing through the phase change stage and effectively controlling the temperature. Experiments show that compared with conventional freezing methods, the three liquid nitrogen treatment groups can show significant advantages in maintaining food quality. In particular, the effect of liquid nitrogen treatment is particularly prominent in maintaining the freshness and nutritional value of meat. In the three-stage variable temperature liquid nitrogen quick freezing process, the liquid nitrogen temperatures (-60°C, -80°C and -100°C) in different phase change stages have a significant effect on the quality of beef. Through the analysis of the experimental results, it is found that when the phase change stage is -60°C liquid nitrogen treatment, the TBARS value (thiobarbituric acid reactant value, used to evaluate the degree of lipid oxidation) of beef reaches the lowest, which indicates that under this condition, the lipid oxidation process of meat is the slowest, thereby effectively extending the shelf life of beef and maintaining its original flavor and taste. In addition, by reducing the oxidation reaction, the integrity of amino acids and vitamins is also maintained to a large extent, which is crucial for consumers' health and nutritional intake. Further observation of the tissue structure of beef by scanning electron microscopy and HE (hematoxylin-eosin) staining technology revealed that the phase transition stage was -60°C liquid nitrogen treatment, the tissue structure of the meat remained good, the cell morphology was intact, and the cells were tightly connected. This shows that liquid nitrogen quick freezing technology can not only slow down lipid oxidation, but also protect the tissue structure of meat at the microscopic level, thereby ensuring the quality stability of food during freezing and storage. These findings provide a strong experimental basis for the application of liquid nitrogen quick freezing technology in the field of meat preservation.

[0029] The present invention maintains -80°C during the pre-cooling and deep-freezing stages. Although a higher temperature of liquid nitrogen is used in the initial stage, the flexible temperature change during the phase change stage and the subsequent temperature recovery strategy make the overall liquid nitrogen consumption more economical. Through reasonable time and temperature management, the freezing process can be carried out efficiently, reducing the consumption of liquid nitrogen and reducing the total cost. This economical use mode of three-stage variable temperature liquid nitrogen not only enables enterprises to save operating costs, but also makes the technology more feasible in large-scale commercial applications.

[0030] The present invention verifies the improvement of meat edible quality by three-stage variable temperature liquid nitrogen quick freezing through a 28-day storage experiment in a -20°C refrigerator. After being treated with liquid nitrogen quick freezing technology, the color of beef is not only effectively maintained, but also its quality shows significant advantages in terms of moisture retention and protein structure. Liquid nitrogen quick freezing can effectively reduce the loss of moisture and nutrients in beef during the subsequent cooking process. Specifically, the beef treated with LNF3 (-80°C / -60°C / -80°C) three-stage variable speed liquid nitrogen quick freezing technology has a cooking loss rate of only 39.20%, which is significantly lower than the beef treated with the conventional -18°C refrigerator ordinary freezing method, the latter of which has a cooking loss rate of up to 46.30%. This comparison clearly shows the superiority of liquid nitrogen quick freezing in maintaining beef quality. In addition, in terms of the content of volatile basic nitrogen and malondialdehyde, LNF3 (-80°C / -60°C / -80°C) three-stage variable temperature liquid nitrogen quick freezing technology also shows significant effects. Compared with the conventional freezing method (CF), the volatile basic nitrogen content of beef treated with LNF3 technology was reduced by 52.73%, which means that the freshness and flavor of the beef were better preserved. At the same time, the malondialdehyde content was also reduced by 12.05%. This change is conducive to reducing the oxidation reactions that may occur during the storage and processing of beef, further improving its edible quality. In summary, liquid nitrogen quick freezing technology has significant advantages in the preservation and quality maintenance of beef. In particular, the three-stage variable temperature liquid nitrogen quick freezing of the LNF3 group can effectively reduce the freezing time and cooking loss of beef, delay the spoilage of proteins and lipids, maintain better muscle tissue morphology, and maintain the fresh appearance characteristics of beef.

[0031] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 Schematic diagram of the experimental design and grouping scheme;

[0034] Figure 2 The freezing curves of yak beef under different freezing treatments and the freezing curves of ordinary refrigerator freezing are shown in the figure;

[0035] Figure 3 Schematic diagram of the change of pH value of yak beef under different freezing treatments;

[0036] Figure 4Schematic diagram of the changes in TVB-N value of yak beef under different freezing treatments;

[0037] Figure 5 Schematic diagram of the change of TBARS value of yak beef under different freezing treatments;

[0038] Figure 6 Schematic diagram of the changes in total thiol content of yak beef under different freezing treatments;

[0039] Figure 7 These are pseudo-color images of yak beef under different freezing treatments. The red area in the pseudo-color image indicates a strong signal (i.e., a large number of hydrogen protons), and the blue area indicates a weak signal (i.e., a small number of hydrogen protons). The distribution changes of light and dark areas indicate the distribution and migration changes of water inside yak beef.

[0040] Figure 8 The scanning electron micrographs of yak meat under different freezing treatments, magnification: 200;

[0041] Fig. 9 Schematic diagram of the changes in optical microscopic images of yak beef under different freezing treatments, magnification: 200. DETAILED DESCRIPTION

[0042] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0043] For example, the beef material used in Examples 1-8 is specifically yak beef, and subsequent experiments are conducted using yak beef material.

[0044] Example 1

[0045] A three-stage variable temperature liquid nitrogen quick freezing and fresh-keeping method for yak beef as described in this embodiment comprises:

[0046] S1: pre-processing the yak beef, such as trimming, removing fascia, and pre-cooling;

[0047] S2: Use three-stage variable temperature liquid nitrogen quick freezing to make the core temperature of the meat reach -18℃;

[0048] S3: After the samples were frozen and stored for 28 days, quality testing and analysis were performed.

[0049] In this embodiment, the step S1 specifically includes:

[0050] S1.1: Select fresh longissimus dorsi muscle of yak beef and remove the surface fascia;

[0051] S1.2: Cut the meat into chunks with a thickness of 2-3 cm and a weight of 150 g;

[0052] S1.3: Use sealed polyethylene bags and pre-cool and store in a refrigerator at 4°C to balance the initial temperature of the meat.

[0053] In this embodiment, the three-stage variable temperature liquid nitrogen quick freezing in step S2 includes a precooling stage, a phase change stage, and a deep cooling stage; specifically, it includes:

[0054] S2.1: Set CF as the control group, and freeze yak meat in a -18℃ refrigerator until the core temperature of the meat reaches -18℃;

[0055] S2.2: Use -80℃ liquid nitrogen treatment to quickly pass through the cooling stage. In the phase change stage, the liquid nitrogen temperature is set to three different treatments of -60℃, -80℃ or -100℃ for 5 minutes to quickly pass through the maximum ice crystal formation area. The -80℃ liquid nitrogen treatment in the phase change stage is defined as LNF1 group, the -100℃ liquid nitrogen treatment is defined as LNF2 group, and the -60℃ liquid nitrogen treatment is defined as LNF3 group;

[0056] S2.3: During the deep freezing stage, the liquid nitrogen temperature is set to -80°C until the core temperature of the meat reaches -18°C, so as to make full use of the cold energy in the first two stages and save liquid nitrogen consumption.

[0057] In this embodiment, step S3 specifically includes:

[0058] S3.1: Use an insertion thermometer placed in the center of the meat, and the temperature is -18°C as the end point of quick freezing; record the temperature curve;

[0059] S3.2: Transfer the samples of CF, LNF1, LNF2 and LNF3 to a -20℃ refrigerator for 28 days and then thaw at 4℃ for quality index determination;

[0060] S3.3: The experimental data were counted, processed, analyzed and plotted using Excel 2019, SPSS26.0 and Origin 2021.

[0061] In this embodiment, the index determination in step S3 includes: pH value determination, thiobarbituric acid value TBARS determination, volatile basic total nitrogen TVB-N determination, L* / a* / b* colorimetry determination, total thiol content determination, moisture distribution determination, scanning electron microscopy observation, and HE staining observation.

[0062] Example 2

[0063] The pH value of each sample was measured

[0064] The pH value is measured with a portable pH meter. Refer to GB5009.237-2016 "National Food Safety Standard for the Determination of pH Value of Food". After calibration with pH standard buffer, insert it directly into the sample to a depth of about 1.5 cm to measure its pH value. Each sample is measured 3 to 4 times, and the results are averaged. The change trend is as follows: Figure 3 As shown. It was found that the pH value of the LNF3 group showed a decreasing trend compared with the ordinary freezing group. This may be because of the large and irregular ice crystals formed in the cells during the ordinary freezing process, which will cause more cell fluid to leak out when thawing. Enzymatic hydrolysis in the cell fluid will further produce amino acids and alkaline substances, eventually leading to an increase in pH. In contrast, liquid nitrogen quick freezing technology can form smaller ice crystals in the sample by significantly shortening the freezing time, thereby reducing damage to the sample structure. Smaller ice crystals reduce the loss of cell contents and thus reduce the chance of enzymatic hydrolysis, which helps to maintain the stability of the pH value of yak beef during frozen storage. Therefore, compared with the traditional air slow freezing method, liquid nitrogen quick freezing technology can more effectively maintain the stability of the pH value of yak beef during frozen storage.

[0065] Example 3

[0066] Thiobarbituric acid value TBARS was performed on each group of samples:

[0067] TBARS is determined by spectrophotometry according to the national standard GB 5009.181-2016 "National Food Safety Standard Determination of Malondialdehyde in Food". The change trend is as follows Figure 4 As shown in Figure 3, there was a significant difference between the LNF3 and CF groups (P<0.05), and the fat oxidation of yak beef in the LNF3 group was slower.

[0068] The TBARS value mainly indicates the content of malondialdehyde, a secondary oxidation product of fat. Fat oxidation is an important factor affecting the quality of meat products and one of the reasons for the spoilage of meat products. Increasing the freezing rate can effectively reduce the mechanical damage to the muscle during the freezing process, reduce the area of ​​muscle tissue exposed to the air, and thus slow down the oxidation rate of fat.

[0069] Example 4

[0070] Volatile basic total nitrogen TVB-N was determined for each group of samples:

[0071] TVB-N is determined by the semi-micro nitrogen determination method in accordance with the national standard GB 5009.228-2016 "National Food Safety Standard Determination of Volatile Basic Nitrogen in Foods". The change trend is as follows Figure 5 shown.

[0072] TVB-N is usually used as an important indicator to measure the freshness of meat during storage. It mainly reflects the degree of protein corruption. The larger the value, the higher the degree of protein corruption. TVB-N is mainly composed of ammonia and primary, secondary and tertiary amines. It is produced by the combined action of endogenous enzymes and spoilage bacteria during the storage of meat, resulting in the degradation of nitrogen-containing compounds such as proteins and amino acids to produce volatile ammonia and amines. The TVB-N content of yak meat frozen by liquid nitrogen under LNF1, LNF2 and LNF3 remained at a similar level, indicating that the effect of liquid nitrogen freezing at different temperatures on the freshness of yak meat was similar. In contrast, the TVB-N content of yak meat treated with LNF3 group was significantly lower than that of CF group (P<0.05). This difference indicates that yak meat under traditional freezing conditions is more prone to corruption during storage, and its freshness is lower than that of yak meat frozen by liquid nitrogen. This may be because the cooling speed of liquid nitrogen quick freezing is fast, the damage to cells is small, and it has a good preservation effect.

[0073] Example 5

[0074] Colorimetric measurement of each group of samples:

[0075] The L* / a* / b* values ​​of the samples were measured using a CR-400 colorimeter, where L*, a*, and b* represent brightness, redness, and yellowness, respectively. The same sample was measured three times at different locations, and the average values ​​were taken. The trend is shown in Table 1. After different freezing treatments of yak beef, it was found that the L* value of the samples in the LNF3 group was significantly improved (P<0.05). This may be because the rapid freezing process caused the muscle tissue to shrink rapidly, forming a large instantaneous temperature difference, so that after thawing, the water migrated to the surface, resulting in light reflection and an increase in the L* value. The a* of the samples in the LNF3 group was slightly higher than that in the CF group, but significantly higher than that of the samples in the LNF1 and LNF2 groups (P<0.05). This indicates that the meat color of the LNF3 group is brighter and more ruddy. Based on the above findings, the color of the yak beef in the LNF3 group is brighter, while the color of the beef in the ordinary refrigerator group gradually becomes duller.

[0076] Table 1 Color changes under different freezing treatments

[0077]

[0078] Example 6

[0079] The total thiol group of each group of samples was determined:

[0080] Take 0.1g beef sample, use the total thiol content detection kit to process it, and measure its absorbance at 412nm. Figure 6As shown. The total thiol content of -20℃ ordinary freezing treatment is 1.60umol / g, which is significantly lower than 2.17, 2.53, and 2.87umol / g of the liquid nitrogen quick freezing treatment groups of LNF1, LNF2, and LNF3. The thiol groups in myosin and actin that constitute myofibrillar protein will continue to undergo oxidation reactions as the storage time increases, resulting in a continuous decrease in the thiol content, so it can be used to indicate the degree of protein oxidation. Under the same storage of 28 days, the LNF3 group can better inhibit protein oxidation than the CF group. This is because the lower the temperature of frozen storage, the higher the thiol content of myofibrillar protein. The thiol plays an important role in the stability of myofibrillar protein and slows down the reaction of myosin and actin to form disulfide bonds. The samples stored in ordinary freezing treatment have a slower freezing speed and form larger ice products, which destroy the structure of myofibrillar protein, expose the internal thiol, and then oxidize to form disulfide bonds.

[0081] Example 7

[0082] Determination of moisture distribution of each group of samples:

[0083] The moisture distribution of beef samples was determined by nuclear magnetic resonance (NMR) imaging analyzer. The samples were cut into (1.0×1.0×1.5) cm3 (length×width×height) and placed in a NMR tube, which was inserted with an NMR probe. The transverse relaxation time (T2) was determined using a CPMG pulse sequence. The NMR measurements were performed under the following conditions: waiting time (TW), 2000 ms; echo time (TE), 0.3 ms; and the number of repeated scans (NS) was set to 8. Each gradient was sampled three times.

[0084] After CPMG testing, samples were analyzed by multi-slice spin echo pulse sequence imaging, and coronal proton density images were acquired by MRI software and modified spin echo multi-slice spin echo sequence. T2-weighted imaging was performed on beef samples with a repetition time of TR = 500 ms and an echo time of TE = 18.14 ms. The water distribution in beef was presented as a surface map with an arbitrary color scale. All grayscale images were converted to pseudo-color images, and the distribution of water was visually presented using Niumag NMR V3.0 image processing software.

[0085] Magnetic resonance imaging (MRI) technology obtains different magnetic resonance signal intensities based on the different resonance frequencies of hydrogen protons in different positions, and converts them into images through spatial encoding technology to study the distribution of water in the sample. The brightness of the image is related to the activity and density of hydrogen protons. The brighter the image, the stronger the activity of hydrogen protons and the higher the density. MRI is often used to analyze the distribution of water in food. In magnetic resonance images with a dark blue background, the H-proton density is colored from high to low in a gradient of red-orange-yellow-green-blue. Blue indicates low-density areas with low water content, while red indicates high water content. Figure 7 As shown, a large number of red areas are evenly distributed in the center and edge of the sample in the LNF3 group, indicating that the moisture content is high and the cell structure is intact, in which different forms of water maintain better stability. A small number of red scattered points are distributed in the edge area of ​​the CF group, indicating that the immobilized water may be converted into free water, and the slow freezing rate leads to more thawing losses and the cell structure is destroyed.

[0086] Example 8

[0087] Scanning electron microscopy (SEM) observations were performed on each group of samples.

[0088] Cut the sample into small pieces and immerse in 10% neutral formalin solution for fixation for 24 hours. Cut 0.5cmx0.5cmx0.2cm (length, width and height) samples along the direction of muscle fibers, dehydrate with a series of alcohol concentrations, and then immerse in xylene. Embed the tissue in paraffin and cut it perpendicular to the direction of muscle fibers at a thickness of 5um. After bleaching, spreading and baking, HE staining is performed, and the slides are sealed with drops of gum. After drying for 24 hours, the slides are observed and photographed under a microscope at 200 times.

[0089] The microstructure of beef was observed by scanning electron microscopy (SEM). The processed beef samples were fixed in 2.5% glutaraldehyde solution at 4°C for more than 1.5 hours, and then cut into small pieces of (5×5×5) mm3 (length×width×height). Next, the samples were dehydrated with an ethanol series (50%, 70%, 90% ethanol); dehydrated with anhydrous ethanol for 3 times, each time for 20 minutes. After the critical point samples were dried, gold was sprayed, and the microstructure of the beef samples was observed by scanning electron microscopy.

[0090] SEM is an effective method for indirectly evaluating the voids of frozen food by observing the microstructure of frozen food after vacuum freeze drying. For fresh samples without freezing, the microstructure of the muscle presents a uniform, dense and neat appearance. However, freezing causes the microstructure of the muscle to change to varying degrees, and the most obvious change is the enlargement of the pore size. Generally speaking, the larger the diameter of the pore, the larger the size of the ice crystal. Among all the samples, the pores of the CF group samples were the largest. This may be due to the slower freezing rate produced by CF, which irregularly distributes large ice products inside and outside the cells, resulting in the expansion of the extracellular space and irreversible mechanical damage to the muscle tissue. The reduction in pore size is due to the higher freezing rate and faster heat exchange produced by the lower liquid nitrogen temperature, which prompts the rapid formation of a large number of small ice crystals in the muscle, causing less damage to the muscle, thus resulting in smaller and more uniform pores after freeze drying. The results show that the temperature regulation of the phase change stage in LNF3 has a certain effect on the microstructure of beef. In summary, LNF3 is a quick freezing mode that can save liquid nitrogen consumption while effectively maintaining the microstructure of muscle.

[0091] Example 9

[0092] HE staining was performed on each group of samples

[0093] The samples (5x5×3mm3) were fixed with Carnoy solution (60% pure ethanol, 30% chloroform and 10% acetic acid, v / v) for 48h. Then, they were gradually dehydrated with 50%, 75%, 85%, 95% and 100% ethanol, each concentration for 30min. The samples were then transparentized with xylene, immersed in paraffin and embedded, and then cut into 4μm thick sections perpendicular to the direction of the muscle fibers with a microtome. After that, the sections were placed on slides, dried in an oven at 55℃, and paraffin was removed from the samples with xylene. The sections were then dehydrated with 50%, 75%, 85%, 95% and 100% ethanol for 3min in sequence, stained with hematoxylin staining solution for 5min, and transferred to 1% hydrochloric acid ethanol solution for uniform staining. The sections were then rinsed with tap water for 15min, stained with eosin staining solution for 1min, rinsed with tap water for 5min, dried and sealed with neutral resin. Finally, the samples were observed with an optical microscope equipped with a digital camera and photographed at 100x magnification under the microscope.

[0094] The integrity of tissue structure is an important indicator of frozen meat quality because it affects the WHC and texture of the meat. The morphological images of beef after different freezing treatments were studied by hematoxylin and eosin (H&E) staining and optical microscopy. Fig. 9As can be seen in the figure, the red area represents the muscle tissue stained with H&E, and the white area refers to the muscle cell space. Generally, fresh samples show complete, multi-faceted, and neatly arranged muscle fibers. However, CF samples showed cell membrane rupture, obvious muscle bundle blurring, and muscle tissue disintegration. This may be due to the irregular large ice pieces formed by slow freezing, which squeezed the muscle cells and caused irreversible mechanical damage to the muscle fibers. LNF treatment can reduce these adverse changes. Fig. 9 As shown, with the LNF3 second freezing stage temperature regulation, the muscle tissue became denser and the muscle bundles were clearer. Compared with the air freezing group, LNF can produce smaller ice crystals and maintain better microstructure, which may be related to the freezing rate and ice crystal size and distribution. This result shows that the LNF3 group can save liquid nitrogen consumption while maintaining better muscle tissue morphology.

[0095] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A three-stage variable temperature liquid nitrogen quick freezing and preservation method for beef, characterized in that: include: S1: trimming the beef, removing the fascia, and pre-cooling and pre-treating; S2: Use three-stage variable temperature liquid nitrogen quick freezing to make the core temperature of the meat reach -18℃; S3: After the samples were frozen and stored for 28 days, quality testing and analysis were performed.

2. The method for preserving beef by three-stage temperature-variable liquid nitrogen quick freezing according to claim 1, characterized in that: The step S1 specifically includes: S1.1: Select fresh beef longissimus dorsi muscle and remove the surface fascia; S1.2: Cut the meat into chunks with a thickness of 2-3 cm and a weight of 150 g; S1.3: Use sealed polyethylene bags and pre-cool and store in a refrigerator at 4°C to balance the initial temperature of the meat.

3. The method for preserving beef by three-stage temperature-variable liquid nitrogen quick freezing according to claim 1, characterized in that: The three-stage variable temperature liquid nitrogen quick freezing in step S2 includes a precooling stage, a phase change stage, and a deep cooling stage; specifically, it includes: S2.1: Set CF as the control group, and freeze the beef in a -18℃ refrigerator until the core temperature of the meat reaches -18℃; S2.2: Use -80℃ liquid nitrogen treatment to quickly pass through the cooling stage. In the phase change stage, the liquid nitrogen temperature is set to three different treatments of -60℃, -80℃ or -100℃ for 5 minutes to quickly pass through the maximum ice crystal formation area. The -80℃ liquid nitrogen treatment in the phase change stage is defined as LNF1 group, the -100℃ liquid nitrogen treatment is defined as LNF2 group, and the -60℃ liquid nitrogen treatment is defined as LNF3 group; S2.3: During the deep freezing stage, the liquid nitrogen temperature is set to -80°C until the core temperature of the meat reaches -18°C, so as to make full use of the cold energy in the first two stages and save liquid nitrogen consumption.

4. The method for preserving beef by three-stage temperature-variable liquid nitrogen quick freezing according to claim 1, characterized in that: The step S3 specifically includes: S3.1: Use an insertion thermometer placed in the center of the meat, and the temperature is -18°C as the end point of quick freezing; record the temperature curve; S3.2: Transfer the samples of CF, LNF1, LNF2 and LNF3 to a -20℃ refrigerator for 28 days and then thaw at 4℃ for quality index determination; S3.3: The experimental data were counted, processed, analyzed and plotted using Excel 2019, SPSS26.0 and Origin 2021.

5. The method for preserving beef by three-stage temperature-variable liquid nitrogen quick freezing according to claim 1, characterized in that: The index determination in step S3 includes: pH value determination, thiobarbituric acid value TBARS determination, volatile basic total nitrogen TVB-N determination, L* / a* / b* colorimetry determination, total thiol content determination, moisture distribution determination, scanning electron microscopy observation, and HE staining observation.

6. Application of the method according to any one of claims 1 to 5 in quick-freezing and fresh-keeping beef.

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