Beef quality assessment of local and imported sources illustrating a contrary view that freezing is the best way of beef preservation using morphometric analysis

The histological analysis of local and imported beef samples throughout storage at various intervals in 4 °C, before and after being frozen at − 18 °C, to detect the changes happened in the microstructure of muscle fibers to evaluate the meat nutritive properties in a step toward rapid evaluation of meat quality. The obtained results illustrated that freezing–thawing step of beef leads to the loss of its muscle fiber structure due to the high moisture content failure, highlighted the idea that imported beef show significant shrinkage in their muscle fibers from the beginning of its purchase to consumers as they seem to be imported as frozen and thawed just before exposed and sold as fresh. Through consumption survey, however consumers prefer local meat, it was detected that 67% of population is eating imported beef with 39.4% more than twice per week. Therefore, consumers’ minds should be changed to depend on locally slaughtered beef on facing their needs of the recommended daily intake of protein.


Introduction
Meat is a main source of many nutrients which are necessary to sustain the normal functioning of the human body.Meat is concentrated sources of high biological value protein which is the main source of nine essential amino acids, as well as micronutrients like iron, selenium, zinc and vitamin B12 needed by the human body.Beef which categorized as red meat is highly pleased by consumers due to their nutritive value, pleasant palatability, and high quality.Therefore, beef intake in a rationale amount should be recommended as part of a healthy diet [1].Beef consumption in Egypt reached 720,000 metric tons (MT), up by 3.5% greater than before confirmed by the United States Department of Agriculture (USDA) official 2019 [2] and it was expected to be 1,354,000 tones per 2029 according to OECD-FAO Agricultural Outlook (https:// data.oecd.org/ agrou tput/ meat-consu mption.htm).Egypt's meat-centric food culture remains unchanged, then due to beef affordability and low Egyptian income, consumers replaced the local meat slaughtered in the country by the imported meat (Brazilian and Indian slaughter).Brazil, Australia, Sudan, and Ethiopia are Egypt's main suppliers for 1 3 imported beef as it is considered the 11th largest country in the world in beef importation due to the shortage of meat production in the country [3].
Meat is a perishable food that can be easily spoiled by the growth of different kinds of spoilage bacteria then pose potential danger of diseases.It has been estimated that 25% of food of plant and animal origin, globally is wasted postharvest or post slaughter, due to microbial spoilage, so that this is actually the most common cause of alterations in food quality [4,5].Storage and handling of meat besides the origin of meat are considered critical reasons for the deteriorative changes and quality problems of the meat.For instance, during freezing and thawing of the meat, there would be a defrosting of muscle fibers, which adversely affects the physico-chemical properties of the product and consequently change their nutritive value for consumer [6].Therefore, it is crucial not only to preserve their initial properties but also to improve them in the process of technological processing.
Meat quality can be detected by many methods, one of such detectors is through the histological analysis of the meat components.Histological examination of meat is performed to determine the degree of aging and freshness by detecting changes in the microstructure of muscle tissue.Today, different bioimaging techniques are available for the microscopic determination of food elements.Commonly, the most used method is optical microscopy which identify all the structures of the meat via their morphological characteristics [7].The topographic histology of meat allows the evaluation of the quality of meat via detecting and measuring the content of tissues (muscle, connective and adipose).In addition, the histological image of the meat can identify and evaluate the composition.In this regard, several histological studies have been published for quality evaluation of hotdog [8], hamburger [9], and other meat products [10,11].It was recorded that 25% of studies made on meat and meat products were depending on histology as a discipline [12] because histological examination provides valid information besides, the low cost of the technique [13].In addition, Histologic methods provide an accurate tool in detecting specific tissue components and architectural alterations induced by various type of meat processing [10,14].
Freezing is among the most used and important methods for food preservation especially for meat and their products to save their qualities until reach the consumers [15].However, freezing followed by thawing affect adversely on the nutritive value of the meat as it causes structural changes in the meat tissue.In addition to this, freezing usually results in a decrease in the diameter of the muscle fiber and in the length of the sarcomeres [16,17].Besides muscular structure change, freezing-thawing process results in chemical change modifications, thus interfering with the overall organoleptic quality of the beef [18].Further freezing and thawing of meat more than once might resulted in change of meat color, loss of moisture and increased oxidation of meat protein in which electrons are transferred from one atom to another.When this occurs in meat, it can lead to a significant deterioration in quality [19][20][21].Consequently, any change in meat capability to hold and sustain moisture leading to a significant effect on the tenderness and juiciness of the meat [21,22].
The purpose of the current study is to assess the degree of cellular damage of beef caused by freezing and thawing process and evaluation of muscle quality induced histologic alterations using the microscopy tool, which is a rapid, easy, inexpensive, accurate histological examination giving an obvious information without interpretation bias and comparing the quality of both local and imported beef samples marketed for consumers in Egypt.The data will change consumers about their habit to use the imported beef as a source of protein in their life.

Sample collection
Beef samples of local and imported slaughtered carcasses sources, were purchased from the local markets in Mansoura City, Egypt, to evaluate the histological changes happened during its storage in fridge temperature (4 °C) before and after sample freezing at − 18 °C for the determination of nutritive value of the meat preserved and detect the effect of freezing on the different source of beef to evaluate their quality.
From each source (local and imported beef samples), six different batches were used for the histological analysis at different intervals as follow; directly after purchase, after 6 h and 24 h after being left in fridge temperature (4 °C), after being totally frozen; sample was taken after complete thawing, then after 6 h and 24 h of storage in fridge temperature (4 °C).From each batch, the sample which taken for the analysis was weighted approximately 5 g each.

Histological technique
The samples were fixed overnight using 10% neutral buffered formalin.Fixed samples were processed in ascending concentrations of alcohol (from 70 to 100%), then xylene and embedded in in paraffin wax to produce blocks [23].The blocks were cut into 5 μm thick sections and stained with hematoxylin-eosin (HE) [24] and were examined using camera (Olympus model C7070WZ) attached light microscope (Olympus CX41), images were captured and analyzed using image J software (version 1.32 J).

Histometric analysis
To evaluate the area of myofiber, and the area in-between myofibers, Histometric analysis was done using the image J software (version 1.32J) [25].Each sample represented by three HE sections, each HE section was examined for the percentage (%) of the extracellular spaces locating in-between the red myofibers of each HE section.On the other hand, a fixed number of myofibers were examined in each section and the myofiber area was measured (µm) (Supplementary Fig. 1).

Statistical analysis
The data obtained were subjected to statistical analysis and expressed as mean ± standard deviation to determine the difference of area of myofiber and the percentage of extracellular spaces in-between myofibers among the different intervals using Kruskal-Wallis test followed by Dunnett's test (p < 0.05).The data were analyzed using Predictive Analytics Software (PASW Statistics 18).

Population survey
Total of 200 populations of various ages had been asked many questions about their beef source and their habit to consume fast food including beef from restaurants and street vendors in Egypt.

Results and discussion
The current study revealed the histological changes occurred to the beef samples of two different sources (local and imported) when stored for certain intervals under fridge storage condition (4 °C) with and without freezing, highlighting the effect of freezing and compare between the quality of beef of both sources.

The morphological features of the muscle fibers of local beef samples during the different intervals (0, 6 h and 24 h) from purchase and after applying freezing
The histological sections of local beef which were directly taken after purchase showed skeletal muscle structure consisting of bundles of regular eosinophilic muscle fibers separated by extracellular spaces (Fig. 1a).After 6 h of storage in fridge temperature (4 °C), the meat autolysis started to begin and was determined by the decrease in myofiber diameter by 120 µm which measured by histometric analysis (Fig. 1b, Table 1).By passing 24 h in fridge, the section of local beef showed a continuous significant (P < 0.05) decrease in the myofiber area by shrinkage degree of 147 µm (Table 1) and significant (P < 0.05) increase in the extracellular spaces (Fig. 1c, Table 1) with an apparent loss of the white lines in between myofibrils.
The aforementioned results agreed with previous study which described the effect of storage on meat histological structure, it stated that after slaughter, meat had few extracellular spaces and more muscle fibers while after 12 days in 4 °C, the muscle fibers shrank and the extracellular spaces increased due to the expel of intracellular water to extracellular spaces [26] Another study showed the degradation of muscle fibers 72 h postmortem and reflected on the histological structure as samples showed uneven staining with fragmented myofibers [27,28].These data revealed that autolysis of muscle fiber happened directly after animal slaughter due to postmortem glycolysis process which is of temporary benefits for meat quality development [29].
Checking the defrosted local beef sample after freezing at − 18 °C and thawed completely histologically, the image (Fig. 1d) revealed myofiber area with diameter of 148.83 µm which is smaller by 130 µm when compared to its analogous examined before freezing (Table 1).After 6 h of leaving the frozen local beef sample in the room temperature, the shrinkage of myofiber continued reaching a diameter of 108.35 µm (Fig. 1e; Table1) followed by slight shrinkage after passing 24 h after thawing reaching 100.69 µm leaving irregular-shaped myofibers with the smallest extracellular spaces detected for the local beef sample (Fig. 1f, Table 1).Interestingly, the extracellular spaces in between the muscle fibers showed significant decrease (P < 0.05) detected by histometric analysis (Fig. 2, Fig. 3) which could indicate the loss of water volume retained during freezing as it was explained before along with the myofibers shrinkage, the fluid directly oozed to extracellular space and easily lost as drip [30].

The morphological features of the muscle fibers of imported beef samples during the different intervals (0, 6 h and 24 h) from purchase and after applying freezing
The imported beef taken directly after purchase without being frozen showed small myofibers with appearance of irregular shaped myofibers (Fig. 2a).By passing 6 h of storing in 4 °C, there was a slight increase in the myofiber area (Fig. 2b) which continued through the whole storage period of 24 h in fridge temperature (Fig. 2c).Analyzing the sample after being frozen, the defrosted muscle fibers shrunk leaving wide extracellular spaces in-between after and within 6 h interval in fridge temperature creating an irregular myofibers (deformed myofibers).However, by leaving the sample    24 h after being thawed in fridge temperature, the regular myofibers totally disappeared and replaced by large irregular eosinophilic myofibers with smaller extracellular spaces in-between (Fig. 2f ).The morphological changes in the muscle structure under the microscope had been confirmed via histometric analysis of the myofibers area (µm) and the extracellular space (%) measurements (Table 2).The myofibers area (µm) was the smallest size where the imported beef sample was defrosted directly after being stored at − 18 °C measuring 88.72 µm which was significantly (P < 0.05) small when compared by all the measure of myofibers area in all the intervals.The extracellular space (%) of the imported beef sample varied throughout the different intervals showing the lowest percentage in sample which stayed for 24 h at 4 °C storage 23.727% giving significant (P < 0.05) difference when compared with other storage intervals for the same sample with and without freezing.The largest extracellular space (P < 0.05) was detected in the imported beef estimated directly after purchasing without any freezing (39.181%), there was no difference between the histometric measure of the extracellular space between interval (0 h) for directly purchased and defrosted sample, such data suggesting that the imported beef had been already subjected to many degrees of freezing-thawing before purchasing for consumers, making them of low quality protein source.Researchers suggested that the drip loss and denaturation of muscle protein depend on storage temperature, they were the lowest at − 18 °C which confirms the unpleasant changes in samples when left in unsaved storage conditions rather than the ultimate storage temperature [31].

The statical significance results of myofibers diameter and extracellular space measurements between local and imported beef samples during the different intervals (0, 6 h and 24 h) from purchase before and after applying freezing
Comparing the results of morphological changes among the different intervals of storage of both local and imported beef samples, there was a significant (P < 0.05) change in the myofibers diameter among all the readings with the freshly purchased local beef samples having the largest myofibers diameter (278.35µm), however, the reading of the myofibers diameters of the corresponding samples after being defrosted were totally changed and the diameters of the local beef sample in the different storage intervals (0, 6, 24 h) significantly decreased while the diameters of the myofibers of imported beef were highly increased (P < 0.05) when compared with the local beef samples (Fig. 3).Such shrinkage of muscle fibers in local beef sample after thawing due to denaturation prove the phenomenon of freezing-induced protein denaturation with various analytical parameters, and evidence from literature showing the relationship between freezing-thawing and denaturation of myofibrillar and sarcoplasmic proteins, respectively [27,28,32] while the drastic changes in the imported sample via different storage intervals at 4 °C resulted from the repeated freezing-thawing operation which leaded to total destruction in muscle fibers [33], that destroy the regular shape of muscle structure giving increase in their diameters reaching 183.19 µm (Table 2).Analyzing the results of extracellular space, (Fig. 4), there was a significant (P < 0.05) difference between the records of local and imported beef sample results in all the intervals before and after freezing (0, 6 and 24 h), respectively, except for the results of each sample after being frozen and thawed for 24 h at 4 °C, there was no significant difference.
From the whole data, freezing-thawing process of local beef which frozen for the first time in our experiment was affected gradually on the beef quality by the natural denaturation of muscle protein and changing the water capacity of the tissue.However, the initial results of histometric analysis data of imported beef sample examined without freezing give an indication that imported beef had been already subjected to repeat freezing-thawing process.Furthermore, the destruction of the muscle fiber, resulted in the histometric analysis of imported beef sample after being frozen, suggesting that imported beef of low quality and of low nutritive value when compared by our local beef sample.Similarly, histological change attributable to the alterations in tissue microstructure, and protein properties resulted from multiple freezing and thawing of meat were studied previously [19,22].

The surveillance results
The consumer survey illustrated that there is no significant difference between choosing among local or imported beef as 52.9% of people prefer local than 47.1% of people who choose imported beef.Regardless the percentage of people who usually choose local beef, around 65.2% of populations are eating fast food including imported food at least once per week, followed by 23.9% of people who not used for fast food and 11% who never eat fast food at all.Such data referred that most people are eating imported beef even they do not choose imported beef because the majority of restaurants and street vendors in Egypt are rely on imported beef in their dishes.

Conclusion
Through simple histological analysis, beef quality can be determined by estimating the morphological structure of muscle fiber and the extracellular space surrounding muscle fibers where the muscle fiber diameter decrease and dramatic change of their shape beside the increase in intermuscular space.The current study confirmed that although the freezing of meat including beef is used, as a method of preservation, it leads to shorten the shelf-life as well as production of low-quality beef via the freezing-thawing step especially in imported beef which is thought to be frozen and sold for consumers as fresh.Therefore, public education about that the consumption of local slaughtered beef is better, as a source of daily intake of protein, than the imported beef which seems to be thawed before selling to them as fresh ones.Further analysis methods are needed to strengthen the idea that most of imported meat purchased in Egypt of low quality.

Fig. 1
Fig. 1 Photomicrograph of local beef sample before and after being frozen at − 18 °C.The sample stained with HE and examined under microscope directly after purchase (a), after 6 h of storage at 4 °C (b), after 24 h of storage at 4 °C (c), after complete freezing followed by complete thawing (d), after 6 h of thawing stored at 4 °C (e), and after 24 h of thawing stored at 4 °C (f); MF = regular myofiber, E = extracellular space, *irregular myofiber; Scale bars = 50 µm

Fig. 2 Fig. 3
Fig. 2 Photomicrograph of imported beef sample before and after being frozen at − 18 °C.The sample stained with HE and examined directly after purchase (a), after 6 h of storage at 4 °C (b), after 24 h of storage at 4 °C (c), after complete freezing followed by complete thawing (d), after 6 h of thawing of stored at 4 °C (e), and after 24 h of thawing stored at 4 °C (f); * = irregular myofiber; Scale bars = 50 µm

Fig. 4
Fig.4 Graph showing the percentage of extracellular space (%) both samples (local and imported beef ) in three different intervals (0, 6, 24 h) before freezing (A) and after being frozen (B); a and b significant different (p < 0.05) for each interval

Table 1 The
4yofiber area diameter (µm) and extracellular space percentage (%) local beef after purchase and after being frozen in three different intervals (0, 6, 24 h) stored at4°C Mean values with different superscripts from "a" to "e" within the same row are significantly different at P < 0.05 .Mean values with different superscripts from "w" to "z" within the same row are significantly different at P < 0.05 Sample examined directly after purchase Sample directly examined after being frozen at − 18 °C

Table 2 The
4yofiber area diameter (µm) and extracellular space percentage (%) imported beef after purchase and after being frozen in three different intervals (0, 6, 24 h) stored at4°C Mean values with different superscripts from "a" to "e" within the same row are significantly different at P < 0.05.Mean values with different superscripts from "w" to "z" within the same row are significantly different at P < 0.05