PHYSICOCHEMICAL, QUALITY CHARACTERISTICS AND MICROBIOLOGICAL EVALUATION OF FROZEN AND CHILLED BEEF BURGER

The present study was planned to compare between frozen and chilled beef burgers. A total of 60 random samples of frozen and chilled beef burgers (30 of each) were collected during the period from May to August 2022 from different supermarkets for frozen beef burgers and refrigerators of markets for chilled beef burgers from Assiut City, Egypt. The samples were investigated for their sensory assessment, microbiological evaluation, physicochemical and keeping quality. To determine whether the results were acceptable, they were compared to Egyptian standards. The results showed that the frozen samples had lower scores of sensory evaluations, but higher in other tests than the chilled samples. Regarding chemical composition, no significant differences between mean values of moisture, fat and ash% of both frozen and chilled samples. The mean values of protein% of chilled samples were higher (15.72±0.66) than frozen samples (12.96±0.52), while the mean values of carbohydrate% were lower. Higher pH and phosphate values (6.39 ±0.028 and 0.373±39.28) in frozen samples in comparison with chilled samples (5.89±0.03 and 0.192±20.64). The obtained mean values of TBA (mg/kg) were 0.46±0.07 and 0.53±0.08 mg/kg, while TVBN were 12.16±0.64 and 11.93± 1.25 % in frozen and chilled samples, respectively. This study indicated defective manufacturing and storage in some chilled and frozen beef burger samples that aren’t of adequate hygienic quality and don’t meet Egyptian requirements. It is important to consider the potential public health risks associated with increased phosphorus (food additive) intake through additions. Regular monitoring and evaluations by researchers should be done for these chemicals


INTRODUCTION
Beef burger patties are among the most well-liked meat items in the fastfood industry (Clonan et al., 2016).Many factors contributed to the consumption of beef burger patties, including the influence of the advertising industry, globalization, shorter meal preparation times, and increased consumer demand for practical goods that are easily accessible and simple to make (Ramos et al., 2021).Fresh minced beef is a common ingredient in many meal preparations all over the world, particularly for burger patty recipes.To prevent bacterial deterioration, minced beef needs to be frozen or refrigerated as soon as it is purchased.Even if the meat is promptly packaged and cooled, the grinding process results in the leakage of tissue fluids, which serve as a rich nutritional source for a variety of bacteria and promote rapid microbial growth.Large amounts of animal fat in meat products speed up the oxidation of lipids, resulting in a shorter shelf life.As a result, the oxidation of lipids during processing and storage affects crucial quality traits like flavor, color, and nutritional value in meat products (Baioumy and Abedelmaksoud, 2021).Fresh or frozen skeletal muscles and the fatty tissue that accompanies them are used to make minced meat.As a result of the irreversible loss of the muscle tissue's cellular structure during the mincing process, minced meat turns into a product that is both incredibly nutritive and perishable.Such a product is susceptible to rapid bacterial growth due to the redistribution of surface contamination across the entire mass.The maximum storage time for raw meat from the moment of slaughter till the time of mincing is still an open issue for the meat industry (EFSA, 3783/2014).Yeast and mold are two examples of the many common microorganisms.Since it increases the likelihood of spoilage and degradation, resulting in significant financial losses and posing a public health threat due to the creation of a wide range of mycotoxins, contamination of meat products with various yeast and mold species is considered to be a real risk (Abd El-Wahab et al., 2021).The goal of the current investigation was to evaluate the physical, chemical, microbiological, and sensory characteristics of beef burgers stored in chilled and frozen conditions.

Collection of Samples:
A total of 60 random samples of frozen and chilled beef burgers (30 of each) were collected during the period from May to August 2022 from local and highly different retail markets of Assiut City, Egypt, for (frozen beef burgers) and refrigerators of markets and butchers for (chilled beef burger).Samples were collected and prepared in areas free from air currents on work surfaces that have been cleaned and sanitized assuring that no foreign sources of microbial contamination were introduced.Sample equipment was sterilized before usage, and it was kept safe from external contamination while in use.Then samples were placed into sterilized containers and placed in refrigerators or freezers.Frozen and chilled samples were transferred immediately with a minimum period of delay to the laboratory in an ice bag.Samples are identified by number and production/sample date.It is recommended that ground beef patties be evaluated within 3 months of frozen storage (AMSA, 2015).

Sensory Evaluation:
The analysis was performed according to (ISO 6658: 1985) and (AMSA, 2015).The test was performed for the different frozen and chilled cooked beef burger samples were assessed by 10-14 members of the Food Hygiene Department (with past experience in burger processing and evaluation) with randomly coded numbers and served warm to panelists to evaluate their sensory characteristics for color, odor, texture (juiciness and appearance), and overall acceptability.A 5-point hedonic scale measuring general acceptability is used, with one denoting "dislike extremely" and five denoting "like extremely."

Cooking properties:
Beef patties were cooked on an electrical grill for 7 min for each side until the internal temperature reached 73°C (Serdaroğlu et al., 2018).

3.2.2.
Cooking loss (%): Beef burgers were weighed before and after cooking to determine the cooking loss (CL %) using a method described by (Bakhsh et al., 2021) by using the following equations: Cooking Loss (%) = [raw weight (g) − cooked weight (g)] raw weight (g) x 100

Cooking yield:
The cooking yield of the beef burgers was determined by the method described by (Gök et al., 2011) and calculating weight differences for burgers before and after cooking, as follows: Cooking yield(%) = Cooked weight (g) Raw weight (g) x 100

Keeping quality characteristics:
5.1.Determination of pH value (Assanti et al., 2021): The pH was obtained at 25°C at the time of calibration, and before measured solutions the pH meter was calibrated with standardized buffer solution at pH 7.0 and pH 4.0, with a portable pH meter (Adwa, Waterproof PH Testers AD11, Romania).(EOS 639/2006).5.3.Thiobarbituric acid reactive substances (TBARS) values were determined according to Radha et al. (2014).

Microbiological examination:
The burgers were subjected to microbiological analysis, aiming to identify the main bacteria in the frozen and chilled beef burgers.

6.2.
Determination of Total psychotropic count (TPsC) in accordance with (ISO 4833:2013-1 protocol).6.3.Determination of Total Yeast and molds was performed using Sabouraud dextrose agar (SDA) supplemented with (Chloramphenicol and Gentamicin) and incubated for 5 days at 28 • C and data were presented as log10cfu/g in accordance to (ISO 21527-2:2008).

Statistical Analysis
Graph Pad Prism version 8.0.2 (263) software was used to conduct the statistical analysis.T-test analysis was used to analyze all of the results.To find significant differences, an unpaired t-test was run with a significance level of p < 0.05 (one-tailed P value).The gathered information was presented as mean ± SE.Trials and measurements were carried out in duplicate.

RESULTS
Table 1: Sensory evaluation for frozen and chilled beef burger samples (30 of each).
The significant difference between the mean of texture and overall Acceptance.Many customers continue to base food sa fety on colour, despite the fact that the int ernal.color of meat is an ineffective indic ator of its safety (Suman et al., 2016).The results in Table 1 demonstrated that frozen samples scored less favorably than chilled samples.
In contrast, the attributes of the chilled be ef burger samples were 3.30± 0.23, 3.20±0.23,2.77± 0.22, 3.20±0.19and 2.97±0.20,respectively.While chilled samples scored "moderately liked" with a mean of 3.10±0.22,which indicates good acceptance of the chilled beef burger samples.Frozen samples showed a slight reduction in the overall acceptability score (2.37±0.17).On average, extra ingredients are added to items in order to influence their quality, shelf life, general acceptance, and the physicochemical reactions that take place when they are frozen (Da-Wen, 2006).

The physical examination of beef burger samples (frozen and chilled).
The Cooking properties of frozen and chilled beef burger samples are shown in Table 2.

WHC of beef burger samples:
The ability of meat to hold onto its own or provide water quickly throughout processing is referred to as the waterholding capacity (WHC) of meat (Mona et al., 2021).There was a significant difference between the means of frozen and chilled samples.The mean WHC% values for the frozen and chilled beef burger samples were 19.27 ± 0.9 and 22.74 ± 1.26, respectively.Proteins that are denaturized during freezing have lower WHCs (Augustynska-Prejsnar et al., 2019;Utrera et al., 2014).

Shrinkage (%) of beef burger samples:
Cooking shrinkage has been linked mostly to meat protein denaturation, which releases water and fat from the beef batter (Pathare and Roskilly, 2016).Shrinkage% increase in chilled beef burgers.Significant difference among means of shrinkage% (P >0.05).The mean values were "12.18 ± 0.82" in frozen beef burger samples, while chilled samples were "21.41 ± 1.56".

Cooking loss (%) of beef burger samples:
The moisture lost during the heating of the product up to a standard core temperature is known as cooking loss (CL) (Godschalk-Broers et al., 2022).Regarding cooking loss (%) of frozen and chilled beef burger samples, highly significant differences among means (P < 0.05), the mean values were 13.54 ± 1.15, and 26.27 ± 1.68 in frozen and chilled beef burger samples, respectively.

Cooking yield (%) of beef burger
samples: According to Aleson Carbonell et al. (2005), yield in meat and meat products is linked to fat and water retention.The mean values of cooking yield % were (84.66 ± 1.6 and 72.77 ±1.8 in frozen and chilled beef burger samples, respectively.A significant difference between means of cooking yield % at both frozen and chilled samples,

Chemical composition (%):
When choosing meat products, consumers must have access to precise information on the chemical and nutritional contents (Erwanto et al., 2012).The nutritional characteristics of the examined meat products are revealed by chemical analysis, and these characteristics are crucial for consumer health and acceptability (Ali et al., 2020).Table 3 revealed the chemical composition % of samples of chilled and frozen beef burgers.

Moisture Content:
Obtained data revealed that the mean moisture contents of chilled and frozen samples, were 61.43±1.16 and 60.73± 0.66%, respectively.There were no significant differences between the chilled and frozen beef burger samples.These results were similar to those found in the beef burger by Ali et al. (2020), "61.02," and by Edris et al. (2012), "61.28±0.17." Because Egypt has its own statutory nutritional standards for meat products, the samples were compared to those standards (E.S, 1688/2005), to determine how acceptably they met those standards.11 (36.6%) of the frozen samples and 12 (40%) of the chilled samples exceeded the Egyptian Standard, which states that beef burger shouldn't have a moisture content more than 60% (E.S, 1688/2005).
The addition of water and non-meat substances, the amount and kind of processing and cooking, the fat percentage (inverse connection), and other variables can all have an impact on the moisture content (Ali et al., 2020).

Protein Content
The main element that gives the meat product its structure is protein (Tornberg, 2005).There was a significant difference in the mean protein.For chilled and frozen samples, the mean values of crude protein contents were (15.72±0.66)and (12.96±0.52),respectively.The mean values of protein percentage of chilled samples were higher than frozen beef burger samples by about 17.56%.These results were lower than those published by Edris et al. (2012) (15.22±0.18)and higher than those reported by Ali et al. (2020) (11.54) for the frozen beef burger.These findings demonstrated that 19 (63.3%) of the frozen samples and 11 (36.6%) of the chilled samples did not comply with the Egyptian Standards (E.S, 1688/2005), which set a minimum protein concentration of 15%.

Fat Content
A beef burger is a product that is enjoyed all over the world; they are typically made of 70% to 75% meat and 25% to 30% fat (Moghtadaei et al., 2018).The mean values of ether extract content for chilled and frozen samples were 12.60±1.19and 10.86±0.86,respectively.Fat analyses did not show a significant difference (p > 0.05) between means and they fulfilled the requirements of the Egyptian Standards (E.S, 1688/2005), which sets a maximum of 20% for lipid content.They also revealed that two (6.6%) of frozen samples and four chilled samples (13.3%) did not meet Egyptian standards.These results were lower than that reported by Ali et al. (2020) "17.13" andby Edris et al. (2012) "19.80±0.19" in the frozen beef burger.
The industry's attempts to save costs and the development of low-fat meat products could have resulted in adulteration, which would have prevented their products from meeting the required standards (Ali et al., 2020).

Ash Content
Ash is a representation of all the minerals contained in food, including sodium, phosphorus, and iron, which can come from salt and spice additions as well as meat used as a raw material (Fernández-López et al., 2006).There is no significant difference between the means (p > 0.05).It is stated that the mean ash concentrations for chilled samples were (2.85±0.13)and frozen samples were (2.89±0.08).These results were lower than those reported for frozen beef burgers by Ali et al. (2020), "4.01," andEdris et al. (2012), "3.36 ±0.07."

Carbohydrate Content
Mainly, carbohydrates in burgers are from the use of starches as ingredients.Starches, such as maize, tapioca, rice, potato, and wheat, have been used as meat filler and water binder and this result could be due to using of cheap ingredients like rusk, bread crumbs, cereal, and soya protein (Joly and Anderstein, 2009).
A significant increase in carbohydrates in frozen burger samples than in chilled samples.The mean values of total carbohydrate contents were (12.55± 0.58) in frozen samples and (7.27± 0.66) in chilled samples.These results were higher than that reported by (Ali et al., 2020) of "6.29".The Egyptian Standards (E.S, 1688/2005) for beef burgers stated that carbohydrates should not exceed 10%.This met the requirements of (EOS, 2005) in chilled samples only and showed that 14 of the frozen samples (46%) were not in compliance with (Egyptian standards 1688/2005).

pH values
One of the most perishable things among the many foods is meat, which has a moderate pH and a high nutritional and moisture content.Meat spoilage and nutrient breakdown are primarily influenced by microbial growth, lipid oxidation, and enzymatic autolysis.As a result, the meat develops unpleasant flavors and odors, forms slime, and becomes discolored, rendering it unfit for human consumption (Pellissery et al., 2020).
Table 4 shows the keeping quality characteristics of beef burgers (frozen and chilled).Highly significant differences (p < 0.05) were obtained in pH values in both frozen and chilled beef burger samples.In frozen samples, burgers registered higher pH values (6.39 ±0.02.) than the chilled sample (5.89±0.03).The results reported by Edris et al. (2012) of "5.97±0.02"were higher than the present study on the frozen beef burger.While results reported by Wesam et al. (2022) in refrigerated beef burger samples were "6.0 ± 0.3" and were slightly higher than the present study in the chilled beef burgers.All pH values in the chilled burger sample were within the typical value "6" in chilled meat established by Egyptian Standard (ES: 3602 /2013), while 53.3% of the frozen sample weren't in compliance with the acceptance limit of pH values for (5.6-6.2) that established by Egyptian Standards (ES: 1522 -2005) for frozen meat.

Food Additive (Phosphates):
Food-grade phosphates are one of the food additives that are frequently used in a variety of products.For a variety of technological reasons, phosphorus additions (E338-341, E343, and E450-452) are being used more frequently in processed and fast food, particularly in the meat industry.They improve food stability, preserve moisture or color, emulsify ingredients, and enhance flavor, in addition to increasing water holding capacity (WHC).Despite the advantages of its technology, it has been estimated that food additives known as "hidden phosphorus" account for 50% of daily phosphorus (P) intake in the Western world (Calvo et al., 2019).Alkaline phosphates are employed in all phosphate blends that are used in meat, and when they are added to somewhat sour meat, the pH of the meat product rises (Long et al., 2011).
The label does not specify the percentage of phosphate that has been analyzed in the samples.(53.3%) from frozen beef burger samples were not accepted according to Egyptian Standards (E.S, 1688/2005).While all chilled samples (100%) fell within the permitted range as indicated by the Egyptian standard specifications that were no more than 0.3%.These variations could be explained by the addition of alkaline phosphates during the production of meat products.This increase in pH will cause electrostatic repulsion between or within the proteins in the meat, leading to a higher WHC (Glorieux et al., 2017).Protein-bound phosphorus found naturally is absorbed more slowly and inefficiently (60%) than phosphorus found in dietary additives, which is promptly and nearly completely absorbed (Wallace et al., 2021).In patients with chronic renal disease and bone health issues, a link between high serum phosphate levels (hyperphosphatemia) and excessive phosphate intake has long been shown (EFSA FAF Panel, 2019).

Lipid oxidation of beef burgers (TBARS):
One of the key issues limiting the quality and acceptability of meat and meat products is lipid oxidation.The oxidative rancidity that develops in beef products during storage is measured using the thiobarbituric acid (TBA) value (malonaldehyde/kg) (Abdelhakam et al., 2019).Both the frozen and chilled beef burger samples showed no significant differences (p > 0.05).In frozen samples, TBARs' mean values were (0.46±0.07 mg MDA/kg), while chilled samples showed TBARs' mean values (of 0.53±0.08mg MDA/kg).Nearly similar results obtained by (Hassanien et al., 2018) were "0.44", while lower than those (Malak and Abdelsalam, 2021) "0.66±0.02".On the other hand, our findings were higher than those published by Edris et al. (2012) for frozen beef burger samples ("0.11 ±0.01") and Wesam et al. (2022) for refrigerated beef burger samples ("0.25± 0.025").According to the Egyptian standards (ES: 3602 /2013) and (ES: 1522 -2005) is not more than 0.9 mg malonaldehyde/kg sample.Four samples (13.3%) of frozen beef burgers and five (16.6%) from chilled samples were recorded above the permissible limit.High levels of bacteria are the cause of meat rotting, while aerobic circumstances result in moisture and protein content, both of these elements can also lead to the oxidation of lipids and proteins, which can jeopardize the safety of food (Tometri et al., 2020).

Total Volatile Nitrogen (TVN mg/100g)
Being meat a perishable product, it undergoes chemical compositional changes during storage as a result of the activity of endogenous enzymes and microbes.A common biomarker of the breakdown of proteins and amines is total volatile basic nitrogen (TVB-N).TVB-N increases with beef storage and is in line with other biomarkers of decomposition (i.e., a function of duration, temperature, packaging, etc.), however, these increases are not always constant (Bekhit et al., 2021).The mean value of TVN value was 12.16±0.64mg% for a frozen beef burger and 11.93± 1.25 mg% for a chilled beef burger.No Significant difference means of TVB-N (p > 0.05) in frozen and chilled beef burger samples.These findings exceeded those of Edris et al. (2012), who reported "10.15±0.32mg%" in the frozen beef burger.All frozen samples fell within the permitted range as indicated by the Egyptian standard specifications in 2005 for total volatile nitrogen concentrations, which were not to exceed 20 mg N/100 g.While four samples (13.3%) of chilled samples were recorded above the permissible limit.

Microbiological examination:
Due to a rapid decline in quality indices and microbiological development, beef burger patties have a maximum shelf life of 3 days at 4°C (Parafati et al., 2019).While some additives with antioxidant functionality are permissible in fresh minced beef preparations, as mentioned in Regulation EU 601 (2014), no additive with antimicrobial activity is allowed.If the storage temperature and duration are not controlled, pathogenic bacteria can grow and produce poisons in beef burgers, making them potentially deadly products (Saleh et al., 2022).Table 5 shows the results of the microbiological analysis of frozen and chilled beef burger samples.According to EOS (2005), the analyzed samples were within the parameters that permitted them to be used for sensory analysis.

Total bacterial count (log10 cfu/g)
The aerobic plate count (APC) or total viable count (TVC) are two of the terms used to describe the standard plate count (SPC), one of the most commonly used tests used to determine the microbiological quality of food (Shaltout et al., 2022).
There was no discernible difference between the total bacterial count (TBC) and mean (log10 cfu/g) in this study in examined chilled and frozen samples (P >0.05).The average values of the chilled and frozen beef burger samples were 4.62 log10 cfu/g and 5.04 log10 cfu/g, respectively.These numbers were therefore deemed to have been met as advised by EOS (2005).

2. Total psychotropic count (log10 cfu/g)
Psychotropic bacteria are the primary cause of meat product spoilage when kept in a refrigerated environment due to their propensity to proliferate at low temperatures.The total amount of psychoactive bacteria can tell you a lot about how well various meat products will keep (Shaltout et al., 2022).No significant difference among means of total psychotropic count (log10cfu/g) in both types of the examined samples (P > 0.05), the mean values for frozen beef burger samples were (3.58± 3.34 log10 cfu/g), whereas the mean values for chilled samples were (3.82± 3.54 log10 cfu/g).Shaltout et al. (2022) looked at 25 samples of beef patties that were ready to eat, and the results showed that the range for the total amount of psychotropic count was > 10 2 to > 10 2 .

Total yeast and mold count (log10 cfu/g)
Fungi can induce three forms of disorders, namely mycosis, mycotoxicosis, and allergies, and are therefore regarded as a major public health risk (Abuzaid et al., 2020).

CONCLUSION
The current study concluded that the investigated beef burgers had a noticeably different set of quality characteristics.In comparison to the Egyptian standards, the majority of them had higher moisture and carbohydrate levels, and some samples had lower protein content.The content of phosphorus from analyzed frozen beef burger samples (53.3%) was not in the permitted range as indicated by the Egyptian standard specifications (2005), this indicated inadequate quality control and improper manufacturing practice.This finding should be taken with caution, as phosphorus from processed meat products is just one part of the total phosphorus intake from various dietary sources.

RECOMMENDATION
Due to the increased consumption of processed meat, high phosphorus intake from additives should be taken into account as a potential public health concern.Decreased usage of these additives as phosphates in processed meat products by meat industries is recommended and produced in accordance with good manufacturing practices (GMP), especially for those products (beef burgers) that are majorly consumed by child populations.Researchers should conduct periodic evaluations and monitoring on a regular basis for these and other additives produced in the country.Also, to prevent excessive shrinkage during cooking, the amount of added fat in the meat product ingredients should be carefully m onitored.These findings suggest that the food industry should move towards using healthier, "greener" technologies for preservation, as this is necessary for the true change of food systems towards sustainability and nutrition sensitivity.2021 Salim, D.A. and El-Roos, N.A.A. (2013)

Table 2 :
Physical examination of frozen and chilled beef burger samples (30 of each):

Table 3 :
Chemical composition (%) of frozen and chilled beef burger samples (30 of each).

Table 4 :
Keeping quality characteristics of frozen and chilled beef burger samples (30 of each).

Table 5 :
Microbiological examination of frozen and chilled beef burger samples (30 of each).
Commission Regulation (EU) No 601/2014 of 4 June 2014 amending Annex II to Regulation (EC) No 1333/2008 of the European Parliament and of the Council as regards the food categories of meat and the use of certain food additives in meat preparations.Off.J. Euro.Union L. 166.Saleh, W.A.M.; El-Desouky, A.E.; Sharoba, A.M. and Osheba, A.S. (2022): Vital Value of Coriander and Fennel Volatile Oils on Quality Beef Burger During Cryopreservation J. of Food and Dairy Sci., Mansoura Univ., Vol. 13 (7):109 -117.