Advances in sustainable food processing technologies

Advances in sustainable food processing technologies: Antimicrobial resistance, process adaptation and consumer acceptance

M.SAJJAD

  • Department of food science and technology, (2020-2024)
  •  University of Agriculture Faislabad (Sub Campus burewala)

abstract

Sustainable food production has become a worldwide priority. Sustainable technologies provide clean-label, nutritious food products and solutions to antimicrobial resistance. Antimicrobial resistance (AMR) remains of major interest for different types of food stakeholders since it can negatively impact human health on a global scale. Antimicrobial-resistant bacteria and/or antimicrobial resistance genes (transfer in pathogenic bacteria) may contaminate food at any stage, from the field to retail. Research demonstrates that antimicrobial-resistant bacterial infection(s) occur more frequently in low- and middle-income countries (LMICs) than in developed countries. Worldwide, foodborne pathogens are a primary cause of morbidity and mortality. The spread of pathogenic bacteria from food to consumers may occur by direct or indirect routes. Therefore, an array of approaches both at the national and international level to control the spread of foodborne pathogens and promote food safety and security are essential. Zoonotic microbes can spread through the environment, animals, humans, and the food chain. Antimicrobial drugs are used globally to treat infections in humans and animals and prophylactically in production agriculture. Research highlights that foods may become contaminated with AMR bacteria (AMRB) during the continuum from the farm to processing to retail to the consumer. To mitigate the risk of AMRB in humans, it is crucial to control antibiotic use throughout food production, both for animal and crop agriculture.Antibiotics have been overused and misused for preventive and therapeutic purposes. Specifically, antibiotics are frequently used as growth promoters for improving productivity and performance of food-producing animals such as pigs, cattle, and poultry. The increasing use of antibiotics has been of great concern worldwide due to the emergence of antibiotic resistant bacteria. Food-producing animals are considered reservoirs for antibiotic resistance genes (ARGs) and residual antibiotics that transfer from the farm through the table. The accumulation of residual antibiotics can lead to additional antibiotic resistance in bacteria.Therefore, this review evaluates the risk of carriage and spread of antibiotic resistance through food chain and the potential impact of antibiotic use in food-producing animals on food safety. This review also includes in-depth discussion of promising antibiotic alternatives such as vaccines, immune modulators, phytochemicals, antimicrobial peptides, probiotics, and bacteriophages.

KEYWORDSAntibiotic alternative; Antibiotic resistance; Farm-to-table; Food safety; Food-producing animal; Residual antibiotic.

INTRODUTION

(1)Sustainability is an essential part of our modern food production system. Carrying out food research that considers environmental, social, and economic factors, is a major objective for food producers and researchers. Strategic development and use of technology can greatly assist in the progression toward a more sustainable food system.
A sustainable future food supply in the face of depleting natural resources, climate change, rapid urbanization, changing demographics, and a growing global population is a global challenge. The demand for resources has paralleled the history and development of mankind. The period of unprecedented growth and the scientific and technological progress after the two World Wars in the twentieth century caused significant depletion to existing resources and the environment.

Increasingly, the focus is on providing sustainable diets that have low environmental impacts and improving the well-being of populations both now and into the future (2). There is an imperative for all parts of the food chain, from production, processing, packaging, storage, to the delivery of food to the consumer, to take steps to make efficient use of resources in each of their operations to ensure healthy diets for an increasing population, against changing demographics, and an increasingly urbanized world.

Food processing is a critical element in the food supply chain. Processed foods have been part of civilization since ancient times. Fire use by humans is related to cooking. Knapped stones in Kenya, dating back to 1.5 million years ago, were identified as being evidence of exposure by humans to high heat (3). Meat has been roasted more than 1.8 million years; bread made 30,000 years ago; beer, wine, and cheese first produced between 7,000 and 5,000 bc, and olive oil and palm oil introduced between 5,400 and 3,000 bc. Other foods available in the bc era include pickles, noodles, chocolate, bacon, fermented flavorings, and sugar, and with many other foods introduced through the ages (4). Even in ancient times, both primary (e.g., drying, milling, oil extraction) and secondary processing (i.e., when products of primary processing are formulated and manufactured into processed foods) were employed to convert produce into safe and palatable foods and to extend shelf life. There have been major brands and processing companies that capitalized on the demand for processed food for the masses through the years. Food processing also creates important opportunities for generating income and employment for communities (5). Processed foods are an integral part of today's diet and a significant contributor to food and nutrition security (6).

Innovative technologies have the potential to improve food production and enhance the quality of new food products to improve consumer acceptance. Gene editing is one of the emerging technologies that have opened up many possibilities for generating crops and animals with improved properties and desired traits (78). Additionally, highly productive food production systems (e.g., hydroponics, aquaponics, and aeroponics) have received attention as alternative farming systems, taking advantages of innovations and advancements in science and technology (910). Increased concerns about environmental sustainability are driving the growing interest in better uses of food wastes, by-products, and ugly produce. Food wastes is one of the major challenges for the global food system as approximately one-third of food produced in the world for human consumption is either lost or wasted each year. Valorization of food by-products and ugly produce (e.g., food products with an abnormal appearance) using smart solutions and technologies can constitute a promising strategy to tackle this challenge (1112).


Overview of the industrial revolutions through a food perspective

The ever-increasing population is putting pressure on natural resources and depleting them rapidly. Technological, environmental, social, and political changes across the globe are creating many new opportunities and challenges for humans. The global population increase is significantly affecting food and water sources (13). The overall industrial revolutions have had a great impact on the many different components of the food industry. The food industry has been continuously updating its processes and products to meet each new revolution (14). Industry 4.0 comprises a diversity of new enabling technologies, as previously mentioned that includes smart sensors, big data, AI, IoT, blockchain, cloud computing, automation, among others. These technologies have important roles in creating modern production processes. The food industry is adopting a customer-orientation as part of a dynamic supply chain. An adaptation of innovative technologies in the different food sectors is important for the sustainability of the production process. New technologies are often also more efficient economically (1516).
Automation is having an increasingly important role in manufacturing to achieve maximum productivity. The use of AI with automated processes is increasing in the food industry (17). AI helps monitor the supply chain and overall production process. IoT includes many technologies that will also affect existing production processes. IoT could also be implemented in the food supply chain to make food safer (18). IoT connects different devices to ensure effective communications between people and things (19). The use of sensor technology and cloud computing devices are important in increasing the efficiency of the food supply chain (2021). The application of Industry 4.0 technologies (e.g., IoT, blockchain, and smart sensors) is also important in reducing food wastage (22). Generally, the digital revolution currently occurring in manufacturing and the food industry, accompanied by greater automation and advanced monitoring methods and processing technologies is likely to have significant roles in enhancing sensory quality and nutritional properties of foods, leading to improved consumer perception and acceptance of these foods.

Consumer acceptance of emerging food trends

Plant-based foods

Current food production practices have been linked to a high prevalence of various chronic diseases as well as significant environmental damage (2324). Over the past century, the modern food and agricultural sectors have contributed to a considerable reduction in world malnutrition and hunger by producing a bountiful supply of inexpensive, safe, and tasty foods. To feed a rising and wealthier global population, more food of higher quality is required. Large-scale production of animal products such as milk, fish, meat, eggs, and their derivatives have been identified as a major contributor to the modern food supply's negative impact on global environmental sustainability (25). Raising cattle for food causes significantly more pollution, water and land use, greenhouse gas emissions, and biodiversity loss than growing plants (and in some cases other animals) for human use (26).
Plant-based (PB) diets are becoming increasingly popular as a strategy to lessen the diet's environmental footprint while simultaneously improving human health and animal welfare. In comparison to omnivores, vegetarians and vegans make up a small percentage of the population, but their numbers have risen in recent years. A side from meat alternatives, non-animal food products are becoming more popular, which creates a business opportunity for the food industry (27). Concerns over the consumption of animal-based food products and their harmful effects on the environment and health have led to an increase in the PB protein business, particularly for innovative items that can replace traditional dairy, egg, and meat products. More people are declaring themselves “flexitarians (vegetarians who occasionally eat animal products),” or opting to consume less dairy, eggs, and meat in favor of more PB meals to help the environment, improve health, or both. According to consumer market research, up to 5 million Americans will have given up meat totally between 2019 and 2020, becoming vegetarians or vegans (28) although data confirming this is not yet available.
Functional PB foods are produced from unprocessed or natural, as well as biotechnologically modified plants. They are considered to have a significant impact on health and wellbeing by reducing disease risks. Many of these functional foods have been related to lower incidences of a variety of health conditions, including diabetes, cardiovascular disease, gout, and cancer. As a result, there is rising interest in functional PB food research and development (2930). Individual PB foods, such as nuts, vegetables, fruits, legumes, whole grains, and coffee, have been shown to be beneficial to the cardiovascular system (31). Significant evidence, on the other hand, links particular animal foods, such as processed and red meat, to an elevated risk of cardiovascular diseases (32, 33), although these results remain controversial. Consumers are increasingly turning to PB milk replacements for health reasons such as lactose intolerance, cow's milk protein allergies, or as a lifestyle choice. PB milk substitutes are generally water-soluble extracts of oilseeds, legumes, pseudo-cereals, or cereals that resemble bovine milk in appearance. As a substitute for cow's milk, they are manufactured by reducing the raw material's size, extracting it in water, and then homogenizing it. Cow's milk replacers can be used as a straight replacement for cow's milk or in some animal milk-based recipes (34) although their nutritional profiles may be quite different and this remains a concern.

Insect-based foods

In response to the increase in the world's population, the existing production of food will have to treble to fulfill the rapidly rising demand for food. Insects are being researched as a new source of animal feed and human food to help meet global food security challenges. Human consumption of insects has several reported advantages including comparable protein levels (35), relatively high levels of unsaturated fat and different nutrients, and a lesser environmental effect due to decreased greenhouse gas emissions (3637). Insects are regarded as more sustainable since they utilize fewer natural resources such as water, feed, and land, and they generate far fewer greenhouse gases and ammonia than bovine and non-bovine animals. They have a high feed conversion ratio because they are cold-blooded, implying that they are particularly efficient at bio-transforming organic resources into insect biomass (3839).
As a result, insect production for human food is increasing in several countries (40). Around 2,000 edible insect species have been identified worldwide. They have been collected from the wild including from Africa, East Asia, and South America, and are used in traditional diets (41). Beetles (31%), caterpillars (18%), ants, wasps, and bees (14%), cricket, locusts, and grasshoppers (Orthoptera) (13%), planthoppers, cicadas, scale insects, true bugs (Hemiptera), and leafhoppers (10%), termites (Isoptera) (3%), dragonflies (Odonata) (3%); flies (Diptera) (2%); and other orders (5%) are the most commonly consumed species globally (42). For example cricket powder was added to pasta to increase its content of protein and minerals and improved the culinary properties and texture (43).
Insect-based food production has been influenced by recent advances and innovations offered by Industry 4.0 technologies. One of them is exploiting the possibilities for engineered insect tissue in cellular culture. Cellular agriculture is a rapidly emerging field that allows for the preparation of such a food system without necessitating changes in customer behavior. The use of insect cell culture in cellular agriculture offers the promise to overcome technical limitations and produce low-input, high-volume, and nutritious food. Insect cells are good candidates for incorporation into cultured meat and other innovative food products due to the robustness of established techniques for culturing insect cells and their ease of immortalization, serum-free growth, have a high-density proliferation, transfection, and a good suspension culture adaptation compared to mammalian cells (44).
Despite their apparent feasibility as a long-term alternative to conventional protein sources, there are still several barriers to their widespread utilization as human food in the West (45). In many Western countries, consumer acceptance remains a hurdle, and insects are usually viewed as unpleasant, even though their flavor has been shown to be mild and tolerable. Consumer disgust can be explained in numerous ways, including social, cultural and religious reasons (46). Therefore, product development to create new insect-based foods, as well as acceptance-boosting strategies, are required (47).

Cell-cultured meat

To satisfy the growing demand for protein for an ever-increasing population, cultured meat is being considered a good substitute for meat. Cellular agriculture is an emerging field for the production of different products. Cultured meat, also known as clean meat or laboratory-grown meat, is a part of cellular agriculture and does not involve any livestock for the production of meat once the initial cells are obtained (484950) although at some point new initial cells are needed.
Cell-cultured meat is produced using tissue-engineering techniques. Different aspects of cultured meat give it an edge over traditional meat such as a lower use of environmental resources, higher nutritional value, lower risk of food-borne diseases, as well as avoiding issues associated with the slaughtering of animals (5152) In the cell-cultured meat process, a biopsy is taken from any living animal from which the stem cells are obtained. The stem cells can proliferate into different types of cells. These cells are cultured in a nutrient medium containing all the required growth factors, nutrients, and hormones. The cells, if directed to muscle growth, continue to grow and form myotubes with a length of about 0.3 mm. These myotubes are then placed in a ring that grows into a small piece of muscle tissue. A schematic diagram for their production. These muscle tissues can further multiply to form more than a trillion strands. These muscle cells continue to grow in size and need to be attached to a scaffold that provides support and orientation (5354).

Hydroponics, aquaponics, and other indoor vertical food production systems

Agriculture-based food production growth is now much lower than the rate of population expansion, which is a concern. As a result, more agricultural production systems must be implemented to improve and achieve expected future food supply needs. Alternative forms of farming systems have become more popular. Hydroponics, aquaponics, and other indoor vertical farming systems are some of the primary sectors where global agricultural output may be improved as growing conditions can be better managed. The pros and cons of hydroponics, aquaponics, and aeroponics systems. Hydroponics is a type of horticulture and a subset of hydroculture that includes mineral fertilizer solutions in an aqueous solvent to grow plants, mainly crops, without soil (55). Any crop may be grown hydroponically, but the most frequent are leaf lettuce, celery, cucumbers, peppers, tomatoes, strawberries, watercress, and various herbs (56). Aquaponics is an indoor vertical farming system combining aquaculture (fish farming) and hydroponics. In aquaponics, farmed fish waste provides nutrients for hydroponically grown plants, which in turn clean the water for the fish. This ensures a closed-loop, long-term feeding supply. Because few pesticides and herbicides are non-toxic to fish, aquaponics production relies on organic pest and weed management (34) Aeroponics is a soilless revolutionary farming system that allows growing plants in the air, where plants' roots are suspended in a mist of nutrient solution (57). This system is well suitable to automation, digitalization, and other advanced technologies. For example, an automated IoT-based aeroponics system, with remote data monitoring, including sensors measuring temperature, humidity, pH value of the water, and the light exposure, has been developed (23).

Status of AMR in the Food Chain

Antimicrobials include antibiotics and related semi-synthetic or synthetic agents that demonstrate antimicrobial efficiency and discriminating toxicity [58]. The emergence of AMR limits the therapeutic possibilities of an antimicrobial, both for clinicians and veterinarians, impacting human and animal health. The WHO issued a report indicating that the antibiotics being developed against pathogens that present the greatest risk to human health are insufficient to control the expanding AMR problems [59]. If not properly regulated, AMR’s impact on mortality is alarming, with mortality increasing from 700,000 to 10 million yearly by 2050 [114].
From 1994 to 2000, AMR contagions were 6, 17, and 22% for the USA, Kuwait, and China, respectively. By 2050, it is predicted that AMR will drop the gross domestic product by 2–3.5%, with a decrease in livestock of 3–8%, substantially impacting the global economy [60]. Cases of MRSA bacteremia decreased by 81% between 2007 and 2013; however, total carbapenemase-producing Enterobacteriaceae increased tenfold between 2009 and 2014 [61]. AMR emergence results in about 25,000 mortalities in the US annually, and the number may vary depending on types of resistance and associated infections [62]. Patient data from 2001 to 2015 in France indicate that multidrug-resistant bacteria (>2 antibiotics) were uncommon (37 of 27,681 patients), with four deaths of which three were attributed to other reasons [63].
India published approximately 2152 studies on AMR of which 1040 (48.3%) were associated with humans, 70 (3.3%) with animals, 90 (4.2%) with the environment, and 11 (0.5%) linked to ‘One Health’. The remaining publications included novel agents, diagnostics, editorials, and miscellaneous subject areas related to AMR [118]. Estimation of the AMR problem in LMICs is based on extrapolation; for example, neonatal sepsis attributed to drug-resistant infection was estimated at 214,500 in the year 2012 based on an estimation of all neonatal deaths attributable to severe infection and drug-resistant infection in first-line management [64].
AMR frequently includes ‘one world’ reflecting the ‘One Health’ approach and a universal problem that links food systems and travel [65]. The ‘One Health’ concept encompasses problems that have inter-relatedness between human health, animal health, food, and the environment and raises common efforts on the part of regulatory agencies to address those challenges [66]. This is exemplified by a research paper that included an animated map displaying the worldwide spread of the ‘New Delhi metallo-β-lactamase 1’ or ‘NDM-1’ resistance gene [67]. Another example is the ‘mobilized colistin resistance 1’ or ‘MCR-1’ gene. The MCR-1 gene was initially isolated from pigs and humans in China [68]. Colistin resistance was <1%, but colistin-resistant K. pneumoniae was linked to high mortality of up to 70% [69]. Researchers reported the occurrence and frequency of antibiotic-resistant S. aureus in 80 samples of meat and chicken. The study was conducted on two swine farms 45% of workers were colonized with the same MRSA strain that was isolated from swine. In the study, S. aureus isolated in 67.5% of the samples were resistant to methicillin, and 87.5% were resistant to bacitracin [70]. A report from India on AMR indicates that more than 70% of E. coli, Klebsiella pneumoniae, and Acinetobacter baumannii isolates and almost 50% of Pseudomonas aeruginosa were resistant to third generation cephalosporins and fluoroquinolones . Additional studies indicated that among Gram-positive bacteria, 42.6% of S. aureus were methicillin-resistant, and 10.5% of Enterococcus faecium were vancomycin-resistant. For Salmonella Typhi and Shigella species, 28 and 82% were resistant to ciprofloxacin, 0.6 and 12% to ceftriaxone, and 2.3 and 80% to co-trimoxazole, respectively. Vibrio cholera showed resistivity rates against tetracycline that varied from 17 to 75% [71]. More than 2.8 million antibiotic-resistant infections occur annually in the US, with more than 35,000 deaths. In 2017, 223,900 cases of Clostridium difficile occurred, and at least 12,800 people died [72].

Microbes Displaying Resistance

The CDC is concerned about the occurrence of community-acquired AMRB. The incidence and development of previously non-antibiotic-resistant microbes remain among the most significant concerns. A CDC report listed 18 AMRB and fungi; some of those microbes are presented in the following section [73]. (74) the selected microbes that developed drug resistivity, as well as the approved antibiotic drugs for the treatment of infection associated with those microbes.

1. Carbapenem-Resistant Acinetobacter (CRA)

CRA causes pneumonia, urinary tract infections, and wound infections. From a food perspective, Acinetobacter is linked to the spoilage of meats and vegetables. Acinetobacter may contain mobile genetic elements that are effortlessly transmitted among bacteria. Some strains produce a carbapenemase enzyme that protects the cell from damage. Acinetobacter is an emerging risk to hospitalized patients, as it is a fomite contaminating common medical equipment in clinical settings. Acinetobacter baumannii is of considerable concern since it cannot be treated with existing antibiotics. In 2017, CRA infected nearly 8500 hospitalized patients, resulting in approximately 700 deaths in the US [75].

2. Carbapenem-Resistant Enterobacteriaceae (CRE)

Enterobacteriaceae include spoilage and foodborne pathogens. They may be isolated from fresh vegetables, soil, and irrigation water. CRE is an imminent concern to patients in healthcare settings. Patients that require devices such as catheters and may take antibiotics for a long duration are at maximum risk of infections with CRE. CRE also harbors mobile genetic materials that can be spread easily between other microbes. Around 30% of CRE carry a mobile genetic component encoding for enzyme production that targets carbapenem antibiotics eliminating these drugs as a treatment option. Some of the most pathologically related Enterobacteriaceae are E. coliKlebsiella pneumoniae, and Enterobacter species which are common infectious agents of the intra-abdominal region and urinary tract infections [27,38].

3. Drug-Resistant Campylobacter

Campylobacter is a major cause of foodborne illness and is associated with raw poultry and unpasteurized milk. Campylobacter commonly causes diarrhea (bloody), fever, abdominal pains, and sometimes sequelae such as irritable bowel syndrome, Guillain-Barre syndrome, and arthritis. Around 29% of all infections are associated with strains that have reduced sensitivity to fluoroquinolones or macrolides (azithromycin), antibiotics used to treat severe Campylobacter infections. Campylobacter spp. are a prominent cause of diarrheal infections and deaths (n = 109,700) in 2010.

4. ESBL Producing Enterobacteriaceae

Foods such as seed sprouts (alfalfa, radish) may be favorable for KlebsiellaKlebsiella is not considered a foodborne pathogen, but foodborne isolates may be ESBL-positive. ESBLs are enzymes that target antibiotics such as penicillins and cephalosporins. CTX-M, a specific ESBL enzyme, emerged in bacteria in the US and spread internationally. The genes encoding the CTX-M enzyme can be transferred to different Enterobacteriaceae species. The combination of CTX-M and ST131 enhances resistance and may spread in combination [78]. E. coli carrying CTX-M genes are common and considered primary contributors to spreading resistance genes across species and/or geographic regions [14,41]. In tropical and subtropical regions, 25 to 50% of infections are linked to ESBL-E, and in the healthy population, the carriage is 20 to 40% in regions endemic to ESBL-E [19,42].

5. Vancomycin-Resistant Enterococcus (VRE)

CDC’s ‘National Healthcare Safety Network’ specified that central line-associated bloodstream infections are most often caused by Vancomycin-resistant Enterococcus faecium. Over 70% of E. faecium are vancomycin-resistant, the antibiotic of choice for treating E. faecium infections. The development of vancomycin resistance may be linked to the egregious use of vancomycin to treat MRSA and C. difficile infections [79]. Enterococcus species may be used in starter cultures, so there is a concern that VRE may be spread by food. However, VRE is not considered a foodborne pathogen.

6. Methicillin Resistant S. aureus

S. aureus is of concern in healthcare facilities as well as in the community. MRSA was initially discovered in 1968 in association with nosocomial infections and has since become community-acquired [80]. S. aureus infections can be challenging to treat since the pathogen may resist methicillin and several other vital antibiotics. S. aureus is a foodborne pathogen that causes food poisoning due to toxin production in food.

7. Drug-Resistant Non-Typhoidal Salmonella

Non-typhoidal Salmonella may be responsible for diarrhea, fever, and abdominal pains. People may acquire Salmonella infections by eating contaminated foods or after contact with the feces of infected people or animals. Antibiotics used to treat patients with Salmonella infections include ciprofloxacin, azithromycin, and ceftriaxone. Infections caused by resistant strains of Salmonella may be more severe and result in higher rates of hospitalization. In 2018, it was reported that Salmonella enterica serovar Infantis was associated with 25% of infections. The majority of infected people had no history of travel but had eaten chicken [81]. Almost all AMR Salmonella infections are foodborne and linked to the consumption of contaminated pork, turkey, and beef [81]. In 2017, 59,066 deaths were due to non-typhoidal Salmonella infection [82]. International traveling has been acknowledged as a contributing risk for Salmonella infection.

Conclusions

While this review is not an exhaustive overview of all emerging food trends, eight of the more pertinent ones, from food technological advances perspectives, were discussed. Each of these emerging food trends has been fostered by the greater use of Industry 4.0 technologies and recent advances in many fields of food science and technology. Innovative solutions based on Industry 4.0 enablers (such as AI, smart sensors, and robotics) can be used to increase agriculture productivity, optimize production conditions, and reduce waste and loss, accelerating the green and digital transition of future food production systems. The interest in traditional animal-proteins alternatives, including plant-based foods and insects and more recent food trends, such as cell-cultured meat, 3D-printed, fortified, and gene-edited foods are likely to continue growing in popularity in response to the increasing consumers' awareness regarding the environmental impact of food choices. With the ongoing rapid technological advances in physical, biological, and digital worlds, other food trends are expected to emerge in the future.
Antimicrobial resistivity is affecting the global population, resulting in health and financial losses. The ‘One Health’ concept is supported by the ‘World Organization for Animal Health’ and WHO, under which suitable approaches can be developed and implemented to control AMR. Currently, the major focuses are on antimicrobial residues in food that may occur due to the indiscriminate use of antibiotics in agriculture. Food and foodstuffs can be contaminated with AMRB at any farm-to-table continuum point. Two major steps need to be monitored to overcome or stop the risk of AMRB in the food chain, i.e., antimicrobial use in foods and AMRB originating from agricultural practices. The developed approaches should be policy-based, enforced for all countries, and entirely backed by government regulations. No action taken by a single country will resolve the AMR problems facing the global food supply, but a collective global approach will surely do so.

Author contributions

Conceptualization, methodology, and writing—original draft preparation: M.SAJJAD, AQSA ZAHID Writing—original draft preparation: AHSAN RASOOL, TALAL KHAN, AHMED RAZA, ALHIDA FATIMA, MUNEEBA RAFIQUE, and INSHA  ASLAM. Writing—review and editing: M.SAJJAD and TALAL KHAN. All authors contributed to the article

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