IMMUNOLOGICAL AND BIOSSENSORY TECHNIQUES FOR DETECTION OF Salmonella spp. IN FOOD DERIVED FROM FISH FARMING – REVIEW

Salmonellosis is the world’s most common foodborne illness. In Brazil, foods contaminated by salmonella lead the statistics. Therefore, the aim of this study is, through biotechnological knowledge, to compile alternative and innovative techniques for the detection of salmonella in foods, such as fish-farming derivatives, immunological and biosensorial techniques. This is a descriptive exploratory data survey of a qualitative nature, aiming at data analysis. Research and data collection were carried out in bibliographic databases: Academic Google, Scielo, CAPES journals and institutional repositories using specific descriptors in Portuguese and English, with words and terms separated by the Boolean operators ‘AND’ and ‘OR’. Some innovative and alternative methods are available to identify the presence of salmonella in food. Immunological and biosensory techniques, despite being less frequent in the scientific literature than molecular methods, are techniques that present high specificity and sensitivity. These techniques have been the most developed alternative methods in fish in recent years. And, they can employ both molecular and immunological techniques in biorecognition, which is characterized as an advantage of not having a requirement for pre-enrichment of the sample. According to the literature found, the techniques covered in this study are quick to respond, which speeds up decision-making by researchers and technicians, which makes the techniques very promising for industrial application.


Introduction
In recent years, the world population has significantly increased its average consumption of fish, having doubled its consumption in the last 50 years (DANTAS FILHO et al., 2021;FAO, 2018;). However, along with the increase in demand for fish, there was an increase in cases of salmonellosis, which harms the marketing of fish and public health (GAZAL et al., 2018). One of the main bacterial agents distributed in the aquatic ecosystem, members of the Enterobacteriaceae and Aeromonadaceae families stand out (EVANGELISTA; LUCIANO, 2021;HUBER et al., 2004). The Enterobacteriales order harbors a large group of pathogenic bacteria, such as the genera Salmonella, Shigella, Edwardsiella and some serotypes of Escherichia coli. Among the bacteria of the Enterobacteriales Salmonella sp. stands out, a ubiquitous bacterium that is able to survive in different environments (McADAM, 2020;OLIVEIRA;VAZ, 2018). Salmonellosis in fish is closely related to its creation, as well as to the environment of its industrialization, as a result of inefficient hygiene practices, equipment and inadequate food handling (FERNANDES et al., 2018).
Salmonella are facultative anaerobic, non-sporeforming Gram-negative, being mostly mobile (except S. Pullorum and S. Gallinarum) by means of peritrichal flagella. Its growth temperature can vary between 5 to 46° C, with an optimal temperature at 37° C (POPOFF; LEMINOR, 2005). According to some molecular studies, the genus Salmonella is divided into two species, Salmonella (S.) enterica and S. bongori. The species S. enterica is subdivided into six subspecies designated by Roman numbers: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV) and indica (VI). The genus Salmonella has 2579 different serotypes (serovars) identified by the Kauffmann-White scheme, based on the bacterial composition of its somatic The test to identify the presence of salmonella in food is a requirement of the health authorities that regulate food safety in their respective countries. Conventional techniques, based on classical cultural methods, are considered sensitive and reliable. However, they depend on a complex sequence of steps and require several days for the result (ANDREWS et al., 2016;EVANGELISTA;LUCIANO, 2021). Recent advances in technologies for detecting and identifying the presence of microorganisms have made available more agile, sensitive and specific alternatives to conventional methods. And, these are often referred to as alternative methods, terms commonly used to describe a variety of tests that include miniaturized biochemical kits, immunological assays, DNA/RNA based tests, and combinations with cultural methods (GAZAL et al., 2018).
According to epidemiological data presented above, salmonella contamination is the most frequent cause of foodborne disease outbreaks. The determination of this pathogen is mandatory in food products for human consumption and can be performed using conventional methods or alternative methods, also called rapid methods (LIEVENS et al., 2011). The development of alternative methods for detecting salmonella is of great importance for food safety and the maintenance of public health. In addition, there is a strong industrial demand for compliance with legislation and the rapid release of food products to the market (DEMERTZIS; ILIADIS, 2015; EVANGELISTA; LUCIANO, 2021).
Given these assumptions, the aim of this study is, through biotechnological knowledge, to compile alternative and innovative techniques for the detection of salmonella in foods, such as fish-farming derivatives, immunological and biosensorial techniques.

Methodology
This study is a bibliographic research carried out by consulting the database of CAPES journals and institutional repositories. The survey of information carried out is characterized as exploratory descriptive, also of a qualitative nature, aiming at the analysis and crossing of data between several articles and literature related to the studied topic PONTES, 2019).
The data survey was carried out from April to July 2021, based on questions raised on the subject, and about 50 studies were consulted, with scientific articles, books and references found in electronic databases and bibliographic bases: Google Scholar, Amazon, Scielo, and others. The criteria adopted for the searches were publications in the last ten years in scientific journals with a consolidated technical and editorial staff, and which have a focus and scope related to the theme. In addition, having a link with a higher education institution and qualis concept (2013)(2014)(2015)(2016) at least B2 in the area of Interdisciplinary assessment.
To collect the information, the following descriptors were searched: Salmonella spp., food infection, salmonellosis, fish chain, food pathogens, fish health, fish microbiology, epidemiology, legislation, detection methods, immunological and biosensorial techniques; in Portuguese and English, with words and terms separated by Boolean operators 'AND' and 'OR' according to the search objectives in each topic of this review article.

Results e Discussion
Techniques for detection of Salmonella ssp.
Molecular tests employ a bacterial nucleic acid sequence as a target for detection and correspond to 47% of validated methods (FSIS, 2016). And, it is the category of alternative methods that has grown the most in recent years. Therefore, as it is a technique often mentioned in the literature, it was not considered in this study.

Immunological Techniques
Experiments based on immunological methods use mono or polyclonal antibodies to identify the presence of salmonella in animal products. Therefore, the antibodies mentioned above can identify antigens in different food matrices . The assays performed through immunological tests involve the ELISA test (Enzyme Linked Immuno in the Sorbent Assay), latex agglutination tests and immunodiffusion assays, as shown in Table 1. It should be noted that the ELISA test is the most widely applied category of assays aimed at detecting salmonella in animal products. What's more, this method is available in a significant number of kits (Food Safety and Inspection Service, 2016) on the market, which are based on different ELISA formats (Figure 1). In a homogeneous class immunological method experiment, labeled antibodies and antigens are mixed freely in the detection system. Therefore, the modulation happens as a conversion in the activity of the marker, but for that, the antigens need to bind to the antibodies. In general, they show a change in the color of the sample SILVA et al., 2018). Generally, immunological assays are heterogeneous, such as a salmonella-specific antigen that binds to its antibody that is located immobilized on a solid food matrix. The antigen-antibody complex is formed and this can be seen by the change in color, which is caused by the enzymatic cleavage of a chromogenic substrate, allowing the identification of the presence of bacteria in the sample to be identified (VALDERRAMA et al., 2015). Regarding the immunological assay reagents, they are unbound components, that is, they are washed away. So, in this way, the response is obtained through the markers, and this is proportional to the amount of analyte in the experimental sample evaluated SILVA et al., 2018).
Experiments with immunological methods can still be considered as competitive or non-competitive. The most administered in laboratories are those used by the sandwich method and the competitive method. In the non-competitive sandwich assay, the primary antibodies are immobilized on a surface VALDERRAMA et al., 2015). Following the addition of the sample with the antigen, a labeled conjugated antibody is added to the system. However, in an experiment with the competitive method, competition occurs between the free labeled antibodies, more specifically in a limited amount. As well as the antigen fixed to a base, or between the labeled antigen from the experimental sample and a limited amount of antibodies SILVA et al., 2018).
In this context, it should be noted that agglutination requires latex particles wrapped with antibodies that react with the antigens on the surface of the bacteria's cells, in this case salmonella, forming visible aggregates for the identification of positive samples. The tests are specific and easy to handle, in addition to their significant reliability (VALDERRAMA et al., 2015). Typically, these tests have been performed as confirmatory analysis techniques, as opposed to screening tests. Alternatively, there are several kit options on the market that have this technique (Food Safety and Inspection Service, 2016). Which employs immunodiffusion reaction, the kit 1-2 Test© (Biocontrol) (SILVA et al., 2018).
As far as the device is concerned, there are two chambers: the first is the inoculation chamber, in which the preenriched food sample is added, and the second is the motility chamber, in which bacterial growth and reaction with flagellar antibodies occur (LEE et al., 2015). A priori, the sample is preenriched for 24 hours. In this way, the sample unit is enriched and inoculated in the inoculation chamber. The inoculated salmonella cells move to the motility chamber, where the antigen-antibody complex is formed (VALDERRAMA et al., 2015). Then, when the result is positive, it evidences the formation of a remarkable three-dimensional immunoband after an average time of 24 to 30 hours (AOAC Official Method 989.13, 1998).
The immunological experiments conducted are comparatively more agile as well as more specific than conventional methods. Furthermore, by associating techniques such as immunomagnetic separation (IMS), with the possibility of automation, it becomes faster and more practical, especially for sampling units available in large quantities (LEE et al., 2015). Among immunological methods, those based on ELISA express specificity and sensitivity comparable to conventional methods and are the most used. ELISA experiments express detection limits between 10 4 and 10 5 CFU mL -1 , levels normally understood after pre-enrichment of the evaluated sample (LEE et al., 2015;. However, it must be admitted that these methods have some limitations for identifying the presence of salmonella, especially in very moist foods such as fish. For in immunological assays it is necessary to conduct a previous enrichment of the sample, in order to obtain the adequate number of cells, in order to increase the analysis time. Furthermore, crossreactions with phylogenetically close antigens possibly occur. As well as antigen variations, sensitivity limits for some food sample matrices, and also the high cost of assay automation (SILVA et al., 2018). But without a doubt, the sensitivity and specificity of these methods strongly depend on the microbiota of the sample, the complexity of the food matrix and the inhibitory substances. Which can be exemplified, heavy metals, antibiotics, polysaccharides, proteins, lipids and other organic compounds .

Biosensory techniques
Biosensors are bioelectronic devices capable of detecting analytes, with agility both quantitatively and qualitatively (FURTADO et al., 2008;SILVA et al., 2018). This technique is developed by two main components, a bioreceptor, a molecule that specifically interacts with the analyte, and a transducer, which transforms the response of the bioreceptoranalyte interaction into an electrical signal (SILVA et al., 2018). There are different types of transducers, the most used are: optical, piezoelectric and electrochemical (FURTADO et al., 2008). Biosensors with electrochemical transducers have been reported more frequently in the detection of pathogens (ARORA et al., 2013).
These devices are widely used in different areas of knowledge and are an agile response alternative for the detection of pathogenic bacteria (VELUSAMY et al., 2010). Furthermore, recent research has shown the development of biosensors capable of detecting the presence of microorganisms or their metabolites in an agile and accurate way with a lower detection limit than conventional methods .
The improvement in the elaboration of biosensors occurs together with the increase in the molecular and biochemical understanding of the analytical response and its supporting technologies (VALDERRAMA et al., 2015). That said, one can currently find miniaturized, affordable and easyto-administer devices on the market. There are nowadays different categories of biosensors that are classified by the type of immobilized biological molecules, by their interaction with the analytic through the analytic response or by the transducer (Figure 2).  Melo et al. (2016) carried out a study on electrochemical immunosensors designed to detect salmonella, whose limits and detection times for the devices are shown in Table 2. In order to compare with molecular and immunological methods, immunosensors stand out more in relation to the period for detection. Table 2 shows the agility in obtaining response from the devices, with detection periods of up to 6 minutes, and low detection limits, with devices capable of detecting up to three mL -1 cells . Such information can be obtained without enriching the sample, which is often necessary for the molecular and immunological methods previously presented.  Dill et al. (1999).
An immunosensor capable of identifying the presence of salmonella in an agile way, and specifically and with a low detection limit was developed (MELO et al., 2016). In that same study, the technique of self-assembled monolayers was used to alter the surface of gold electrodes, using the thiol cysteamine. Furthermore, Protein A was applied for the oriented immobilization of the primary antibody through covalent bonds. It should be emphasized here that the biosensor response curve expressed a qualitative behavior with a detection limit of only 10 CFU mL -1 and an identification period of 125 min (GONÇALVES et al., 2014).
Furthermore, cross-reaction tests against Escherichia coli and Citrobacter freundii strains expressed high specificity of the device developed (GONÇALVES et al., 2014). The good application capacity of the immunosensor in foods, such as fish and milk, was proven (BRITO, 2020;MELO et al. 2016), and it can be evaluated in other food matrices (BENETTI, 2009;MATACA, 2014;MONTEIRO, 2018;SILVA JÚNIOR, 2017). It is noteworthy, according to Mataca (2014), Monteiro (2018) and Brito (2020), biosensory techniques have been the most developed alternative methods in fish in recent years.
Before concluding, it is important to point out that these alternative methods were designed to detect a specific target. In ways that make them faster and more suitable for application in quality control and food safety. That is, agile screenings are usually carried out in a considerable number of samples, in order to detect a specific analytical (SILVA et al., 2018). However, positive results detected by alternative methods are considered only presumptive. Therefore, they need confirmation by an official standard method (GONÇALVES et al., 2014;. Finally, the alternative methods covered in this work can be certified by independent institutions to validate the identification of microorganisms in food (AOAC, AFNOR, MicroVal, NordVal) or regulatory agencies (FSIS, FDA BAM, EFSA, ANVISA, ISO) (GONÇALVES et al., 2014). Furthermore, they can only be administered in food matrices for which they have been certified, adopting the validation conditions, which guarantees the reliability of the method used by the researcher and/or applicator (LEE et al., 2015).

Conclusions
Some innovative and alternative methods are available to identify the presence of salmonella in food, based on different biotechnological techniques. The techniques highlighted by this work are immunological and biosensorial. Because, despite these methods being less frequent in the scientific literature than molecular methods, they are techniques that present high specificity and sensitivity.
It should be noted that biosensory techniques have been the alternative methods most developed in fish in recent years. And, they can employ both molecular and immunological techniques in biorecognition, which is characterized as an advantage of not having a requirement for pre-enrichment of the sample. According to the literature found, the techniques addressed in this work are quick to respond, which speeds up decision-making by researchers and technicians, which makes the techniques very promising for industrial application in the detection of bacteria in moist foods.