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Exploring the nutritional potential of L-methionine in fish nutrition

Published: October 7, 2020
By: Bruno Tadeu Marotta Lima, Camilo Ivan Camilo Ospina-Rojas, Tatiana Garcia Diaz, Ramalho J. B. Rodrigueiro. / Animal Nutrition Service - CJ do Brasil, Av. Engenheiro Luís Carlos Berrini, 105 – 04571-010 – São Paulo, SP, Brazil.
INTRODUCTION
Like other animal production systems, aquaculture has developed into a highly globalized trade-dependent industry, therefore as global fish farming continues to increase, the need for formulating nutritionally balanced and cost-effective diets with high-quality protein source is important (Furuya et al., 2010). In fact, nutritional value of protein in the diet for fish is influenced by its amino acids(AA)  compositions,  therefore,  the quality of the protein source used and the composition of AA in the diet are two indispensable requirements that influence the growth and health of fish (Furuya et al., 2010; Wilson, 2003). Indeed, it is established that excess of AA in the diet will result in the catabolism of the AA with associated ammonia excretion and loss of energy (Lloyd et al., 1978). Hence, provide AA in the diet, to meet physiological need, improves fish protein conversion efficiency and minimizes nitrogen pollution, due to reducing of waste excreted (Cho et al., 2001; Liebert et al., 2007; Watanabe, 2002). Reduction in performance, protein retention, increased nitrogen excretion and oxidation of AA, as a consequence of the decrease in digestion, absorption and metabolism, can be attributed to the deficient or unbalanced profile of AA in the diet (Aragão et al., 2004; Rawles et al., 2006).
Formulation of practical diets is not such a simple process, as it depends on the AA composition of the ingredients and many other factors. In addition, a wide variation in the AA content can be found due to the diversity and availability of ingredients on the market (Furuya et al., 2010). Hence, dietary supplementation with synthetic AA depends on the potential use of each ingredient used in the diet, mainly on the content of nutrients and availability of AA, as well as the stages of the utilization process, such as digestion, absorption and use of each nutrient (Berge et al., 2004; Wilson et al., 2002). 
In this context, since methionine (Met) is one of the most important AA for fish and has been used for a long time by aquaculture as first limited AA in aquafeed, this article reviews the information on L-methionine and its supplementation importance and considers their applications in fish nutrition.
METHIONINE
Methionine is a sulfur-containing aliphatic, nonpolar α-amino acid. As an essential amino acid (EAA), cannot be synthesized de novo by fish, which depend on dietary methionine (Li et al., 2009). Methionine plays an important role in the biochemical and physiological function in fish. It is mainly used to synthesize protein and donor of methyl groups in the form of S-adenosyl-methionine  (SAM)  (Bender, 2003;  2014; Mai et al.,  2006).  SAM  itself is relevant to the biosynthesis of bioactive compounds in fish such thymidine, carnitine, epinephrine, melatonin, phospholipids, purine, coenzyme A and active sulfate, pyrimidine, creatine, which all have several important physiological role in fish body, and also polyamines (spermine and spermidine), which are related to cell proliferation and growth (Baker et al., 1969; Baker, 2006; Brosnan et al., 2006; Chen et al., 2018; Duan et al., 2012; Harpaz, 2005; Kasper et al., 2000; Mai et al., 2006; Murray et al., 1996; Tulli et al., 2010; Twibell et al., 2003; Wu et al., 2005; Zhou et al., 2006). In addition, Met supplementation also increases digestive enzyme activity in fish (Chen et al., 2019).
Methionine is an important precursor of cysteine in fish, which is a constituent of glutathione syntheses, an important antioxidant cellular (Azeredo et al., 2017). Glutathione metabolism is markedly altered in response to infection (He et al., 2017; Malmezat et al., 2000). In addition to its role,  it has an important function in protecting against oxidative damage through the glutathione peroxidase system, which has been well characterized in several tissues (Cowey et al., 1992). According to Keembiyehetty et al. (1995), supplementation of Met in diets for fish considerably increased liver levels of glutathione.
Methionine, through cysteine conversion, is also a precursor of taurine, which plays an important antioxidant compound in fish (Boonyoung et al., 2013; Khan, 2014; Yokoyama et al., 2001). According to Yokoyama et al. (1992), Met supplementation showed an increase in taurine level in the muscle of rainbow trout. In addition, Met is also an important substrate for the synthesis of choline and, like phosphatidylcholine and acetylcholine, which play essential role in nerve function and leukocyte metabolism (Duan et al., 2012; Li et al., 2007). Moreover, as expected, methionine deficiency results in poor growth performance and others severe problems in animal health (Table 1).
Exploring the nutritional potential of L-methionine in fish nutrition - Image 1
SOURCE OF METHIONINE IN FISH NUTRITION
Methionine is basically produced from chemical or fermentation process (Anusree et al., 2015). Chemical synthesis produces a racemic D and L isomers of methionine: DL-methionine (DL-Met; 99% pure; 50 percent D-methionine and 50 percent L-methionine), and Methionine hydroxy analogue calcium salt (MHA-Ca). L-methionine is an organic and natural isomer (L-Met; 99% pure), produced through a biologically precursor fermentation process (Willke, 2014).
The fermentation process has many advantages, among them, it produces only the L-form amino acids, avoiding possible additional processing steps (Sugimoto, 2009). In addition, fermentation process provides an eco-friendly alternative source to the chemical process, which generally uses hazardous sources, as it uses carbon (glucose, sucrose, mannose, xylose, arabinose, galactose). and fructose) as substrates for the production of amino acids (D'Este et al., 2018; Desai et al., 2010; Ikeda, 2003; Sabri et al., 2013; Zuend et al., 2009). In this context, since the discovery of the fermentative process to produces L-amino acids in the 1970s, research on AA fermentation has increased and several attempts have been made to commercialize the production of methionine by submerged fermentation (Kase et al., 1974; Kinoshita, 1959; Willke, 2014). Since then, several attempts to produce Met by fermentation have been reviewed by Roy et al. (1989), Mondal et al. (1996), Gomes et al. (2005), Kumar et al. (2005), and Shim et al. (2016).
The biological efficiency of MHA-Ca depends on its metabolism and the efficiency of its conversion to L-methionine. Since MHA-Ca is not considered as true AA, its metabolism and absorption by the intestine can be impaired as it lacks amino group in their structure, which can serve as a recognition site for the carrier protein (Baker, 2006). Several of the studies carried out with different species of fish,  channel catfish  (Robinson et al.,  1978), sunshine bass (Keembiyehetty et al., 1995; Keembiyehetty et al., 1997), rainbow trout (Cheng et al., 2003), red drum (Goff et al., 2004), and hybrid striped bass (Kelly et al., 2006), also support the notion that MHA-Ca can be used as a source of methionine by fish, but that its relative biological efficiency is lower in relation to D and L isomers on an equimolar basis.
Both Meth isomers can be metabolized by different species of fish with similar efficiencies (Baker, 2006; Ma et al., 2013; Poston, 1986). However, since all AA used in protein synthesis must be in the L configuration, then, it is necessary to convert the D-isomer form into its respective L-isomer (D'Mello, 2003). Hence, D-methionine isomer needs to be deaminated and converted to keto-analogue by d-methionine oxidase and subsequently reamined to L-met. L-methionine is the natural isomer metabolically active in its current form and used efficiently by fish (Baker et al., 1969; Powell et al., 2017). According to Poston (1986), fish fed the L-methionine isomer showed most efficiently performance and produced the greatest weight gain in comparison with other Meth sources.
D-Met form is not used directly by the cells of the gastrointestinal tract until the conversation to L-Met by the enzyme d-amino acid oxidase (DAO). The quantity and expression of these oxidases are considered very low in young animals, consequently, they have higher concentrations of free D-amino acids compared to adult animals. Serious damage can be caused if the D-amino acids ingested are not metabolized by these enzymes, and are accumulated in the tissues (D'Aniello et al., 1993). DAO efficiency depends on the species and age of the fish (Sarower et al., 2003). Some fish species may have lower DAO enzyme activities in the early stages of life (Chen et al., 2007). In this context, L-Met is the only biologically functional form of methionine that is easily used by the intestinal cells of young animals.
In an experiment carried out by Poston (1986), it was observed that fish fed with the L-methionine isomer showed better feeding efficiency in relation to other sources of methionine (Figure 1). In addition, according to the second order predictive polynomial relationships between weight gain and each source of methionine in the diet (Figure 2) support the data in figure 1, indicating that the growth response was greater in trout fed L-methionine and lower in those fed with other sources of methionine.
Exploring the nutritional potential of L-methionine in fish nutrition - Image 2
 
Exploring the nutritional potential of L-methionine in fish nutrition - Image 3
Hence, it is important to recognize the differences in the use of each Met isomer between each different species (D'Mello, 2003). The bioavailability and effectiveness of different sources of Met in fish may be related to different species or the lack of sensitivity of the bioassays, and also depends on the efficiency of absorption of the intestine and the activity of specific enzymes necessary for the conversion of isomeric forms into L-Met (Dibner et al., 1984; Dibner et al., 1992; Guo et al., 2020). In this context, L-Met is a key in utilization for better fish performance, intestinal development, and digestive tract due to the higher bioavailability and efficiency.
IMPORTANCE OF L-METHIONINE IN TASTE SENSITIVITY
The ability of fish to locate and capture food in the water depends on the sensitivity and specificity of the animals' sensory systems to detect food, and the specificity of each food present in the environment. Therefore, sensory systems are intrinsically involved in the fish's appetite and eating behavior (Caprio et al., 2015). According to Hara et al. (1993), amino acids cause important chemical signals, which stimulate various behavioral reactions through the taste in fish. The taste receptors in fish are located along the skin surface of the entire body, especially on the head, fins and tail of the fish. The receptors present variable responses to the different amino acids present in the fish's diet. These responses are particularly important for different species of fish, since they influence the animal's feeding behavior (Lane et al.,  1982;   Yoshii et al., 1979). Electrophysiological studies are usually indicated in the evaluation of taste receptors, and reveal that these receptors respond differently to each of the different amino acids present in the diet of fish (Caprio et al., 2015). 
Generally, the response of gustatory chemoreceptors in fish is more efficient than most naturally occurring amino acids in organisms, that is, amino acids in the L-form (Yoshii et al., 1979). According to Yoshii et al. (1979), none of the tested D-isomers was as effective as their corresponding L-isomers in the eel (Anguilla anguilla). In addition, studies carried out with rainbow trout (Oncorhynchus mykiss) and eel (Anguilla anguilla), confirm that the feeding behavior was stimulated, mainly, by the L-isomers evaluated in the fish (Adron et al., 1978; Mackie et al., 1983). 
Taste receptors tend to have a better response to the most preferred food components of fish. Thus, as previously explained, L-meth is considered one of the most limiting amino acids in fish nutrition, therefore, fish tend to have a greater preference for this amino acid. According to the taste response spectra for the different amino acids examined electrophysiologically, L-meth showed a powerful taste electrophysiological threshold in Tuna (Thunnus orientalis) (Kohbara et al., 2006). Thus, these data indicate that there is a greater efficiency of L isomers in relation to D isomers in fish, therefore, in addition to being an important stimulant in fish, L-methionine contributes to increase the attractiveness of diets for animals. 

APPLICATION OF L-METHIONINE IN AQUAFEED
In the last years, aquaculture has seen rapid growth in production volume and economic performance in recent decades, and today it is an important supplier of a quality protein source and employment (FAO, 2020). As a result of increased scale production, it also increases the likelihood that the industry will face emerging biological, economic and social challenges that may influence the ability to maintain fish production in an ethical, productive and ecological way (Føre et al., 2018). Therefore, it is important that the sector finds a sustainable alternative to overcome the negative environmental impacts caused by the use of limited and non-renewable sources (Ayer et al., 2009). In addition, in the context of declining fossil resources and stronger environmental restrictions, alternative, more sustainable processes based on natural resources are increasingly gaining interest in the population. In organic agriculture, especially poultry and pig farming, the supply of methionine by chemical process has become a problem, and the use of this synthetic methionine in organic farming is prohibited in major countries. For this reason, the search for low-cost sources of L-Met, which meets the rules of organic agriculture, has recently intensified, especially L-met from non-GMO fermentative (Willke, 2014). 
Fish, as in other animals, does not seem to have a true protein requirement, but a well-balanced mixture of EAA in the diet is essential for physiological processes as well as for growth and homeostasis. Therefore, a more precise approach with an ideal proportion of AA expressed based on digestible values can play an important role in fish nutrition, in addition to minimizing nitrogen excretion and minimizing environmental impacts (Furuya et al., 2010). 
In fact, the efficiency for protein synthesis in fish is determined by the availability of the most limiting AA at the site of protein anabolism (Verstegen et al., 2003). According to Furuya et al. (2010), determining the first limiting AA, commonly Met, may reduce total crude protein in fish production by reducing feed costs, as well as environmental impact, if economic growth can be maintained with less nitrogen input to fish feed. Therefore, determining the requirement of AA in the diet for fish will allow the formulation of the feed with greater precision, which meets the limiting requirements of AA. In this way, the balance of dietary AA for fish is of extreme importance due to the significant effects of these nutrients to reduce fish nitrogen losses and pollution and also improve feed conversion rates, while maintaining rapid growth (Furuya et al., 2010; Small et al., 1999). 
Likewise, methionine is often considered the first limiting AA in many protein sources that are normally used in aquafeed, mainly in vegetal protein source (Nguyen et al., 2009b; Nunes et al., 2014; Zhou et al., 2006). Several studies have effectively demonstrated the effects of increased intake of synthetic methionine on growth performance, increased body protein and decreased body fat in fish, in addition to several other important physiological functions, and determined the requirement of methionine for fish growth Tilapia is an economically important freshwater fish and one of the most cultivated fish in the world, representing 2.82% of total aquaculture production and produced in 125 countries. In the last two decades, its production increased by more than 350%, reaching 5,714,901 t in 2017 (El-Sayed, 2019). Several studies have been carried out on the methionine effects in fish nutrition over the years, and a lot of progress has been achieved on the role of Met in tilapia nutrition to optimize the nutritional value of these AA and on the growth performance, protein deposition and many physiological process (Table 2). 
The wide variation observed in the methionine requirement among tilapia species and strains may be due to the genetic potential, fish size, age, laboratory condition including feeding regime, feed allowance, water temperature, stock density, feed ingredients sources or physiological state (Ahmed et al., 2003; Kim et al., 1992; Rodehutscord et al., 1997; Tacon et al., 1985; Wilson, 2003).
Exploring the nutritional potential of L-methionine in fish nutrition - Image 4
FISH MEAL REPLACEMENT
Aquaculture has been continuously challenged to improve economic profits while maintaining environmental sustainability. Aquaculture feeds are among the most expensive animal feeds on the market due to the use of high levels of specific and expensive ingredients, in addition to the highly technical processing used (Furuya et al., 2010). One of the strategies used to improve fish production is to reduce feed costs and the environmental impact, reducing the protein content in the diet (Diógenes et al., 2016). As protein is the most expensive component of animal feed, the inclusion of its ideal amount will allow us to develop economical dietary formulations (Ahmed et al., 2003; Craig et al., 2017; Nunes et al., 2014). Therefore, it is important to incorporate cheaper alternative protein ingredients available in the formulation of fish feed, with care regarding the balanced amount of EAA and, therefore, reduced the cost of feeding (Azaza et al., 2008; Kaushik et al., 2004; Keembiyehetty et al., 1995; Nguyen et al., 2009b).
In the aquaculture industry, fish meal (FM) is traditionally used as a source of reference protein in fish diets, primarily for its high protein content with well-balanced AA, high palatability and good energy source, essential fatty acids, minerals and vitamins (Furuya et al., 2010). Since most of this FM comes from fisheries, the development of novel alternatives to reduce the dependence of farmed fish derived from wild-caught fish is needed (Hardy, 2010). 
The market price of FM has increased by almost three times in the last decade due to the restrictions on fishing for wild fish (Beal et al., 2018). This can be explained due to the strong market demand for FM by the aquaculture and livestock sector residing in the main importing countries and, in particular, in China, and also the reduction in FM production and availability (Tacon et al., 2008b). However, the future use of these finite and valuable commodities is likely to change yet again in view of the dramatic price increases observed in the case of FM in last years (Martinell et al., 2020). In fact, the growth in aquaculture production based on the use of FM under these conditions is fundamentally unsustainable, especially if it is still used as the main source of protein for fish diets (Guo et al., 2020). 
In view of this situation, much progress has been made in the search for selected alternatives to replace this finite and overpriced source in aquaculture diets (Gatlin III et al., 2007; Kaushik et al., 2008; Tacon et al., 2008a). Replacement FM as the main source of protein by proteins of plant source is one of the main sustainable goals in the future and to meet the growing needs of aquaculture, in addition to potentially reducing the levels of nutrients in wastewater (Trushenski et al., 2006). Due to its large-scale production worldwide,  vegetable protein represents an abundant source of protein that could potentially be used in the fish` diets without compromising their health, growth, or survival. Several sources of vegetable proteins are commonly used in aquaculture foods, including grain meal (wheat and gluten), vegetables (peas, beans, peanuts and lupins), and most commonly oilseeds (soy, rapeseed, sunflower, seed cotton) (NRC, 2011). The recent concept of reducing the use of FM by vegetable protein sources, has aroused great interest in research on this concept (Figueiredo-Silva et al., 2015; Liebert et al., 2007). 
There are basically two main factors that should be considered when formulating fish based on foods with high levels of vegetable meal: energy density, as some meals have a high carbohydrate content, which has little nutritional value for species carnivorous and AA content, because some EAA, such as lysine and methionine, are generally deficient in vegetable protein sources (Gatlin III et al., 2007). 
Among the various sources of vegetable proteins already mentioned, soy products have been wildly used to replace FM, due to their high protein content and global availability (García-Ortega et al., 2016; Gatlin III et al., 2007; Kissinger et al., 2016; Zhao et al., 2010). Soybean constitute one of the largest volumes of vegetable protein, with large annual production and good acceptance by most of cultivated fish species, making them a reliable and viable alternative to FM in fish diets (FAO, 2018; Kaushik et al., 2008). In addition, the use of soy-based products has been suggested as an efficient way to reduce phosphorus pollution in salmon farms, due to its low concentration in this mineral (Bergheim et al., 1995). Soy-based products are successfully used as a partial or total protein source in several fish diets, such as rainbow trout (Burr et al., 2012; Escaffre et al., 2007; Kaushik et al., 1995; Mambrini et al., 1999), catfish (Kumar et al., 2017; Webster et al., 1995), sea bass (Kaushik et al., 2004; Wang et al., 2017), grouper (García-Ortega et al., 2016; Millamena, 2002), tilapia (El-Saidy et al., 2002; 2003; Figueiredo-Silva et al., 2015; Webster et al., 2016; Zhao et al., 2010), Japanese sole (Abdul Kader et al., 2012), Atlantic salmon (Carter et al., 2000), cobia (Chou et al., 2004; Lunger et al., 2007; Zhou et al., 2005), sea bream (Biswas et al., 2007; Hernández et al., 2007; Takagi et al., 2001), and longfin yellowtail (Kissinger et al., 2016). 
Soybean meal (SBM) is considered one of the most nutritious vegetable proteins ingredients for fish and is well acceptable to totally substitute FM in diets for fish by supplementation of EAA (Furuya et al., 2004a). At the Figure 3 is possible to observe the levels of the main amino acids in the SBM, demonstration of low methionine content.
Supplementation with Met was shown as an efficient strategy to replace FM by Met-limiting SBM in diets for tilapia (Figueiredo-Silva et al., 2015; Shiau et al., 1987), rainbow trout (Boonyoung et al., 2013), sea bass (Tulli et al., 2010), sunshine bass (Keembiyehetty et al., 1997), and by soy protein concentrate (SPC) in diets for rainbow trout (Cheng et al., 2003; Gaylord et al., 2009; Kaushik et al., 1995), red sea bream (Takagi et al., 2001), and African catfish (Fagbenro et al., 2004). But whether soy proteins products, as SBM and SPC, can effectively replace FM in fish diets is still controversial, since methionine is often considered to be the first limiting amino acid in both ingredients in fish diets (Belghit et al., 2014; Berge et al., 2004; Furuya et al., 2010). However, when compared to FM, SBM has a low digestible methionine level. Several studies have demonstrated the success of replacing FM with SBM in tilapia diets when the EAA profile was balanced by AA supplementation (El-Saidy et al., 2002; Figueiredo-Silva et al., 2015; Furuya et al., 2004a; Nguyen et al., 2009b). Therefore, the use of SBM in fish diets is limited by its AA profile, which is deficient in the EAA both lysine and methionine (Gatlin III et al., 2007). Practical diets generally contain high levels of vegetable proteins often require supplementation with methionine to satisfy the total sulphur amino acid (TSAA) requirements for fish (Keembiyehetty et al., 1997).
Exploring the nutritional potential of L-methionine in fish nutrition - Image 5
Supplementing vegetable-based diets using methionine has been shown to improve the nutritional value of these diets, resulting in a significant effect on performance and protein deposition in different fish species, such as sunshine bass (Keembiyehetty et al., 1997), rainbow trout (Belghit et al., 2014; Gaylord et al., 2009; Rodehutscord et al., 1995; Rumsey et al., 1983), sea bream (Takagi et al., 2001), and tilapia (Figueiredo-Silva et al., 2015). The accumulation of muscle proteins is the main objective in fish nutrition, therefore, optimizing the supply of methionine in the diet requires precise knowledge of the role of this SAA, especially in muscle (Belghit et al., 2014). The balance of the AA profile of the vegetable protein-based diet with its respective limiting EAA significantly improved whole-body protein accretion and voluntary feed intake for rainbow trout (Kaushik et al., 1995; Mambrini et al., 1999), sea bass (Kaushik et al., 2004), Atlantic salmon (Espe et al., 2006), sea bream (Dias et al., 2009), sole (Silva et al., 2009), and tilapia (El-Saidy et al., 2002; Figueiredo-Silva et al., 2015; Furuya et al., 2004a). 
It is well known that feed can represent up to 70% of operational costs in fish production, and nowadays special attention is given to tilapia nutrition, with an emphasis on the partial or total replacement of fish meal by less expensive vegetable protein sources (El-Sayed, 1999). In diets for tilapia it is very common to use high levels of soybean meal, and the total substitution of fish meal is acceptable, as long as the diets are supplemented with synthetic amino acids, mainly with methionine. In addition, Nile tilapia uses synthetic AAs with great efficiency and presents excellent performance results when supplemented with methionine (Furuya et al., 2004a). 
Several studies are carried out with tilapia, as it is a model fish for testing AA efficient in aquafeed, and several researchers examined the effects of increased L-methionine intake and observed improvement in growth performance and determined the need for methionine for growth (He et al., 2017; Jackson et al., 1982; Liebert, 2009; Nguyen et al., 2009a). These studies generate additional information on the requirement for L-methionine and this in a contextual way definitely contributes to the development of economical and balanced AA aquafeeds. 
According to (Shiau et al., 1987), with methionine supplementation, FM can be replaced up to 67% by SBM without any adverse effect on weight gain and feed conversion rate (Figure 4). Therefore, given the assumption, L-methionine supplementation proved to be an efficient strategy to replace FM with alternative protein sources limiting Met in diets for rainbow trout (Cowey et al., 1992; Kaushik et al., 1995), African catfish (Fagbenro et al., 2004), tilapia (Shiau et al., 1987), and sea bass (Azeredo et al., 2017; Machado et al., 2015; Thebault et al., 1985; Tulli et al., 2010). 
Exploring the nutritional potential of L-methionine in fish nutrition - Image 6
In order to reduce FM in fish diets, a practical example was proposed by designing a balanced diet to meet the requirement for optimal growth for Nile tilapia (Oreochromis niloticus) according to Furuya et al. (2004b). Assuming that are available L-methionine and four protein sources, soybean Meal, soy protein concentrate, meat and bone meal and fish meal. By adopting an approach based on replacement of FM, protein content and Met can be meet by increasing others protein ingredients, and also increasing levels of synthetic Met in the diet. This formulation approach based on fish meal replacement can reduce the cost of the diet by 46% for tilapia (Table 3).
Exploring the nutritional potential of L-methionine in fish nutrition - Image 7
CONCLUSION
Taken together, the present review demonstrated that methionine is an indispensable and essential amino acid for fish, used to synthesize protein, donor of methyl groups and as a precursor of several important substances. While various sources of methionine can be used in fish diets, L-methionine supplementation can be considered a key strategy for juvenile fish that do not have mature enzymes responsible for converting D-Isomer to L-Isomer. L-Methionine is an important amino acid to replace FM content that support the growth rates for the sustainable and economical production of fish. Also, L-methionine supplementation plays an important role in health and other physiological processes vital to fish homeostasis.

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Bruno Tadeu Marotta Lima
Fenchem Inc.
Iván Camilo Ospina-Rojas
CJ Bio
Tatiana Garcia Diaz
Ramalho Rodrigueiro
CJ Bio
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