Polysaccharides from seaweed as ingredients in marine aquaculture feeding: alginate, carrageenan and ulvan

Excessive use of antimicrobials in aquaculture can select for resistant bacteria that may pose a risk to public health. For this reason, alternatives to the use of these compounds are currently being sought. Recent studies indicate that the use of functional ingredients in marine aquaculture, such as certain polysaccharides derived from algae, may be related to an improvement in the immune system of cultured organisms, reducing the risk of infections and, therefore, the use of antimicrobials. . Seaweeds are rich in polysaccharides that have various beneficial effects such as immunostimulant and prebiotic activity, making them promising functional compounds and a good alternative to the use of antibiotics. The objective of this review has been to describe three algae polysaccharides: alginate, carrageenan and ulvan, capable of improving the health status of organisms cultivated in marine aquaculture, as well as to compile recent studies that relate these compounds with different beneficial effects. produced in marine fish farming.
Fish farming has increased globally in recent years due to an increase in fish consumption and declining natural reserves. After 4 decades of continuous growth in aquaculture production, more than half of the fish consumed in the world is supplied by this activity (APROMAR 20101). Currently, the design of diets for aquaculture feeding is aimed not only at providing the necessary nutrients for optimal development, but also at providing functional ingredients that improve the health status of the fish (Burr et al. 2005). In addition, the nutritional status of the fish and stress will influence its immune status and therefore its defense against diseases (Gatlin et al. 2006).
The intensification of production in aquaculture allows to optimize profitability, but it can also increase the susceptibility to diseases in cultured organisms, since water quality deteriorates and stress conditions increase. As in other production systems, antimicrobial agents have been widely used in aquaculture (tetracycline, amoxicillin, quinolones, etc.) to treat infections caused by various pathogens.
Due to the abuse of antimicrobials in aquaculture, bacteria in the aquatic environment can develop resistance that can be transferred to other bacteria, thus creating a potential risk to public health, due to the development of acquired resistance in bacteria in the aquatic environment that can infect to humans and that these resistant bacteria can act as a reservoir of resistance genes and disseminate them, ultimately being incorporated into human pathogens. In the first case, resistant bacteria can reach humans through consumption of aquaculture products, through drinking water, or through direct contact with water or aquatic organisms (Schwarz et al. 2001, FAO/OIE/ WHO 2006).
Due to the above, several alternatives to the use of antimicrobials in aquaculture have been proposed, such as the use of vaccines, the increase in non-specific defense mechanisms, as well as the use of prebiotics, probiotics and immunostimulating substances (Irianto & Austin 2002, Gatesoupe 2005, FAO/OIE/WHO 2006).
Due to the above, several alternatives to the use of antimicrobials in aquaculture have been proposed, such as the use of vaccines, the increase in non-specific defense mechanisms, as well as the use of prebiotics, probiotics and immunostimulating substances (Irianto & Austin 2002, Gatesoupe 2005, FAO/OIE/WHO 2006).
​Functional food has been defined as that which has a proven beneficial effect on one or several specific functions in the body, beyond the usual nutritional effects, this fact being relevant for improving health and well-being and/or reducing risk. of disease (ILSI 19992). In the present work, 3 polysaccharides derived from algae (alginate, carrageenan and ulvan) are proposed as functional ingredients in marine aquaculture to improve the health status of organisms and thus reduce the use of antibiotics. The main objective of this work was to carry out a review of the current studies on the use of marine algae and/or their polysaccharides, as functional ingredients in aquaculture, as well as to describe
the beneficial effects that these compounds can bring to farmed marine fish. This work focuses mainly on the addition of polysaccharides obtained from brown algae (alginate), red algae (carrageenan) and green algae (ulvan) to marine fish feed.
Seaweeds contain bioactive substances such as polysaccharides, proteins, lipids, and polyphenols with varied antibacterial, antiviral, and antifungal activity, among others (Castro et al. 2006). These biological activities give algae great potential as a supplement in fish feed. The cell wall of algae contains, among other elements, abundant polysaccharide matrix formed by neutral and acid sugars that can also be found in terrestrial plants. However, in the latter, the carbohydrates are not sulfated and it is these groups that allow the formation of molecules with different structures and give them beneficial properties (Castro et al. 2006).
Alginates are sulfated polysaccharides present in the cell wall of brown algae (Phaeophyceae class) in species such as Macrocystis pyrifera and Ascophyllum nodosum (Khotimchenko et al. 2001), and there are also certain bacteria capable of synthesizing alginate extracellularly (for example, the genera Azotobacter and Pseudomonas) (Draget 2005). Alginates are structured in linear polymer chains, composed of mannuronic acid monomers and its epimer, guluronic acid, linked by (1-4) bonds. The monomers can be organized in homopolymeric packages rich in guluronic acid, or rich in mannuronic acid and in heteropolymeric packages alternating the two acids (Brownlee et al. 2005). The percentage of these 3 packets depends on the origin of the alginate, the age of the tissue and other factors. Manuronic acid is found in young algae, and in senescent ones this acid is transformed into its epimer, guluronic acid, due to the enzyme C5-epimerase. In mature tissues, mannuronic acid is located mainly in the extracellular spaces, while guluronic acid is found in cell walls (Khotimchenko et al. 2001).
2ILSI North America Technical Committee on Food Components for Health Promotion (1999). Food Component Report. ILSI Press, Washington, DC, USA.
The physiological effects that occur due to the consumption of alginate in humans and terrestrial mammals have been widely demonstrated, observing a reduction in blood cholesterol, prebiotic activity, mobilization of fatty acids, reduction of blood glucose, reduction of enzymatic activity in the intestine, preventive effect against cancer, increased feeling of satiety, reduction of blood pressure and stimulation of the immune response (Hoebler et al. 2000, Vaugelade et al. 2000, Warrand 2006). Furthermore, recent studies suggest that certain alginates may enhance the repair of intestinal mucosal damage (Brownlee et al. 2005, Warrand 2006).
Wang et al. (2006) investigated the prebiotic effect of alginate in vitro and in rats, comparing it with fructooligosaccharides and concluded that it was capable of increasing Bifidobacteria and Lactobacilli counts more significantly than fructooligosaccharides.
Carrageenan is a generic term for a complex family of polysaccharides that are extracted from different red algae. It is a polysaccharide composed of alternating b-D-Galactose residues linked by a 1,3-linkage and a-D-Galactose linked by 1,4 bond. There are 3 types of commercial carrageenan for the food industry: kappa-, iota and lambda that differ in the amount and position of the sulfate group (Warrand 2006).
The physiological effects demonstrated so far for this polysaccharide are: repair of intestinal damage, stimulation of the immune system of fish (Fujiki et al. 1997, Castro et al. 2004), antiviral activity especially against human papillomavirus (Buck et al. 2006, Roberts et al. 2007) and modification of the composition of the intestinal microbiota since these compounds are not metabolized in the colon (Jimenez-Escrig et al. 2000, Warrand 2006). On the other hand, Lahaye & Kaeffer (1997) observed that the addition of iota-carrageenan to the diet of rats favored the proliferation of the intestinal mucosa. Carrageenans have been used mainly as an immunostimulant supplement in aquaculture, an activity that is developed in the next section of this work.
Ulvano are water-soluble polysaccharides extracted from the cell walls of green algae, mainly from species belonging to the Ulva – Enteromorpha complex (Lahaye & Robic 2007, Robic et al. 2009). It is mainly composed of rhamnose, glucuronic and iduronic acids, and xylose, being most often distributed in repeating disaccharide units (Robic et al. 2009).
Green algae carbohydrates are not broken down by human digestive enzymes or by colonic bacteria (Paradossi et al. 2002). Ulvan may serve as a stabilizer and promoter as it binds to growth factors involved in intestinal mucosal growth and repair (Warrand 2006).
In different studies, the various functional properties of these polysaccharides have been demonstrated in vitro; for example, an antitumor activity (Kaeffer et al. 1999), antioxidant (Qi et al. 2005), immunostimulant (Castro et al. 2004, 2006; Leiro et al. 2007), antiviral activity (Schaeffer & Krylov 2000) and activity anticoagulant (Mao et al. 2004). In vivo studies have also observed other beneficial activities, such as the production of mucin in the colon of rats (Barcelo et al. 2000) and the modulation of lipid metabolism, decreasing hyperlipidemia in rats (Sathivel et al. 2008). Recent studies evaluating the polysaccharides from these algae on turbot phagocytes have shown stimulation of the immune system response (Castro et al. 2004, 2006).
In recent years, many studies have been carried out on the use of different substances as supplements in fish production and which allow the use of antimicrobials to be progressively replaced (Burr 2005, FAO/OIE/WHO 2006, Ringø et al. 2010a, b). These compounds can be classified as immunonutrients (compounds that serve as a substrate or energy source for the immune system), immunostimulants (regulate the immune system response by sending signals to the neuro-immuno-endocrine system) and compounds and/or organisms living modulators of the colonic flora (Burr et al. 2005, Gatlin et al. 2002).
The beneficial effects that can be produced by adding polysaccharide compounds from marine algae to the diet of marine aquatic organisms are mentioned below. Table 1 shows some of the recent studies, carried out in vivo, in which these polysaccharides are evaluated and that they present beneficial effects in marine aquaculture organisms. As can be seen in said table, most studies focus on the use of alginate, with few evaluating the functionality of carrageenan and none that of ulvan.
The addition of small amounts of algae to fish feed produces an increase in growth, feeding efficiency, protein synthesis (decreasing protease activity) and lipid stores in muscle (Nakagawa 2010).
In relation to alginate from Ascophylum nodosum, some authors demonstrated its potential to modify lipid metabolism, thus Yone et al. (1986) and Nakagawa et al. (1997) used it as an additive in the feed of Japanese sea bream (Pragus major) at levels of 2.5% and 5%. These authors found an increase in the proportion of muscle protein, improving the absorption and assimilation of dietary protein.
Various studies have shown that the addition of alginate to the diet of marine aquaculture fish produces an improvement in growth and feeding efficiency (Conceição et al. 2001, Yeh et al. 2008, Ahmadifar et al. 2009, Jalali et al. 2009). In addition, Conceição et al. (2001) observed a 3-fold increase in the retention of newly synthesized proteins in turbot specimens that were supplemented with alginate.
Immunity encompasses all the mechanisms and responses used by the body to defend itself against pathogens. Fish have a less specific immune system, compared to mammals, with a shorter response, a smaller repertoire of immunoglobulins and mmunological memory and a weak mucosal response (Trichet 2010). In recent years, the study of compounds with immunostimulating activity has been of interest, among which are microbial products such as b-glucans, lipopolysaccharides, peptidoglycans, bacterial fermentation products and some herbal extracts in aquaculture, since they increase the host’s immune response. , which can be a good alternative to the use of antibiotics (Li 2006). The biological effects of immunostimulants are dependent on receptors on target cells that recognize them as potential high-risk molecules and trigger defense pathways. There are more and more studies that support the benefit of using immunostimulants in aquaculture since they induce protection against diseases due to the increase in the immune response (Bricknell & Dalmo 2005, Jaafar et al. 2011).
Various studies demonstrate the immunostimulating activity of algae and their polysaccharide compounds in aquaculture (Bagni et al. 2005, Cheng et al. 2007, 2008; Chiu et al. 2008, Yeh et al. 2008, Ahmadifar et al. 2009, Harikrishnan et al. al. 2010). In vitro studies carried out with turbot phagocytes have shown that polysaccharides extracted from algae such as Ulva rigida and Chondrus crispus (Castro et al. 2004, 2006) produced a greater response from the immune system.
On the other hand, there are more and more in vivo studies that reiterate the ability of polysaccharide compounds from different algae to increase the response of the immune system. The immunostimulating activity of alginate has been demonstrated in different marine fish; such as halibut Hippoglossus hippoglossus L. (Skjermo & Bergh 2004), seabass Dicentrarchus labrax (Bagni et al. 2000) and different species of grouper e.g., Epinephelus coicoides, Epinephelus fuscoguttatus, Epinephelus brneus (Cheng et al. 2007, 2008; Chiu et al. al. 2008, Yeh et al. 2008, Harikrishnan et al. 2011), leading to increased fish survival. Carrageenan and ulvan as modulators of the immune system response in marine fish have been less studied; even so, the results obtained so far are promising. Two studies conducted with different types of carrageenan in E. coicoides and E. fuscoguttatus found positive results for resistance to Vibrio alginolyticus infection (Cheng et al. 2007, 2008). Although the use of immunostimulants in aquaculture has obtained good results in various investigations, there are two positions regarding their effects when applied in early stages of fish development. There are researchers who think that they can be added to the feed of larvae of aquaculture organisms with minimal impact on the development of the immune system of these animals and others who believe that their early administration can be detrimental to the development of the fish’s immune system (Bricknell & Dalmo 2005).
In homeothermic animals, the microbiota of the gastrointestinal tract participates in the digestive function and also acts as a protective barrier against pathogens. Fish also harbor different bacteria in their gut capable of inhibiting the colonization of pathogens (Nayak 2010). On the other hand, studies with microorganism-free animal models have observed that said intestinal microbiota is involved in the proliferation, maturation and epithelial immunity of fish (Rawls et al. 2004, Rekecki et al. 2009).
Due to the role that the intestinal microbiota has in nutrition, growth, immunity, intestinal balance and resistance to diseases in aquatic animals (Kesarcodi-Watson et al. 2008), the modulation of the intestinal microbiota is presented as one of the alternatives to the use of antimicrobials in aquaculture (Ringø et al. 2010a, b). The addition of prebiotics and probiotics are two ways by which changes in the intestinal microbiota can be induced. The main beneficial effects related to such bacterial changes are an improvement in growth and an increase in the immune system response (Nayak 2010).
Prebiotics are non-digestible ingredients that beneficially affect the host, selectively stimulating the growth and/or activity of certain colonic bacterial populations (Gibson & Roberfroid 1995). Its use has been very wide in the production of birds and other terrestrial mammals; however, in aquaculture more studies would be necessary to confirm this effect.
The gastrointestinal tract of invertebrates and vertebrates provides a suitable habitat for various microorganisms that play an important role in the health and nutrition of the host. However, the composition of the anaerobic microbial populations that inhabit the intestine of fish It is little known, so research is needed to provide more information in this regard. Such flora is essential to characterize the intestinal microbial community of fish and thus evaluate the necessary dietary supplements to stimulate the production of beneficial bacteria in farmed fish (Burr et al. 2005, Ringø et al. 2010b). In addition, the intestinal bacterial populations are different in freshwater and marine fish, so in marine fish those belonging to the genera appear as dominant bacteria.
Photobacterium, Pseudomonas and Vibrio, while in freshwater fish species of the genera Aeromonas, Pleisomonas Bacteroides, Fusobacterium, Eubacterium and the Enterobacteriaceae family dominate (Ringo et al. 2010a).
On the other hand, it is known that certain microbial populations of the gastrointestinal tract, such as lactic acid bacteria, are capable of producing antibacterial compounds that can considerably reduce the counts of pathogenic species in the intestinal microbiota of fish, thus improving their development (Verschuere et al. 2000, Burr et al. 2005). These lactic acid bacteria are part of the normal intestinal microbiota of fish from the first days of life, although they are not dominant (Ringø & Gatesoupe 1998, Ringø et al. 2005). Various factors regulate microbial populations (e.g., lactic acid bacteria) in the gastrointestinal tract, to mention the concentration of dietary polyunsaturated fatty acids, competition for nutrients, presence of chromic oxide, salinity, and host stress. (Ringø & Gatesoupe 1998). The survival of these intestinal microbial communities depends on the availability of substrate, which will be used to generate different products such as short-chain fatty acids, amino acids, polyamines, growth factors, vitamins and antioxidants essential for the activity of the intestinal mucosa (Fric 2007). ).
Seaweed-derived polysaccharides have traditionally been used as thickening agents in the food industry. However, due to their complexity that makes them resistant to degradation by human intestinal enzymes, they are a substrate for intestinal bacteria and could be proposed as potential prebiotics (Ramnani et al. 2012). Alginates, carrageenans and ulvan, being polysaccharide compounds, may have prebiotic activity, exerting a positive selective effect on the intestinal microbiota of fish. Furthermore, dietary carbohydrates play an important role in the immune response through their interactions with gut microbiota and gut-associated lymphoid tissue (Trichet 2010).
In relation to another of the alternatives proposed for the modulation of the intestinal microbiota, Gram & Ringø (2005), studying the modulation of the intestinal microbiota of fish, proposed the following definition of probiotics: ‘live microorganisms that added to food or the environment environment (water) increase the
host viability. Probiotics are currently used in aquaculture to control some diseases in fish, although their mode of action is poorly understood (Ringø et al. 2010a). Several options for their action mechanisms have been considered: displacing potential pathogens by producing substances that inhibit their growth, competing for nutrients or space, altering microbial metabolism and/or stimulating the host’s immune system (Irianto & Austin 2002, Gómez & Balcázar 2008, Kesarcodi-Watson et al. 2008).
The main advantage of prebiotics over probiotics is that the former are natural ingredients and their incorporation into the diet does not require special precautions, therefore authorization as a food additive is easier to obtain (Gatesoupe 2005). However, while the goal of prebiotics is to stimulate beneficial flora, some opportunistic pathogenic bacteria may acquire the ability to utilize these substances or their breakdown products, if administered continuously (Gatesoupe 2005). It is therefore necessary prior to its application to carry out studies in certain fattening phases of different fish species (Gatesoupe 2008). In addition, in the use of probiotics, the species used become predominant in the gastrointestinal tract only during dietary treatment, making it necessary to take into account that there is little chance of colonizing the intestine, if the bacterial species used does not belong to the intestinal microbiota. characteristic of a fish species. Therefore, it is proposed to stimulate the growth of indigenous microbial species by supplementing the diet with indigestible carbohydrates that act as prebiotics (Mahiouet al. 2006).
Although there are not many studies focused on the use of polysaccharides (alginate, carrageenan and ulvan) as functional ingredients in marine aquaculture, the results obtained so far indicate that they could be a promising ingredient to improve the health status of marine fish, since that beneficially affect their growth, feeding efficiency and survival, in addition to having prebiotic and immunostimulant capacity. After carrying out this review, there is a clear need to carry out studies that delve into the composition of the intestinal microbiota populations.

Of marine fish to more effectively assess its modulation.
For future research and taking into account, on the one hand, the limitations of probiotics and, on the other, the beneficial effects that polysaccharide compounds from algae can bring to aquatic organisms, there is sufficient evidence to believe that any of the three compounds (alginate, carrageenan and ulvano) could be added together with a bacterial species, previously selected as probiotics, to the diet of aquaculture fish to study the symbiotic effect (polysaccharide and prebiotic). One could even consider the encapsulation of said bacteria with alginate for better administration. However, it is important to take into account the parallel study of the quality of the meat of fish fed with polysaccharides, as well as consumer acceptance due to the possible changes that occur in their sensory characteristics, due to the impulse of aquaculture. to cover the current demand for fish consumption.

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