Polysaccharides from seaweed as ingredients in marine aquaculture feeding: alginate, carrageenan and ulvan
Excessive use of antimicrobials in aquaculture can select resistant bacteria that can 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 algae-derived polysaccharides, may be related to an improvement of the immune system of cultured organisms, reducing the risk of infections and, therefore, the use of antimicrobials. Seaweeds are rich in polysaccharides that possess several beneficial effects such as immunostimulant and prebiotic activity, which make them promising functional compounds and a good alternative to the use of antibiotics. The aim of the present review was to describe three algal polysaccharides: alginate, carrageenan and ulvan, capable of improving the health status of organisms cultured in marine aquaculture, as well as to compile recent studies that relate these compounds to different beneficial effects produced in marine fish farming.
Fish farming has increased worldwide in recent years due to an increase in fish consumption and the decrease of natural stocks. 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 aquaculture feed diets is aimed not only at providing the nutrients necessary 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 thus its defense against disease (Gatlin et al. 2006).
Intensification of aquaculture production can optimize profitability, but it can also increase susceptibility to disease in cultured organisms, as 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.
1APROMAR. 2010. Annual report on marine aquaculture in Spain. Business Association of Marine Culture Producers. [online] http://www.apromar.es
Source: Patricia Peso-Echarri1, Carmen Frontela-Saseta1, Carlos A. González-Bermúdez1, Gaspar F. Ros-Berruezo1 and Carmen Martínez-Graciá1
(Aeromonas hydrophila, A. salmonicida, Edwardsiella tarda, Pasteurella piscicida, Vibrio anguillarum and Yersinia ruckeri) (Angulo 1999). However, the frequent use of antibiotics is controversial due to the emergence of microbial resistance, the potential impact on environmental bacterial communities and the risk of residues of antimicrobial agents in aquaculture products for consumption (Li 2006, FAO/OIE/WHO 2006, Kesarcodi-Watson et al. 2008).
Due to the abuse of antimicrobials in aquaculture, bacteria in the aquatic environment can develop resistances that can be transferred to other bacteria, thus producing a potential risk to public health, due to the development of acquired resistance in bacteria in the aquatic environment that can infect humans and to the fact that these resistant bacteria can act as a reservoir of resistance genes and spread them, ultimately incorporating themselves into human pathogens. In the first case, resistant bacteria can reach humans by consumption of aquaculture products, through drinking water, or by 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 enhancement of non-specific defense mechanisms, as well as the use of prebiotics, probiotics and immunostimulants (Irianto & Austin 2002, Gatesoupe 2005, FAO/OIE/WHO 2006).
Functional food has been defined as a food that has a proven beneficial effect on one or more specific functions in the body, beyond the usual nutritional effects, being this fact relevant for improving health and well-being and/or reducing the 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 review 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 algae and/or their polysaccharides can have on the health of the organisms.
the beneficial effects that these compounds can provide to cultured marine fish. This work is mainly focused 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 various antibacterial, antiviral and antifungal activities, 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, an abundant polysaccharide matrix formed by neutral sugars and acids that can also be found in terrestrial plants. However, in the latter, 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).
CHARACTERISTICS OF ALGINATE
Alginates are sulfated polysaccharides present in the cell wall of brown algae (class Phaeophyceae) in species such as Macrocystis pyrifera and Ascophyllum nodosum (Khotimchenko et al. 2001), 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 monomers of mannuronic acid and its epimer guluronic acid linked by (1- 4) bonds. The monomers can be arranged 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 packages 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 algae this acid is transformed into its epimer, guluronic acid, due to the enzyme C5-epimerase. In mature tissues mannuronic acid is mainly localized in the extracellular spaces, whereas guluronic acid is found in the 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 of alginate consumption in humans and terrestrial mammals have been extensively demonstrated, including blood cholesterol reduction, prebiotic activity, fatty acid mobilization, blood glucose reduction, reduction of enzyme activity in the intestine, cancer preventive effect, increased satiety, reduction of blood pressure and stimulation of the immune response (Hoebler et al. 2000, Vaugelade et al. 2000, Warrand 2006). In addition, 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 able to increase Bifidobacteria and Lactobacillus counts more significantly than fructooligosaccharides.
CHARACTERISTICS OF CARRAGEENAN
Carrageenan is a generic term for a complex family of polysaccharides extracted from different red algae. It is a polysaccharide composed of alternating residues of b-D-Galactose linked by 1,3-linkage and
a-D-Galactose linked by a 1,4 bond. There are 3 types of commercial carrageenan for the food industry: kappa-, iota and lambda which 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 following section of this work.
CHARACTERISTICS OF ULVAN
Ulvan 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, most often found distributed in repeating disaccharide units (Robic et al. 2009).
Green algal carbohydrates are not degraded by human digestive enzymes or colon 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).
Different studies have demonstrated in vitro the various functional properties of these polysaccharides; for example, 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 anticoagulant activity (Mao et al. 2004). Other beneficial activities have also been observed in in vivo studies, such as mucin production in the rat colon (Barcelo et al. 2000) and modulation of lipid metabolism decreasing hyperlipidemia in rats (Sathivel et al. 2008). Recent studies evaluating polysaccharides from these algae on turbot phagocytes have shown stimulation of the immune system response (Castro et al. 2004, 2006).
BENEFICIAL EFFECTS RELATED TO THE USE OF ALGAL POLYSACCHARIDES AS A SUPPLEMENT IN MARINE AQUACULTURE
In recent years, many studies have been carried out on the use of different substances as supplements in fish production and that 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 into immunonutrients (compounds that serve as a substrate or energy source for the immune system), immunostimulants (they regulate the immune system response by sending signals to the neuro-immuno-endocrine system) and compounds and/or living organisms that modulate the colonic flora (Burr et al. 2005, Gatlin et al. 2002).
The beneficial effects that the addition of polysaccharide compounds from seaweeds to the diet of marine aquatic organisms can produce are mentioned below. Table 1 presents some of the recent in vivo studies evaluating these polysaccharides and showing beneficial effects in marine aquaculture organisms. As can be seen in this table, most of the studies focus on the use of alginate, with few studies evaluating the functionality of carrageenan and none evaluating the functionality of ulvan.
EFFECT ON GROWTH AND FEEDING EFFICIENCY OF AQUATIC ORGANISMS
The addition of small amounts of algae in fish feed produces an increase in growth, feed efficiency, protein synthesis (decreasing protease in protease activity) and lipid deposits in muscle (Nakagawa 2010).
In relation to alginate from Ascophylum nodosum, some authors demonstrated its potential to modify lipid metabolism, as Yone et al. (1986) and Nakagawa et al. (1997) used it as a feed additive for 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.
Several studies have shown that the addition of alginate to the diet of marine aquaculture fish results in improved growth and feed efficiency (Conceição et al. 2001, Yeh et al. 2008, Ahmadifar et al. 2009, Jalali et al. 2009). Furthermore, Conceição et al. (2001) observed a 3-fold increase in the retention of newly synthesized proteins in turbot specimens supplemented with alginate in the diet.
ACTIVITY ON THE IMMUNE SYSTEM
Immunity encompasses all the mechanisms and responses used by the organism to defend itself against pathogens. Fish have a less specific immune system compared to mammals, with a shorter response, smaller immunoglobulin repertoire and a weak immunological memory and mucosal response (Trichet 2010). In recent years, the study of compounds with immunostimulatory activity, including microbial products such as b-glucans, lipopolysaccharides, peptidoglycans, bacterial fermentation products and some herbal extracts in aquaculture, has been of interest, since they increase the host immune response, which may be a good alternative to the use of antibiotics (Li Li, 2010).
to the use of antibiotics (Li 2006). The biological effects of immunostimulants are dependent on receptors on target cells that recognize them as potentially high-risk molecules and trigger defense pathways. An increasing number of studies support the benefit of the use of immunostimulants in aquaculture as they induce protection against diseases due to increased immune response (Bricknell & Dalmo 2005, Jaafar et al. 2011).
Several studies demonstrate the immunostimulatory 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. 2010). In vitro studies with turbot phagocytes have shown that polysaccharides extracted from algae such as Ulva rigida, and Chondrus crispus (Castro et al. 2004, 2006) produced an enhanced immune system response.
On the other hand, more and more in vivo studies reiterate the ability of polysaccharide compounds from different algae to increase the immune system response. The immunostimulant activity of alginate has been demonstrated in different marine fish; such as halibut Hippoglossus hippoglossus L. (Skjermo & Bergh 2004), sea bass 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. 2008, Yeh et al. 2008, Harikrishnan et al. 2011), producing 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 obtained positive results in resistance to Vibrio alginolyticus infection (Cheng et al. 2007, 2008). Although the use of immunostimulants in aquaculture has obtained good results in several investigations, there are two positions about their effects when applied in early stages of fish development. There are researchers who believe that they can be added to the feed of larval 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).
CHANGES IN THE INTESTINAL MICROBIOTA. PREBIOTIC ACTIVITY
In homeothermic animals, the microbiota of the gastrointestinal tract is involved in digestive function and also acts as a protective barrier against pathogens. Fish also harbor in their gut different bacteria capable of inhibiting the colonization of pathogens (Nayak 2010). On the other hand, studies with animal models free of microorganisms have observed that this 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 of gut microbiota in nutrition, growth, immunity, intestinal balance and disease resistance in aquatic animals (Kesarcodi-Watson et al. 2008), modulation of gut 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 improved growth and increased immune system response (Nayak 2010).
Prebiotics are non-digestible ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of specific bacterial populations in the colon (Gibson & Roberfroid 1995). They have been widely used in the production of poultry and other terrestrial mammals; however, in aquaculture more studies are needed to confirm this effect.
The gastrointestinal tract of invertebrates and vertebrates provides a suitable habitat for diverse microorganisms that play an important role in host health and nutrition. However, the composition of the anaerobic microbial populations inhabiting the gut of fish is poorly understood, and 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 dietary supplements needed to stimulate the production of beneficial bacteria in aquaculture fish (Burr et al. 2005, Ringø et al. 2010b). In addition, the intestinal bacterial populations are different in freshwater and marine fish, thus in marine fish the dominant bacteria are those belonging to the genera
Photobacterium, Pseudomonas and Vibrio, while in freshwater fish, species of the genera Aeromonas, Pleisomonas Bacteroides, Fusobacterium, Eubacterium and the family Enterobacteriaceae 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 gut microbiota of fish from the first days of life, although they are not dominant (Ringø & Gatesoupe 1998, Ringø et al. 2005). Several factors regulate the microbial populations (e.g., lactic acid bacteria) in the gastrointestinal tract, for example, the concentration of polyunsaturated fatty acids in the diet, competition for nutrients, the 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 indispensable for intestinal mucosal activity (Fric 2007).
Polysaccharides derived from marine algae 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 substrates for intestinal bacteria and could be proposed as potential prebiotics (Ramnani et al. 2012). Alginates, carrageenans and ulvan, being polysaccharide compounds may possess prebiotic activity, exerting a positive selective effect on the intestinal microbiota of fish. In addition, dietary carbohydrates play an important role in the immune response through their interactions with the intestinal microbiota and the lymphoid tissue associated with the intestine (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 the feed or to the environment (water) increase the viability of the host’. 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 mechanisms of action have been considered: displacing potential pathogens by production of substances that inhibit their growth, competing for nutrients or space, altering microbial metabolism and/or stimulating the host 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, making authorization as a food additive easier to obtain (Gatesoupe 2005). However, although the purpose of prebiotics is to stimulate beneficial flora, some opportunistic pathogenic bacteria may acquire the ability to use these substances or their degradation products if they are administered continuously (Gatesoupe 2005). It is therefore necessary, prior to their application, to carry out studies in certain fattening stages of different fish species (Gatesoupe 2008). Furthermore, in the use of probiotics, the species used become predominant in the gastrointestinal tract only during the 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 non-digestible carbohydrates that act as prebiotics (Mahious et 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 they beneficially affect their growth, feeding efficiency and survival, in addition to having prebiotic and immunostimulant capacity. After this review, there is a clear need for further studies on the composition of the gut microbiota populations of marine fish in order to evaluate in a more detailed manner of marine fish in order to more effectively evaluate its modulation.
For future research and taking into account on the one hand the limitations of probiotics and on the other hand the beneficial effects that polysaccharide compounds from algae can provide to aquatic organisms, there is sufficient evidence to believe that any of the three compounds (alginate, carrageenan and ulvan) 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). The encapsulation of such bacteria with alginate could even be considered for a better administration. However, it is important to take into account the parallel study of the quality of the flesh of fish fed with the polysaccharides, as well as consumer acceptance due to possible changes in their sensory characteristics, due to the aquaculture drive to meet the current demand for fish consumption.