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alimentos funcionales, alimentos marinos, PUFAS, biotecnología marina

alimentos funcionales, alimentos marinos, PUFAS, biotecnología marina



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    marine biotechnology advances towards applications in new functional foods☆ marine biotechnology advances towards applications in new functional foods☆ Document Transcript

    • Research review paper Marine biotechnology advances towards applications in new functional foods☆ Ana C. Freitas a,b, ⁎, Dina Rodrigues b , Teresa A.P. Rocha-Santos a,b , Ana M.P. Gomes c , Armando C. Duarte b a ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu, Portugal b CESAM & Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal c CBQF/Escola Superior de Biotecnologia, Catholic University, Rua Dr. António Bernardino de Almeida, P-4200-072 Porto, Portugal a b s t r a c ta r t i c l e i n f o Available online 29 March 2012 Keywords: Marine Biotechnology Functional foods Ingredients The marine ecosystem is still an untapped reservoir of biologically active compounds, which have consider- able potential to supply food ingredients towards development of new functional foods. With the goal of increasing the availability and chemical diversity of functional marine ingredients, much research has been developed using biotechnological tools to discover and produce new compounds. This review summarizes the advances in biotechnological tools for production of functional ingredients, including enzymes, for the food industry. Tools involving biotechnological processes (bioreactors, fermenta- tions, bioprocessing) and those involving genetic research designated as molecular biotechnology are dis- cussed highlighting how they can be used in the controlled manipulation and utilization of marine organisms as sources of food ingredients, as well as discussing the most relevant shortcomings towards applications in new functional foods. © 2012 Elsevier Inc. All rights reserved. Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1506 2. Marine functional ingredients and their sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1508 3. Functional foods incorporating marine ingredients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509 4. Marine biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1509 4.1. Biotechnological processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1510 4.1.1. Cell factories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1510 4.1.2. Bio-processing technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1511 4.2. Molecular biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512 4.2.1. Marine metagenomic approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1512 4.2.2. Transgenic approach. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513 5. Final considerations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513 Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1513 1. Introduction Nowadays consumers are increasingly aware of the relationship between diet, health and disease prevention. It is well known that consumption of foods such as fruits, vegetables, cereals and marine foods rich in polyunsaturated fatty acids (PUFAs) beyond meeting basic nutritional needs, is also fundamental for health promotion and disease risk reduction (Shahidi, 2009). Research studies in the past years have correlated diet and some chronic diseases, therefore highlighting the enormous potential of foods in the prevention and progression of chronic diseases such as atherosclerosis (Casós et al., 2008), cancer (Trottier et al., 2010) and symptoms relief in osteoar- thritic patients (Ameye and Chee, 2006). Despite today's consumers being increasingly conscious of food safety, quality and health related issues, populations from the so called developed countries, namely populations from the European Union (EU) and United States of America (USA), still have much to do in what concerns strategies to fight modern age diseases such as cardiopathies, obesity, osteoporosis, Biotechnology Advances 30 (2012) 1506–1515 ☆ This manuscript has been submitted for publication in Biotechnology Advances. It is not to be reproduced or cited without the written permission of the authors. ⁎ Corresponding author at: ISEIT/Viseu, Instituto Piaget, Estrada do Alto do Gaio, Galifonge, 3515-776 Lordosa, Viseu, Portugal. Tel.: +351 232910017; fax: +351 232910193. E-mail address: (A.C. Freitas). 0734-9750/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2012.03.006 Contents lists available at SciVerse ScienceDirect Biotechnology Advances journal homepage:
    • cancer, diabetes, allergies and stress (Cencic and Chingwaru, 2010). Some of these diseases are highlighted by the European Food Safety Authority (EFSA) Food-based Dietary guidelines (Anon, 2008) as the most frequent diet-related health problems such as cardiovascular diseases, overweight/obesity, dyslipidemia, hypertension and type 2 diabetes. Additionally, the increasingly aging populations require dif- ferent foods and diets for healthy active aging and improved well- being (Roberts and Rosenberg, 2006). Based on the health criterion and on foods with specific health- enhancing characteristics, there has been a growing interest in re- search, development and commercialization of functional foods, nutraceuticals, and dietary supplements around the world (Shahidi, 2009). Up to date there is no standard definition for functional foods—the term is generally used to refer to a food, and according to Health Can- ada, a functional food is similar to a conventional food, that is con- sumed as part of a usual diet which either provides physiological benefits or reduces the risk of chronic disease beyond basic nutrition- al functions ( According to Food Agriculture Organization (FAO; Anon, 2007), functional foods are those foods similar to conventional food in appearance, intended to be consumed as part of a normal diet containing biologically active compounds which offer potential for enhanced health or reduced risk of disease. For nutraceutical and/or dietary supplements, no consensual defini- tion is also found and there is still ambiguity about the regulatory re- quirements related to nutraceuticals (Shirwaikar et al., 2011). In certain countries, functional foods and nutraceuticals are still used in- terchangeably (Shahidi, 2009) since some nutraceuticals are used as conventional foods or as sole items of a meal or diet. From our point of view, and in agreement with some authors (Ameye and Chee, 2006), nutraceuticals or dietary supplements should be defined as functional ingredients sold as powders, pills, and other medicinal forms not associated with food. Nevertheless, a common aspect is mandatory in functional foods, nutraceuticals or dietary supple- ments: in all of them, the main focus is on improving health and re- ducing disease risk through prevention towards improvement of quality of life and well-being contributing to an increased healthy longevity (Shahidi, 2009). Although no exact definition exists for functional foods, it is clear that it belongs to foods that besides their nutritional effects, have demonstrated benefit to one or more functions of the human organism, improving the state of health or well-being, and reduc- ing the risk of disease. Functionality is inherent to a feature intro- duced in the food matrix in order to improve health or reduce any adverse health effect, accomplished by: i) eliminating or promot- ing a chemical change on a deleterious ingredient; ii) addition of new health-promoting food ingredients or probiotic microorgan- isms; iii) addition of an existing health-promoting food ingredient, increasing its concentration in a fortification process; and iv) in- creasing the bioavailability or stability of the health-promoting food ingredient. Sources of functional ingredients exist in many different reservoirs that may be found both in terrestrial and marine environments. The terrestrial environment (p.e., fruits, vegetables, cereals and mush- rooms) as a reservoir of bioactive compounds is by far more explored than the marine environment (p.e., fish, sponges, macro- and micro- algae). Although many functional marine ingredients are presently known, it is believed that other marine ingredients remain to be eval- uated and new sources yet to be discovered. The marine environment is a major reservoir of bioactive compounds that have potential to be applied in several phases of food processing, storage and fortification (Rasmussen and Morrissey, 2007). The characteristics of marine envi- ronment such as various degrees of salinity, temperature, pressure, and illumination impart particular interest on compounds derived from marine organisms. In order to increase the availability and chemical diversity of marine functional ingredients, more research is applying biotechnological tools to discover and produce marine compounds (Baerga-Ortiz, 2009). In this review, we will focus on advances in biotechnology to dis- cover, produce or transform compounds from marine sources to be incorporated as functional ingredients in potential functional foods. Genetic research, exploring new advances in marine biotechnology, will also be a target for discussion. The functional ingredients should be dietary, but not obligatory nutrient, biologically active components present in unmodified whole food or added to a food vehicle (Anon, 2007). The interest of functional ingredients has been enhanced by the recent advances in genetics namely on nutritional genomics (Box 1), which include the nutrigenomics and the nutrigenetics (Shirwaikar et al., 2011), since food may play a role for the individual needs and predispositions. Box 1 Nutritional genomics. Nutritional genomics requires a deep knowledge of nutrition, genetics, biochemistry and the “omic” technologies (Fenech et al., 2011). This new science branch includes nutrigenomics and nutrigenetics. Nutrigenomics is the study of interaction of dietary components with the genome and the changes taking place in the metabolism as a result of that (how nutrients affect the gene expression), whereas nutrigenetics is focused on un- derstanding how the genetic constitution modulates the re- sponse to nutrients and the use of individual genetic data to develop functional foods or nutraceuticals to be more compati- ble with health (De Caterina, 2010; Subbiah, 2007a,b). This in- formation supports the thesis that a selected diet according to genome could reduce the genetic risk of suffering certain dis- eases (Plaza et al., 2008) leading to “personalized nutrition”; a preventive approach for optimizing health, delaying diseases or diminish it intensity or severity (Fenech et al., 2011) since all diseases have a genetic predisposition (Simopoulos, 2010). Andrade et al. (2010), studied the interaction between genes and dietary habits and detected that a diet rich in PUFAs resulted in the benefic effect of increasing high density choles- terol which was only observed in individuals that were not car- riers of E*4 allele of the APOE gene. Recent reviews about Nutrigenetics and Nutrigenomics highlight the state of the art on nutrition research and its application to dietetic practice (Fenech et al., 2011; Simopoulos, 2010). References Andrade FM, Bulhões AC, Malif SW, Schuch JB, Voigt F, Lucatelli JF, et al. The influence of nutrigenetics on the lipid profile: Interac- tion between genes and dietary habits. Biochem Genet 2010;48:342–355. De Caterina R. Opportunities and challenges in Nutrigenetics/ Nutrigenomics and Health. In: Simopoulos AP, Milner JA, edi- tors. Personalized Nutrition. World Rev Nutr Diet, Basel, Karger 2010;101:1–7. Fenech M, El-Sohemy A, Cahill L, Ferguson LR, French TC, Tai ES, et al. Nutrigenetics and Nutrigenomics: Viewpoints on the current status and applications in nutrition research and prac- tice. J Nutrigenet Nutrigenomics 2011;4:69–89. Plaza M, Cifuentes A, Ibánez E. In the search of new functional food ingredients from algae. Trends Food Sci Technol 2008;19:31–9. Simopoulos AP. Nutrigenetics/Nutrigenomics. Annu Rev Public Health 2010;31:53–68. Subbiah MTR. Nutrigenetics and nutraceuticals: the next wave riding on personalized medicine. Transl Res 2007:149:55–61. 1507A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • 2. Marine functional ingredients and their sources Marine resources are a source of high value-added compounds with nutraceutical value to be used as functional ingredients: omega-3 oils, chitin, chitosan, fish protein hydrolysates, algal constit- uents, carotenoids, collagen, taurine and other bioactive compounds (Kadam and Prabhasankar, 2010), are examples of such compounds that can be added at different stages, from processing to storage, of the food production process. Supplementation of foods with function- al or bioactive ingredients has become an increasingly interesting way to develop new functional foods for the so called health- conscious consumers. In Fig. 1 the main marine functional ingredi- ents, their inherent functionality and potential food applications are displayed. Enhancement of antioxidant activity and immunity stimulation are the most studied health benefits and have driven consumers to be more aware that diet can serve both nutrition and health promo- tion goals. Food products containing marine derived oils rich in omega-3 fatty acids, chitin, chitosan, etc. are some food products that are being commercialized in several markets around the world including United States, Japan and some countries of Europe (Kadam and Prabhasankar, 2010). Algae, in particular, edible algae sometimes referred as seaweeds, are a very interesting natural source of compounds with biological activity, that may be used as functional ingredients, and considering their great taxonomic diversity, research on identification of biologically active com- pounds from algae can be seen as an almost unlimited source. Moreover, such extracts are virtually fat and calorie-free, making them increasingly sought for commercial purposes. Macroalgae, i.e. Sargassum species, have been found to be good sources of dietary fiber and carotenoids with an- tioxidant activity and play important roles in the prevention of neurode- generative diseases (Chandini et al., 2008; Ganesan et al., 2008; Je et al., 2009; Kadam and Prabhasankar, 2010). Food reserves of brown algae are typically complex polysaccharides and higher alcohols; many bioac- tive metabolites have been isolated from these algae with different phar- macological activities such as cytotoxic, antitumor, nematocidal, antifungal, anti-inflammatory and antioxidant (Gamal, 2010; Je et al., 2009). Algins, carrageenans and agar are examples of polysaccharides derived from algae that are widely used as thickeners and stabilizers in foods as well as for gels (Rasmussen and Morrissey, 2007). Sulphated fucans, such as fucoidans from brown algae, carrageenans from red algae and ulvans from green algae, have been known to act as modula- tors of coagulation as well as reveal antithrombotic, anti-inflammatory, antioxidant, anticancer and antidiabetic activities, among others (Pomin, 2009; Wijesekara et al., 2011). Soluble polysaccharides from algae have tremendous potential as dietary fiber for human nutrition and are being evaluated as new possible prebiotic compounds (Gupta and Abu-Ghannam, 2011). The biological importance of macroalgae as well as of their pigments can be found in recent reviews by Gamal (2010) and Pangestuti and Kim (2011). In addition, Holdt and Kraan (2011) have not only presented an exhaustive description of bioactive compounds that can be found in seaweeds and their application in func- tional foods but have also discussed important and emerging legislation issues in their recent review. Microalgae are considered important producers of some highly bioactive compounds found in marine resources; they can be used to improve food nutritional profile due to their richness in PUFAs and pigments such as carotenoids and chlorophylls (Gamal, 2010). Fish and fish wastes are known sources of bioactive ingredients: i) calcium from fish bones; ii) fish oils rich in PUFAs from fish livers; iii) protein hydrolysates of high biological value, peptides with anti- hypertensive activity and amino acids, such as taurine, which have anti- oxidant activity and positive effects on cardiovascular system, from fish proteins; iv) vitamins, antioxidants and minerals (Dragnes et al., 2009; Guerard et al., 2010; Kadam and Prabhasankar, 2010). An overview of the bioactive peptides derived from marine organisms as well as their Functional ingredient FunctionalityMarine sources Potential food application Shrimp prawn crab - Reduction of lipid Chitin, chitosans chitooligosaccharide derivatives Fish and Salmon, sardines, tuna, Crustaceans Shrimp, prawn, crab, squid, lobster, cuttlefish absorption - Antitumor activity - Antibacterial and antifungal activities - Anti-Alzheimer’s activity - Gelling agents - Emulsifying agents - Food preservatives - Dietary fibre Proteins: collagen, gelatin, albumin, protamine; Bioactive peptides Amino-acids: Taurine Fish and fish wastes Salmon, sardines, tuna, herring, trout, lingcod, catfish, cod, mackerel D salina S maxima - Anticoagulant activity - Antioxidant activity - Antibacterial activity - Anti-hypertensive activity - Blood pressure reduction - Stabilizing and thickness agents - Protein replacements - Gelling agents Lipids: omega-3 fatty acids Microalgae D. salina, S. maxima, C. vulgaris, N. frustulum, B. sinensis - Cardiovascular diseases reduction - Arthritis and hypertension disease reduction - Visual and neurological improvement - Used in bread and confectionary products - Fish oils capsules with potential use in other foods Pigments: carotenoids (astaxanthin, lutein, fucoxanthin), phycobilins, chlorophylls Phenolic compounds: phlorotannins Fungi R d l b l Phycomycetes - Vitamin precursors - Anti-carcinogenic activity - Antioxidant, anti- inflammatory and anti-aging activities - Risk reduction diabetes (II) - Food colorants - Food antioxidants Polysaccharides: algins, carrageenans, agar, fucans, exopolysaccharides p Algae/ Seaweed Red algae, brown algae, U. pinnatifida, P. cruentum, L. japonica, B. gelatinum - Risk reduction diabetes (II) - Anticoagulant activity - Antibacterial, antiviral and antifungal activities Anti inflammatory activity - Gelling agents - Stabilizing , thickness and emulsifying agentsp y Cyanobacteria/ Extremophiles C. capsulata, Nostoc spp., Alteromonas spp., Vibrio spp. - Anti- inflammatory activity g - Food gums Fig. 1. Main marine functional ingredients with potential food applications, sources and inherent functionality (Hurst, 2006; Ngo et al., 2011; Rasmussen and Morrissey, 2007). 1508 A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • biological (antihypertensive, antioxidant, anticoagulant, antimicrobial) activities was recently published by Kim and Wijesekara (2010). Chitin, the second most abundant natural polymer, is extractable from crustaceans (Honorkar and Barikani, 2009). Chitosan, a chitin de- rivative resulting from processing shells and bones from crab, shrimp, cuttlefish, etc., is a biodegradable and biocompatible polymer with antibacterial activity which can be used as a food preservative. Its abil- ity to absorb fat emphasizes its potential use as an anti-cholesterol agent (Hayes et al., 2008b). Biological activities of chitosan and chitoo- ligosaccharides (hypocholesterolemic, antimicrobial, immunity- enhancing, antitumor, anticancer, antioxidant, etc.) and the potential application in food, among other applications are reviewed by Xia et al. (2011). Chlorophyll a, phycocyanins, and phycoerythrin are pigments of interest found in cyanobacteria which are also named as blue-green algae. These microorganisms present a secondary metabolism that produces several compounds: some are problematic for public health since they are strong hepatotoxins or neurotoxins whereas others re- veal potential biological activities such as anticancer, antibacterial, antifungal, and immunosuppressive properties; these have all been reviewed by Gamal (2010). Owing to their rich chemical composition, which depends on microalgae species, they can be used as a nutri- tional supplement or represent a source of food natural colorants as soon as their safety is assured (Mata et al., 2010; Spolaore et al., 2006). Phycocyanin is a blue, light-harvesting pigment in cyanobacte- rium with antioxidant properties (Eriksen, 2008). Marine bacteria and fungi are being considered as new sources for marine natural products (Duarte et al., 2012; Imhoff et al., 2011). Ma- rine fungi (yeasts) with high concentration of γ-amino-butyric acid (GABA), a promising functional and healthy food ingredient, has been reported by Masuda et al. (2008). Marine extremophylic bacteria are also of particular interest since they have metabolic pathways adapted to various extreme marine environments; the extremophiles are still a huge source of unknown and uncultivated bacteria; many microbial exopolysaccharides and enzymes from extremophiles have potential and unique properties (Laurienzo, 2010). Diatoms are also particular organisms that besides being photosynthetically robust or- ganisms with a high degree of flexibility to adapt to different environ- ments (from the Equator to the Ice sea), which is of interest for biotechnological applications, they are able to produce substances for functional foods (Bozarth et al., 2009); the most relevant function- al ingredients are PUFAs such as EPA, ARA, DHA and other omega-3 fatty acids. 3. Functional foods incorporating marine ingredients Besides the scientific interest for the use of marine functional in- gredients, there are various challenges ahead that have to be over- come to use them in new functional foods. Foods should have good sensorial characteristics in order to be accepted by the consumer since very few consumers are willing to compromise taste for health- iness in food (Honkanen, 2009). To our knowledge there is still a lack of research in the application of such functional/bioactive ingredients in foods as well as the scientific validation of their technological and biological feasibility. Fernandes et al. (2008) studied the incorporation of commercial chitosans and chitooligosaccharides, of crab shells ori- gin, in food such as milk and apple juice to ascertain the influence of food components on their antimicrobial activity, evaluating in parallel the acceptance by a sensory panel. The reported results are an exam- ple of the difficulties to overcome in the development of new func- tional foods. Although the antibacterial effect against pathogenic Staphylococcus aureus and Escherichia coli was dependent on bacteria Gram nature and chitosan's molecular weight, their addition to apple juice led to some unpleasant off-flavors which increased in magnitude with chitosan molecular weight. In fact, the incorporation of marine ingredients in foods of different nature may be a problem because it potentially leads the consumer to expect a negative influence on food because it is a result of mixing products of different nature (Honkanen, 2009). Several studies have shown the need for a high correlation between functional ingredient origin and food vector in order to promote functional food acceptance (Siegrist et al., 2008). The design of functional foods based on incorporation of marine ingredients has been more successful in bakery and pasta products and was recently reviewed by Kadam and Prabhasankar (2010). Incorporation of edible seaweed wakame (Undaria pinnatifida) up to 20% had sensorial acceptance resulting in improved amino acid and fatty acid profiles, increase of antioxidant activity, and higher content of fucoxanthin and fucosterol in seaweed pasta (Prabhasankar et al., 2009). Fucoxanthin, which was not affected by food processing, and its metabolites have been reported to have antioxidant, anti- cancer, anti-obesity and anti-inflammatory activities (Myashita and Hosokawa, 2008). Dietary ingestion of wakame has also been reported to reduce blood pressure (Chandini et al., 2008). Enrichment of foods such as bread and other bakery products with fish oils is widely and extensively used around the world; due to their increased content in eicosapentaenoic acid (EPA) and docosahexae- noic acid (DHA), these products are considered functional foods lead- ing to reduction of cardiovascular diseases (Kadam and Prabhasankar, 2010). Omega-3-enriched eggs are commercialized in several coun- tries around the world. Recently, design of functional spaghetti enriched with long chain omega-3 fatty acids was attempted by Verardo et al. (2009) and Iafelice et al. (2008). In Japan, several foods (soybean paste, potato chips, and noodles) with added chitosan are available as cholesterol-lowering functional foods (Borderías et al., 2005). According to Kadam and Prabhasankar (2010), more and constant efforts in research and design of novel functional foods based on marine functional ingredients are needed for reducing health problems through the diet. The introduction of marine compounds in human diet will always be a complex subject due to several types of constraints such as diet type and habits that are related to cultural and ethnic aspects of a population, consumer ideas and fears about sea pollution and also legislation itself. For example there are different views with respect to marine sources as functional foods. As a simple example, edible seaweeds are a product with a very long tradition in human diet in Japan, China and Korea, and in the USA as a consequence of the migra- tion phenomenon from east to west, whereas in Europe, although France has placed great effort in getting these approved for human consumption, some countries still present legal obstacles that may delay approval (Holdt and Kraan, 2011). Taking all this into account, consumer acceptance of new functional foods with marine bioactive compounds will certainly be dependent on the balance between habits and traditions, their perception about the real health benefits of functional food and as previously mentioned organoleptic issues. Legislation is another bottleneck for new functional foods with ma- rine bioactive compounds since it is continuously changing or being updated with the publication of new regulations and in some aspects these are very complex to be applied in any particular country. Addi- tionally, the commercialization of bioactive compounds or functional foods with health claims implies an extensive scientific dossier, to prove the scientific evidence, which is highly expensive, and burden- some (Holdt and Kraan, 2011) and the niche market targeted may not be sufficient to cover the economical investment. 4. Marine biotechnology Biotechnology covers a wide array of different techniques and ap- plications. As well established, the Convention on Biological Diversity (CBD), defines biotechnology as “any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use” ( biotech/fao-statement-on-biotechnology/en). Marine biotechnology 1509A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • is an emerging field focused on research and development of techno- logical applications of living marine organisms as well as their deriv- atives and bioprocesses. In a broader definition, marine biotechnology is based on the use of marine organisms or their components to pro- vide goods or services. The marine derived products have been uti- lized to several areas such as health, environment remediation, new industrial processes, new pharmaceuticals and also on food supply which is the area of particular interest of this review. Nowadays, more researchers are applying the tools of biotechnology to the dis- covery and production of marine natural products (Baerga-Ortiz, 2009). In this review, advances on biotechnological tools for production of functional ingredients including enzymes for food industry use, updating information published by Rasmussen and Morrissey (2007), will be divided in two subsections: I) Tools involving biotechnological processes (bioreactors, fermentations, and bioprocessing) and, II) Tools involving genetic/molecular approach and genetic manipulation which could be designated as molecular biotechnology. Metabolic en- gineering in marine microorganisms by genetic manipulation is be- coming an area of especial scientific interest due to the application of more known marine genomes. 4.1. Biotechnological processes 4.1.1. Cell factories The cultivation of marine organisms, often designated as cell fac- tories, has been practiced by Man for ages. Macroalgae or seaweeds, and microalgae are mainly cultivated by two approaches: i) Open systems, or ii) Closed and artificial systems; each one of them with benefits and limitations (Fig. 2). The main advantage of outdoor cultivation is related with use of solar energy to produce biomass. Production of added value compounds with microalgae in a profit- able way is possible because they are photosynthetic organisms and therefore the biomass production with functional ingredients directly from solar irradiation at high photosynthetic efficiencies is attractive and environmentally sound. Open culture systems in- volve lower costs since it is cheaper to build and operate, more du- rable, and generally offer a high production capacity (Mata et al., 2010). For example, for the production of β-carotene, one of the most interesting cell factories identified is the unicellular alga Dunaliella salina, because of its high capacity to accumulate high concentration of carotenoids (Lamers et al., 2008). Culturing macroalgae in the vicinity of fish farms or in coastal waters has been seen as an environmental friendly way to reduce water eutro- phication (Sanderson et al., 2008). On the other hand, the influence of less controlled external factors is one of the main drawbacks pointed to open systems; temperature and light fluctuations due to weather conditions (Masojidek and Torzillo, 2008) influence di- rectly cell growth rates and therefore the production of secondary metabolites. Possibility of pollution and of contamination as well as evaporation losses and CO2 diffusion, are other constraining fac- tors (Harun et al., 2010; Rasmussen and Morrissey, 2007). Bearing in mind that the production of bioactive compounds from algae normally demands the use of monocultures and controlled cul- tivation systems to achieve higher levels of production efficiency (Mata et al., 2010) led to the development of closed-culture systems (large scale bioreactors). Bioreactors provide better control of growth parameters (such as nutrients, temperature, pH, dissolved CO2 and O2), prevent contaminations, allow higher algae cell concentration, higher volumetric productivities, (Harun et al., 2010; Mata et al., Open ponds Carotenoids Biotechnological processes Systems Products Raceway ponds Sh ll d Cell factories Cultivation Open ponds or tanks Carotenoids Photo- bioreactors Biomass Flat Tubular Shallow ponds Circular tanks - Macro-algae - Micro-algae - Diatoms - Cyanobacteria Reactors Carotenoids Biomass Polysaccharides Omega-3 fatty acids bioreactors Horizontal Vertical Helical, etc. Fermenters Heterotrophic Mixotrophic M i Enzymatic hydrolysis Batch reactors Bio-processing Proteins Peptides Mixotrophic Clarification Filtration Purification Drying Concentration Marine sources - Fish and fish by- products - Shellfish wastes Membrane bioreactor with ultrafiltration Batch reactors Peptides Amino acids Chitin, Chitosans, COS Purification Marine sources - Fish and fish by- d t Subcritical Traditional Extraction processes Enzymes Chitosans, COS Carotenoids P l h id Solvents Pressing methods Pressurized hot water products -Shellfish wastes -Algae fluid extraction Supercritical fluid extraction Polysaccharides Omega-3 fatty acids Phenols Pressurized ethanol CO2 Fig. 2. Main biotechnological processes as well as extraction techniques applied to explore marine resources for added value products namely functional ingredients. 1510 A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • 2010) but with higher costs and energy requirement than the simpler open systems such as ponds which remain a competitive cultivation option (Harun et al., 2010). An interesting comparison between photobioreactors (PBRs) and ponds for several culture conditions and growth parameters is available in Mata et al. (2010), who also discuss the advantages and disadvantages of continuous and batch operation modes. Study cases of cultivation in open pond systems have been reported; algae biomass and lutein production by micro- or macro- algae are reported in Table 1. Outdoor cultivation of microalgae for carotenoid production is reviewed by Del Campo et al. (2007), who highlight that within open systems, one of the best choices is the use of open shallow ponds made of leveled raceways running as sim- ple loops or as meandering systems. More recently, Fernández-Sevilla et al. (2010) published a review on the potential of microalgae as lu- tein source and compared this to the production of lutein based in marigold oleoresins (current process to obtain lutein from marigold petals), indicating that microalgae (Murielopsis spp. and Scenedesmus almeriensis) could compete with marigold because of their lutein con- tent and biomass productivity. Microalgae cultivation has become even more attractive because of the possibility to easily control the growth conditions in closed sys- tems such as PBRs, together with the well-known biochemical diver- sity of these organisms. Photobioreactors are either flat or tubular (Fig. 2), can adopt a variety of designs and operation modes offering in general, higher productivity and better biomass quality (Del Campo et al., 2007). Tubular reactors consisting in translucent tubes, which yield a large illumination surface, are considered more suitable for microalgae outdoor cultivation (Harun et al., 2010). A description on PBRs based on both design and mode operation is available in Mata et al. (2010). The bioreactor engineering for the generation of marine bacterial products was reviewed by Lang et al. (2005) and updated by Zhang et al. (2011); they describe the innovative bioreac- tor technology in marine microbiology research which may in the near future, between other possibilities, lead to the search and pro- duction of new functional ingredients. According to Zhang et al. (2011) and depending on the research objective, different practices and reactors could be applied. If researchers want to mimic the natural environment conditions, the high-pressure reactors are suitable, since most of the marine environment is under pressure; these reactors could be associated with analytical instruments to monitor essential parameters of the mimicked environment (temper- ature, metals, CO2, and sulfide); To stimulate, enrich, cultivate or produce metabolites of interest where low substrate concentration and long incu- bation periods are of interest, rotating disk bioreactors are the more appropriate. In addition to production of high cell concentrations, it is also impor- tant to obtain metabolites at profitable concentrations. Techniques such as stress cultivation, designated as milking process, have been attempted to increase produced amounts or to induce the production of secondary metabolites. Milking microalgae for their secondary metabolites allows the reuse of the culture and their continuous removal increasing produc- tivity of algal cultures (Rasmussen and Morrissey, 2007). Stress condi- tions such as nitrogen limitation, high salt concentrations, high light intensities, and low temperatures could promote higher levels of carot- enoids produced by microalgae (Wijffels, 2008). However it is still un- known how and to what extent stress could be applied since small variations in growth conditions results in different metabolites concen- tration. Fernández-Sevilla et al. (2010) discuss the effect of stress factors on lutein production by microalgae referring that there is margin for im- provement in lutein content in Murielopsis spp. but more work is needed to know about the effect of stress factors and their interactions. As a result of metabolism flexibility of some strains of algae or cya- nobacteria that are able to grow heterotrophically in fermenters, het- erotrophic and mixotrophic cultivation are subjects of interest and research (Spolaore et al., 2006). Heterotrophic cultivation may be pre- ferred over photoautotrophic cultivation, since glucose can be used as a source of carbon and energy which is generally less costly and a more controlled process (Rasmussen and Morrissey, 2007). Eriksen (2008) reviews the photoautotrophic, mixotrophic and heterotrophic production of phycocyanin, a blue pigment with antioxidant properties, by two cyanobacteria (Arthrospira platensis and Galdieria sulphuraria). 4.1.2. Bio-processing technologies Bio-processing technologies involving enzyme-mediated hydroly- sis to produce marine ingredients are other approaches to explore Table 1 Examples of micro and macroalgae, diatoms and cyanobacteria cultivation in outdoor open and closed systems, published between 2007 and 2012. Cultivation method System and characteristics Species Products/functional ingredients References Open Open tank in semi continuous culture Muriellopsis spp. Lutein Blanco et al. (2007) Open raceway pond Spirulina platensis Algae biomass Grobbelaar (2007) Open tank Ulva lactuca Seaweed biomass Robertson-Andersson et al. (2008) Open tank Spirulina platensis Phycocyanin Silveira et al. (2007) Closed Bubble column photobioreactor Scenedesmus almeriensis Lutein Sánchez et al. (2008) Airlift flat plate photobioreactor Porphyridium cruentum Polysaccharide Liquin et al. (2008) Helix tube photobioreactor Dunaliella salina β-carotene Zhu and Jiang (2008) Horizontal tube photobioreactor Pavlova viridis Eicosapentanoic acid Hu et al. (2008) Heterotrophic Nitzschia laevis Eicosapentanoic acid Chen et al. (2008) Airlift tubular photobioreactor Spirulina platensis Biomass Ferreira et al. (2012) Blanco AM, Moreno J, Del Campo JA, Rivas J, Guerrero MG. Outdoor cultivation of lutein-rich cells of Muriellopsis sp. in open ponds. Appl Microbiol Biotechnol 2007;73:1259–1266. Ferreira LS, Rodrigues MS, Converti A, Sato S, Carvalho JCM. Kinetic and growth parameters of Arthrospira (Spirulina) platensis cultivated in tubular photobioreactor under different cell circulation systems. Biotechnol Bioengineer 2012;109:444–450. Grobbelaar JU. Photosynthetic characteristics of Spirulina platensis grown in commercial-scale open outdoor raceway ponds: what do the organisms tell us? J Appl Phycol 2007;19:591–8. Hu C, Li M, Li J, Zhu Q, Liu Z. Variation of lipid and fatty acid composition of the marine microalga Pavlova viridis (Prymnesiophyceae) under laboratory and outdoor culture con- ditions. World J Microbiol Biotechnol 2008;24:1209–1214. Liquin S, Changhai W, Lei S. Effects of light regime on extracellular polysaccharide production by Porphyridium cruentum cultured in flat plate photobioreactors. Bioinformatics and Biomedical Engineering. ICBBE 2008:1488–1491. Robertson-Andersson DV, Potgieter M, Hansen J, Bolton JJ, Troell M, Anderson RJ et al. Integrated seaweed cultivation on an abalone farm in South Africa. J Appl Phycol 2008;20:579–595. Sánchez JF, Fernández JM, Acién FG, Rueda A, Pérez-Parra J, Molina E. Influence of culture conditions on the productivity and lutein content of the new strain Scenedesmus almer- iensis. Process Biochem 2008;43:398–405. Silveira ST, Burkert JFM, Costa JAV, Burkert CAV, Kalil SJ. Optimization of phycocyanin extraction from Spirulina platensis using factorial design. Bioresource Technol 2007;98:1629–1634. Zhu Y, Jiang J. Continuous cultivation of Dunaliella salina in photobioreactor for the production of β-carotene. Eur Food Res Technol 2008;227:953–9. Chen G, Jiang Y, Chen F. Variation of lipid class composition in Nitzschia laevis as a response to growth temperature change. Food Chem 2008;109:88–94. 1511A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • marine biological resources permitting an added value use of 30 to 50% by-products resulting every year from 140 million tons of fish and shellfish (Guerard et al., 2010). There is a great interest to convert marine food by-products into valuable functional ingredients such as bioactive peptides, chitosan and chitooligosaccharide (COS) (Hayes et al., 2008a,b; Kim and Wijesekara, 2010; Manni et al., 2010; Ngo et al., 2009). The enzyme-mediated hydrolysis is a relatively in vitro simple process based on contact between enzymes and marine substrate solution at op- timum values of pH and temperature in a batch reactor. Once this first stage is finished, enzymes are inactivated and several other steps, such as clarification and filtration, are necessary in order to obtain the functional ingredients (Fig. 2). Membrane bioreactors equipped with ultrafiltration membranes or multi-step recycling membrane re- actors combined with ultrafiltration membranes system, are technolo- gies developed for bio-processing and efficiently recovering bioactive functional ingredients with desired molecular size and functional prop- erties (Ngo et al., 2011). The development of membrane biotechnology has been applied for separation, fractionation and recovery of marine ingredients in order to obtain more pure compounds with efficient yield ratios. Different approaches have been applied depending on the chemical structure of the target compounds: the potential of mem- brane processes using microfiltration and ultrafiltration to obtain food grade phycocyanin (82% recovery with 1.0 purity) from Spirulina sp. to be applied in the food industry was reported by Chaiklahan et al. (2011); marine peptides with antiproliferative activity have been frac- tionated and concentrated using ultrafiltration and nanofiltration membrane systems (Vandanjon et al., 2007). A review on membrane applications in functional foods and nutraceuticals has recently been published by Akin et al. (2012). Extraction technologies, especially those considered clean process- es such as methods involving water extraction, will also be considered in this revision since it completes the technological approach to ex- plore marine resources for functional ingredients. As previously men- tioned marine organisms have all developed diverse and unique characteristics that allow them to survive under conditions with vary- ing degrees of salinity, pressure, temperature and illumination. Marine extracted enzymes, other valuable products from marine organisms, can provide numerous advantages over traditional enzymes used in food processing due to their activity and stability under unusual and ex- treme reaction conditions (Rasmussen and Morrissey, 2007). Proteases, amidases, lipases, and polysaccharide degrading enzymes such as chit- inases, alginate lyases, agarases, and carrageenases, are some of the ma- rine enzymes of potential interest (Zhang and Kim, 2010), some of them target of isolation, characterization and optimization by protein engi- neering (Sarkar et al., 2010). A review on research status on marine mi- crobial enzymes is available in Zhang and Kim (2010) whereas a synopsis of different bioprocess engineering systems for production of marine enzymes is available in Sarkar et al. (2010). Due to marine biotechnology tools, scientists have been extracting and identifying molecules that promote survival in such environ- ments; new and improved extraction methods are still being devel- oped. Hayes et al. (2008a) reviewed methods of chitin extraction from shell wastes coupled with the novel enzymatic and microbial hydrolysis methods; alternatives to chemical chitin extraction pro- cess have been researched such as fermentations with proteolytic and chitinolytic bacteria. Traditional methods using organic solvents with restrictions for application in food industry or pressing methods have been used in fish oil extraction with good results when applied to fish by-products with high-oil content (Rubio-Rodríguez et al., 2010). New environmentally clean technologies to produce food in- gredients have been researched in the last years such as supercritical fluid extraction (SFE) and subcritical water extraction (SWE); these ex- traction methods are based on the use of non-toxic solvents, and also ca- pable of high levels of selectivity with lower periods of extraction; the possibility of direct coupling with analytical chromatographic techniques such as gas chromatography or supercritical fluid chromatography is another pointed advantage (Herrero et al., 2006). The mostly used su- percritical fluid is carbon dioxide that is considered a green non-toxic, cheap and non flammable solvent which allows to process omega-3 fatty acids at lower operation costs, and is easy to separate from the processed products at room temperature (Rubio-Rodríguez et al., 2010). Supercritical fluids have better transport properties than liquids and are good solvents which depending on pressure and temperature can solve different types of solutes (Herrero et al., 2006). In fish oil, these properties can be used to separate free fatty acids or to obtain concentrates according to molecular weight or degree of saturation. According to Akin et al. (2012), membrane separation coupled to su- percritical fluid technologies is a novel separation technology with great potential to be applied to functional food industry. SWE is based on extraction with hot water under high pressure and has been applied to extract antioxidants from microalgae (Herrero et al., 2006). Lipids from microalgae can be extracted by pressing, sol- vent extraction, supercritical fluid extraction and ultrasounds (Harun et al., 2010). A new extraction method based on the off-line combina- tion of pressurized-liquid with solid phase extraction was described to extract bioactive phenolic acids from algae (Onofrejová et al., 2010). 4.2. Molecular biotechnology 4.2.1. Marine metagenomic approach Although standard culture techniques account for a small fraction of the marine microbiota diversity, culture-dependent approaches contin- ue to provide new chemical structures with biological activities and provide a promising approach for the search of new compounds (Imhoff et al., 2011). Furthermore, the knowledge of the marine ge- nome especially of uncultured organisms, will allow to screen for new genes and obtain new compounds from marine microbial resources and therefore it is important that more marine microorganisms are studied in genome programs. From the genome information it is possi- ble to know whether an organism has the potential to produce new compounds by secondary metabolic pathways. This is a potential tool to explore since the number of genes encoding biosynthetic enzymes in bacteria and fungi is more than the known secondary metabolites (Scherlach and Hertweck, 2009). However, it seems that most biosyn- thetic gene clusters are either silent or cryptic pathways, especially under standard laboratory culture conditions (Imhoff et al., 2011; Scherlach and Hertweck, 2009) Metagenomics—the study of genetic information of an environ- mental sample containing uncultured and diverse microbial popula- tions—is a genetic approach (Fig. 3) in which genome fragments from a sample containing microbiota community are extracted, ana- lyzed, transferred/cloned and expressed in suitable hosts, normally in E. coli, as well as the screening of clones for production of new en- zymes or bioactive compounds (Kennedy et al., 2010; Wijffels, 2008). Once the genes involved in the biosynthesis of bioactive com- pounds are known, it becomes possible to enable their expression and produce them in a more efficient way (Wijffels, 2008). The appli- cation of metagenomic approach provides the opportunity to study and exploit the enormous microbial diversity present in the marine environment, enabling to access the protein-coding genes with bio- technological interest (Kennedy et al., 2008). According to Lorenz and Jürgen (2005), it is possible to express large fragments of DNA as well as screen large clone libraries for functional activities. Large scale DNA-based approaches confirm the huge microbial diversity in the oceans and according to Kennedy et al. (2010), sequence-based metagenome databases in combination with bioinformatic as well as functional-based methods to mining novel genes encoding enzymes from microbial marine organisms will be powerful tools to open new frontiers. Functional metagenomics focuses on genes recognizable for their function and examples of enzymes isolated from marine sources based on this approach include esterases, lipases, chitinases, etc. (Kennedy et al., 2010). 1512 A.C. Freitas et al. / Biotechnology Advances 30 (2012) 1506–1515
    • 4.2.2. Transgenic approach Optimization of metabolites production with functional properties has been achieved by genetic modification which enhances physio- logical properties of algae or other cell factories. Efforts involving the expression and activity of biosynthetic enzymes in other host are the bases of recombinant technology that involves recombination of DNAs of different origins in one host. Recombinant production of omega-3 fatty acids such as EPA and DHA by E. coli has demonstrated great potential and an alternative way to produce PUFAs by fermenta- tion (Amiri-Jami and Griffiths, 2010). A powerful driving force with algae is the possibility of using genetically modified algae (transgenic algae) as bioreactors or cell factories for expression products (such as carotenoids, PUFAs, and enzymes) or to transform photoautotrophic to heterotrophic algae by introducing a gene for sugar transport (Hallman, 2007). The conversion of photoautotrophs to heterotrophs is of biotechnologi- cal interest since algae cultivation dependent of light is expensive and less efficient (Rasmussen and Morrissey, 2007). According to Hallman (2007), green, red, and brown algae as well as diatoms are some examples of successfully transformed algae, the majority by nuclear transformation. The potential interest of using transgen- ic algae is related to the control of algae as a bioreactor orientated to overproduction or to produce new metabolites by forcing them to express specific genes (Spolaore et al., 2006). However the bio- technological use of transgenic algae faces some problems and challenges, namely competitiveness, public acceptance, regulatory issues and biosafety. Metabolic engineering controlling specificity of biosynthetic en- zymes to accept new substrates or improve pathway efficiency is an- other approach to create new metabolites or increase productivity. Research in the metabolic pathways and regulatory mechanisms is of interest to develop commercial strains (ex. mutated Dunaliella bar- dawil) with higher capacity to produce antioxidant carotenoids (Rasmussen and Morrissey, 2007). 5. Final considerations Advances in biotechnological tools used for identification, pro- cessing and extraction have been responsible for a positive trend in the number of available food ingredients with potential functional properties, derived from marine sources. Biotechnology enables a tai- lored production of value-added biological compounds by different organisms under specific controlled growth conditions using well designed bioreactors. Despite the advances, challenges remain related to improved cultivation techniques for certain marine organisms which must be more efficient and profitable. More genetic research is still necessary, although many huge steps have been taken in the last years. The real challenge is to overcome the bridge between the findings of new strains and transgenic strains and their commercial application to produce food functional ingredients. Acknowledgments This work was supported by the Portuguese Science Foundation (Fundação para a Ciência e Tecnologia), through individual research grants attributed to Ana C. Freitas (SFRH/BPD/73781/2010), Dina Rodrigues (SFRH/BD/77647/2011), and Teresa A. P. Rocha-Santos (SFRH/BPD/65410/2009). References Akin O, Temelli F, Köseoğlu S. Membrane applications in functional foods and nutra- ceuticals. Crit Rev Food Sci Nutr 2012;52:347–71. Ameye LG, Chee WSS. Osteoarthritis and nutrition. From nutraceuticals to functional foods: a systematic review of the scientific evidence. Arthritis Res Ther 2006;8: R127. Amiri-Jami M, Griffiths MW. Recombinant production of omega-3 fatty acids in Escher- ichia coli using a gene cluster isolated from Shewanella baltica MAC1. J Appl Micro- biol 2010;109:1897–905. 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