The Innovation of Technology for Microalgae Cultivation

Innovation of Technology 18 The for Microalgae Cultivation and Its Application in Functional Foods and the Nutraceutical Industry Akira Satoh, Masaharu Ishikura, Nagisa Murakami, Kai Zhang, and Daisuke Sasaki CONTENTS 18.1 Introduction 313 18.1.1 About Yamaha Motor Co Ltd 313 18.1.2 Some Background on the Research and Development into Microalgal Biotechnology by Yamaha Motor 314 18.2 An Overview of Microalgae Mass Culture in Functional Foods and the Nutraceutical Industry 314 18.3 Research and Development into the Commercial Production of Microalgae at Yamaha Motor 316 18.4 Astaxanthin Raw Material Manufacturing Process 318 18.4.1 Outline of the Factory and Manufacturing Process 318 18.4.2 Quality Assurance System Under Good Manufacturing Practice Conditions 320 18.5 Astaxanthin and its Source, Hematococcus Alga 321 18.5.1 Astaxanthin 321 18.5.2 Structure and Source of Astaxanthin 322 18.5.3 Antioxidative Activity of Astaxanthin 322 18.5.4 Haematococcus Algae 322 18.6 Benefits of Astaxanthin for Human Health Management 323 18.6.1 Astaxanthin and Atopic Dermatitis 323 18.6.2 Astaxanthin and Brain Health 323 18.6.3 Astaxanthin and Metabolic Syndrome 324 18.7 Concluding Remarks 326 Acknowledgment 326 References 326 18.1 18.1.1 INTRODUCTION ABOUT YAMAHA MOTOR CO LTD Yamaha Motor Co Ltd was a spin-off from its parent musical instrument company, Nippon Gakki Co Ltd (now Yamaha Co Ltd.) and became an independent company in 1955, supplying the latest 313 © 2010 Taylor and Francis Group, LLC 314 Biotechnology in Functional Foods and Nutraceuticals motorcycles in Shizuoka, Japan At the time more than 100 motorcycle companies existed in Japan; now only four companies exist, including Yamaha Motor Yamaha Motor is mainly concerned with manufacturing and supplying motorcycles, marine products, and many other types of power products worldwide Approximately 90% of its sales come from motorcycles, marine products, and power products: only 10% of all their sales are generated in Japan, the remaining 90% being overseas orders In 2006, Yamaha Motor Co Ltd pioneered a new business concerned with biotechnology, which Yamaha Motor originally developed to provide bulk astaxanthin as a functional food ingredient 18.1.2 SOME BACKGROUND ON THE RESEARCH AND DEVELOPMENT INTO MICROALGAL BIOTECHNOLOGY BY YAMAHA MOTOR In 1997, a meeting was held in Kyoto, Japan, to discuss the international convention on the stabilization of greenhouse gas concentration in the atmosphere At the meeting, many advanced countries agreed to reduce the emissions of six greenhouse gases, including CO2 In Japan, the Kyoto meeting was considered very important for many people and companies, alerting them to the effects of global warming Environmental issues concerning global warming set Yamaha Motor on the road to researching and developing carbon dioxide reduction and fixation technology In addition, they began to develop environmentally friendly power products, such as lower CO2 emission engines and fuel cells Yamaha Motor became interested in the biofixation of CO2 using algal photosynthesis, which has a CO2-fixing capability far superior to that of higher plants In the creation of an efficient biofixation system based on microalgae mass culture, Yamaha Motor began developing new conceptual photobioreactors using fluid dynamics technology that was previously utilized in the field of marine products (see Section 18.3 for details) Throughout the research, they accumulated specific technology for microalgae mass cultivation, including several photobioreactors in which each microalgal cell could be supplied with adequate light photons, dissolved CO2, and the nutrients required for maximum photosynthesis The successful development of high-density mass cultivation technology led them to a further challenge in microalgal biomass production, producing astaxanthin on a commercial scale as a functional foods for the nutraceutical industry 18.2 AN OVERVIEW OF MICROALGAE MASS CULTURE IN FUNCTIONAL FOODS AND THE NUTRACEUTICAL INDUSTRY Microalgae, including cyanobacteria, are microscopic photoautotrophs in which inorganic compounds and sunlight energy are converted into biomass Applied research on algal culture and biomass began in the late 1940s (Burlew, 1953) This research included alternative and unconventional protein sources, photosynthetic gas exchange for space travel, aquaculture feed, wastewater treatment, renewable energy sources, biological fixation of greenhouse CO2, and production of recombinant biopharmaceuticals (Borowitzka, 1995; Becker, 2004; Spolare et al., 2006; Eriksen, 2008) It was discovered that microalgae synthesize a variety of valuable substances, including carbohydrates, lipids, vitamins, pigments, and other biological active compounds (Borowitzka, 1995; Becker, 2004; Pulz and Gross, 2004; Spolare et al., 2006; Cardozo et al., 2007) Microalgal cultivation technology and its subsequent biomass and products have received much research attention in the last 5–6 decades due to their potential as possible commodities and other industrial applications However, only a limited number of microalgae have been found suitable to produce competitively priced products for use in functional foods and the nutraceutical industry (Vonshak, 1990; Borowitzka, 1999; Ben-Amotz, 2004; Cysewski and Lorenz, 2004; Hu, 2004; Iwamoto, 2004; Pulz and Gross, 2004; Spolaore et al., 2006) These include: Chlorella, mainly cultured in Japan, Taiwan, and Germany, with an annual production of 2000 tons dry weight; Arthrospira (Spirulina) and Aphanizomenon flos-aquae, mainly cultured in China, India, USA, and Japan, with an annual production of 3000 © 2010 Taylor and Francis Group, LLC Innovation of Technology for Microalgae Cultivation and Its Application 315 and 500 tons dry weight, respectively; Dunaliella, mainly cultured in Australia, Israel, and the United States, with an annual production of 1200 tons dry weight and which is used as a source of β-carotene; Haematococcus, mainly cultured in the United States, Israel, Sweden, and Japan, with an annual production of 300 tons dry weight, used as a source of astaxanthin; Crypthecodinium and Schizochytrium, mainly cultured heterotrophically in the United States, with an annual production of 240 and 10 tons, respectively, used as a source of docosahexaenoic acid (DHA) The bottleneck of the microalgal business is price competitiveness, and this depends on difficulties with microalgal cultivation These problems are derived from the growth and metabolic characteristics of a selected strain (e.g., growth rate, cellular content of a target product, optimum conditions, and stress tolerance), the culture system used (ponds and photobioreactors), and the sustainability of the culture and hence target product (e.g., contamination risks, productivity, quality, and seasonal effects) Before discussing the commercial production of microalgae at Yamaha Motor, details of the technology and problems are briefly mentioned For further details on microalgae, culture systems, and their advantages and problems, readers are referred to the book edited by Richmond (2004) and reviews by Borowitzka (1999), Lee (2001), Pulz (2001), and Tredici (2004) Commercial cultivation of microalgae is mainly performed under outdoor conditions using open-air systems (circular and raceway ponds) and natural sunlight, due simply to the economics of production One of the major problems with open-air systems of outdoor cultures is contamination risk, that is, difficulties in maintaining a monoalgal culture (species control) and prevention of bacterial and/or protozoal overgrowth (sterility) An extreme culture environment is therefore necessary to reduce the contamination risk, and a major reason why only a limited number of microalgae have been successfully mass cultured under outdoor conditions using open-air systems and marketed commercially Examples include, Dunaliella, which requires high saline culture conditions, Spirulina, which requires high alkaline conditions, and Chlorella, which grows well in nutrient-rich media Another major technical problem affecting sustainable productivity in microalgal cultures is the difficulty of supplying an adequate amount of light to each algal cell Light irradiation reaching the surface of the culture is decreased by increasing either the distance from the surface or the cell density, due to mutual shading, leading to limited light energy for algal growth In open-air systems, therefore, pond depth must be less than 50 cm and cell density at harvest is low (less than 0.6 g dry weight/L) and therefore a very large culture area is required for commercial-scale production of algal biomass—hundreds of hectares, provided by a number of ponds, each stretching to 1000 m2 Mixing, temperature and gas transfer are also important factors for algal growth, but these factors are difficult to control in open-air systems Microalgal culture technology has developed a closed system using a photobioreactor to overcome the problems discussed above, especially contamination risk and light limitation, and consequently to achieve higher cell density In typical photobioreactors, a series of transparent tubes, flat plate chambers, cylinders, or sleeves are positioned vertically, horizontally, coiled, or at a desired angle Mixing and temperature control are also improved in comparison with open-air systems An optical path in the photobioreactor is a precise parameter which can be altered through the diameter of tube or thickness of the chamber, to control algal growth and productivity in photoautotropic cultures In general, a shorter optical path produces a higher cell density, while culture volume per reactor decreases, resulting in a larger surface area-to-volume ratio The high cost of bioreactors and difficulties in scale-up are the two bottlenecks of photobioreactors, which limits successful use of commercial scale photobioreactors in the microalgal business (Tredici, 2004) Both ponds and outdoor photobioreactors have been used for autotrophic cultivation of Haematococcus pluvialis, used in astaxanthin production (Olaizola and Huntley, 2003; Cysewski and Lorenz, 2004; Del Campo et al., 2007) Constant irradiation of sufficiently high light and temperature control are required, since light intensity and temperature are the most critical factors affecting the growth of H pluvialis and astaxanthin accumulation (Margalith, 1999) In addition, © 2010 Taylor and Francis Group, LLC 316 Biotechnology in Functional Foods and Nutraceuticals sterility is a severe problem in the cultivation of Haematococcus, compared with other successfully mass-cultured algae (Dunaliella, Spirulina, and Chlorella), since no selective culture environment to prevent bacterial and/or protozoal overgrowth is currently available for this alga (Margalith, 1999) A culturing technology which enables both increased astaxanthin production and sterile culture conditions is required An indoor cultivation system has recently been developed to manufacture an astaxanthincontaining H pluvialis algal biomass, namely the “YAMAHA High-efficiency Photobioreactor.” The development of this high-efficiency photobioreactor is described in the next section 18.3 RESEARCH AND DEVELOPMENT INTO THE COMMERCIAL PRODUCTION OF MICROALGAE AT YAMAHA MOTOR As described in Section 18.1.2, Yamaha Motor became engaged in the development of outdoor photobioreactors for the biofixation of CO2 using algal photosynthesis In order to achieve high-density algal cultures with high photosynthetic activity, we at Yamaha Motor focused on the “flashing light effect,” also called “intermittent illumination,” which had been reported to be a means of utilizing a larger fraction of the sunlight shining on a given area (Emerson and Arnold, 1932; Weller and Franck, 1941; Rieke and Gaffron, 1942; Tamiya and Chiba, 1949; Kok, 1953) The “flashing light effect” is a situation in which cells in a high-density culture are exposed to light and dark periods in turn at high frequencies by mixing Cell mixing in a reactor was found to be very important but it was suggested that mixing performance would depend on the shape of the reactor As a novel approach to the design and screening of photobioreactors, we applied computational fluid dynamics to analyze the mixing performance of various shapes of photobioreactor (Sato et al., 2002, 2005; Tomita et al., 2005a, 2005b) In these studies, we made several types of photobioreactor including dome-, parabola-, pipe-, and diamond-type Side views of the dome- and pipe-type photobioreactors are shown in Figure 18.1 These photobioreactors were then evaluated in terms of light reception, global mixing, and local mixing, followed by an evaluation of algal biomass productivity using Chlorococcum littorale, which is regarded as an extremely high-CO2 tolerant algal strain, suitable for high-density culture (Kodama et al., 1993) A computational fluid dynamics analysis of the pipe-type photobioreactor is shown in Figure 18.2 This shape of bioreactor was found to be the best at light reception and biomass productivity: 20.5 g/m2/day in dry weight was observed during winter days in Japan This pipe-type photobioreactor was then applied to the outdoor cultivation of Chaetoceros calcitrans, FIGURE 18.1 The side view of a dome-type (a) and pipe-type (b) photobioreactor at Yamaha Motor © 2010 Taylor and Francis Group, LLC Innovation of Technology for Microalgae Cultivation and Its Application FIGURE 18.2 317 Computational fluid dynamics analysis of the pipe-type photobioreactor a valuable algal strain widely used as a feed in marine hatcheries A biomass productivity of 37.3 g/m2/day was observed for Chaetoceros calcitrans The culture volume of the pipe-type photobioreactor could be increased up to approximately 200 L In addition, the simultaneous use of artificial light and sunlight to increase algal productivity was made possible by inserting fluorescent lamps into the inner cavity, since the space could be sealed completely after the insertion of lamps to prevent rainwater invasion However, further enlargement of the reactors was not possible due to the strength of the structural material and difficulties in cleanup and sterilization The pipetype photobioreactor is therefore applicable for small-scale on-site production of live microalgae for aquaculture feeds and we have indeed marketed live Chaetoceros calcitrans cells since 2002 (Yamauchi, 2003) Our next R&D project was mass cultivation of H pluvialis for production of a more valuable compound, astaxanthin Due to the scale-up problems of the pipe-type photobioreactor, we decided to use a vertical flat-plate-type reactor for this project, since previous research had shown this type of reactor to have excellent light illumination efficiency, leading to superior algal growth (Zhang et al., 2001) Our first efforts involved outdoor cultivation of H pluvialis (Figure 18.3), however, as described in the previous section, bacterial and/or protozoal overgrowth was a severe problem due to the lack of a selective culture environment In addition, biomass productivity and astaxanthin yield were very unstable and depended on weather conditions and seasonal differences, since algal growth and astaxanthin accumulation are greatly influenced by light availability and temperature (see Section 18.2) In order to establish a stable industrial manufacturing process, we moved to indoor cultivation using artificial lights, and this resulted in the development of the “YAMAHA High-efficiency Photobioreactor.” High-quality and high-quantity astaxanthin-containing algal oil have thus been produced since October 2006, and the manufacturing process was given a health food raw material Good Manufacturing Practice (GMP) certification Details of our manufacturing process (GMP approved) are described in the next section © 2010 Taylor and Francis Group, LLC 318 Biotechnology in Functional Foods and Nutraceuticals FIGURE 18.3 An outdoor vertical flat-plate-type photobioreactor at Yamaha Motor, used in the cultivation of H pluvialis 18.4 18.4.1 ASTAXANTHIN RAW MATERIAL MANUFACTURING PROCESS OUTLINE OF THE FACTORY AND MANUFACTURING PROCESS A factory dedicated to the manufacture of astaxanthin raw material (astaxanthin-containing dried algal powder of H pluvialis), namely the “Fukuroi Factory II,” was completed in October 2006 as the first and only factory capable of producing the raw material in Japan The proprietary indoor cultivation system, demonstrating practical use of a number of the “YAMAHA High-efficiency Photobioreactors” is the main production facility (Figure 18.4) This facility is operated in an FIGURE 18.4 The indoor cultivation system known as the “YAMAHA High-efficiency Photobioreactor” in the Fukuroi Factory II © 2010 Taylor and Francis Group, LLC Innovation of Technology for Microalgae Cultivation and Its Application 319 unattended manner at night, enabling highly controlled batch cultures under continuous illumination, with lower labor costs The factory is located on a site of approximately 37,000 m2 in the Kuno Industrial Park of Fukuroi city, Shizuoka prefecture The building area is approximately 1800 m 2, with 3300 m2 total floor space At present, the production capability is approximately 20 tons of dried algal powder per year, however, the factory is designed as a unit of standardized equipment, making it possible to increase the production capability easily and quickly in response to market expansion by increasing the number of units required The manufacturing process consists of several steps before shipment: cultivation, separation, drying, packaging, and quality inspection (Figure 18.5a) The process is entirely optimized under the concept of “manufacturing high-quality and safe products.” Briefly, at the cultivation step, the bioreactors are continuously illuminated with optimized synthetic light during a culture period and maintained at the desired temperature to maximize astaxanthin accumulation in algal cells The quality of water is also carefully considered for optimum astaxanthin production In order to minimize the risk of contamination, the reactors are isolated in a class 100,000 clean room (Figure 18.4); the cultivation and all operations are performed in the same or a more strictly controlled environment under sanitary quality control Great care is also taken in the system design and operations at Astaxanthin oil Shipping inspection Packaging (b) Concentration Extraction Shipping inspection Packaging (a) Drying Separation Cultivation FIGURE 18.5 A production flow scheme for astaxanthin raw material production (a) and subsequent astaxanthin-containing H pluvialis oil (b) © 2010 Taylor and Francis Group, LLC 320 Biotechnology in Functional Foods and Nutraceuticals subsequent steps of harvesting the algal cell culture up to packaging the dried algal powder, both to protect the astaxanthin from degradation or deterioration by bacteria, excess heat, oxygen, and light, and to prevent contamination from foreign bodies In our manufacturing process, raw material with an astaxanthin content of over 5% is consistently produced (Zhang, 2005, 2007), which is much higher than that obtained from outdoor cultivation, where it reduces to less than 2% in the winter season (personal communication) This stable supply of highly sterilized biomass with a high astaxanthin content is advantageous, not only to maintain low extraction costs during downstream processing, but also as a quality and safety guarantee for further downstream business partners and end-users 18.4.2 QUALITY ASSURANCE SYSTEM UNDER GOOD MANUFACTURING PRACTICE CONDITIONS GMP is the rule employed to produce high-quality and safe products, by which a manufacturing system is guaranteed constant quality throughout the whole process, from the raw material stock to product shipment Although the GMP was originally imposed as a legal duty for medicine manufacturers, similar GMP conditions have been applied to manufacturers of cosmetics, food additives, and health foods in recent years In February 2005, the Japanese Ministry of Health, Labor and Welfare published a voluntary inspection guideline on GMP in the manufacturing of health foods and the safety of raw materials (No 0201003) We designed and improved our factory to meet the GMP requirements and in July 2007 the Fukuroi factory II was given a health food raw material GMP certification by the Japanese Health Food Standards Association (JIHFS) Our quality assurance system had thus established a reliable GMP system, certified by an outside organization Our GMP is composed of three basic principles: keeping mistakes to a minimum level; preventing pollution and product loss; and designing a management system for guaranteed high-quality products The indispensable requirements to achieve this GMP are listed below, where (1) through (4) are germane to management side while (5) through (8) refer to equipment The complete preparation of various standard operation procedures (SOPs), operation manuals and operational records; correct operation of equipment strictly observing these SOPs and manuals In addition, correct storage of the operation records, for easy access A traceability system using serial numbers for each product lot An education and training system, and skills improvement for all staff and workers at the plant Execution of a rigorous plant self-check system, based on our GMP, at the end of every run Clean-room installation, complete control of air-conditioning facilities and purification control in all production processes Manufacturing environment maintenance to prevent cross-contamination and foreign body mixing Installation of appropriate inspection equipment for both the process control and product standard test Running the automatic production monitoring system over 24 h Astaxanthin raw material manufactured under GMP conditions in the Fukuroi factory II is shipped to a medicine manufacturer for further extraction to prepare astaxanthin-containing H pluvialis oil (Figure 18.5b) The extraction process to produce astaxanthin oil also fulfills both the production system and quality assurance system in accordance with the principles of GMP Our GMP-grade astaxanthin oil product (Figure 18.6) is prepared using an advanced quality assurance system equal to that used in orally administered medicine © 2010 Taylor and Francis Group, LLC Innovation of Technology for Microalgae Cultivation and Its Application FIGURE 18.6 321 Yamaha Motor’s GMP-grade astaxanthin oil product 18.5 ASTAXANTHIN AND ITS SOURCE, HEMATOCOCCUS ALGA 18.5.1 ASTAXANTHIN Astaxanthin, a natural lipophilic tetra terpenoid with a deep red color, is a carotenoid like β-carotene and lycopene and is widely distributed in nature, especially in marine organisms including salmon, salmon roe, shrimp, crab, and microalgae (Hussein et al., 2006) It is a xanthophyll from the carotenoid group, with oxygen-containing functional groups, and also possesses hydroxyl and oxo functional groups In nature, astaxanthin is found in its esterified form or binding form, bound to proteins, because these forms are more stable than the dialcohol form Plants, algae, and microorganisms can synthesize de novo carotenoids; however, animals lack the ability to synthesize these compounds and so must acquire them from their diet Many carotenoids are known as provitamin A as they can be cleaved at the central C15=C15′ double bond and converted into vitamin A in vivo (Goodman and Huang, 1965; Olson and Hayashi, 1965) In fish, many xanthophylls, including astaxanthin, are reported to be converted reductively to retinol in vivo (Katsuyama and Matsuno, 1988) In contrast, human astaxanthin is reported to be cleaved asymmetrically at the C9 position and is therefore regarded as a non-provitamin A carotenoid (Kistler et al., 2002) Xanthophyll esters are thought to be hydrolyzed prior to absorption (Zaripheh, 2002) Only nonesterified astaxanthin is detected in serum after the ingestion of esterified astaxanthins (CoralHinostroza et al., 2004; Odeberg et al., 2003) Xanthophylls are mixed with bile acid to make a micelle, and are absorbed as a micellar solution by the intestinum tenue after intake The absorbed xanthophylls are then incorporated by intestinal mucosal cells into chylomicra and released into the lymph In the lymph, chylomicra-containing xanthophylls are digested by lipoprotein lipase, reducing their size, and xanthophylls reach the liver as chylomicra remnants In the liver, they are incorporated into lipoprotein, which is synthesized in the liver, and translocated to each tissue It was recently reported in humans that blood xanthophylls translocate more easily to red blood cells than to plasma when compared with hydrocarbon carotenoids (Nakagawa et al., 2008) It is suggested that the difference in the chemical nature of xanthophylls and hydrocarbon carotenoids causes the difference in their in vivo behavior © 2010 Taylor and Francis Group, LLC 322 Biotechnology in Functional Foods and Nutraceuticals Due to their high antioxidant properties and other functions (see Section 18.6), many astaxanthincontaining nutraceuticals with potent effects on human health are coming onto the market 18.5.2 STRUCTURE AND SOURCE OF ASTAXANTHIN The astaxanthin molecule has a symmetric configuration and two chiral centers; two carbon atoms adjacent to hydroxyl functional groups are chiral There are three enantiomeric isomers of astaxanthin, (3S, 3′S), (3R, 3′R), and (3R, 3′S) Chemically synthesized astaxanthin is a mixture of 1:2:1 of (3S, 3′S), (3R, 3′S), and (3R, 3′R) enantiomer, respectively This is mainly used in the field of aquaculture as a reviver and is not used as an ingredient in human neutraceuticals A green microalga (H pluvialis), a red yeast (Phaffia rhodozyma), and crustacean by-products are commercially available as natural sources of the astaxanthin pigment These sources are often used in the nutraceutical industry because of recent natural food trends and safety concerns The forms of astaxanthin in these natural sources are slightly different from each other Astaxanthin from Haematococcus is the (3S, 3′S) isomer (Renstrom et al., 1981) and is almost esterified with fatty acid to form mono- or diesters (Johnson and An, 1991) In contrast, Phaffia rhodozyma is reported to synthesize the (3R, 3′R) isomer (Torissen et al., 1989), which is mainly unesterified (Andrewes and Starr, 1976) Haematococcus alga is considered to be the most efficient natural source of astaxanthin (Hussein et al., 2006) and is presently used as the main source of natural astaxanthin (see Section 18.5.4) 18.5.3 ANTIOXIDATIVE ACTIVITY OF ASTAXANTHIN Carotenoids are generally known to possess powerful antioxidative activity, thought to be because of their long conjugated polyene system (Nishida et al., 2007) The stable structure and strong antioxidative activity of astaxanthin is considered to be due to the conjugation of the oxo group to the polyene system Astaxanthin is reported to show a strong quenching effect against singlet oxygen, with potency more than 100-fold higher than that of α-tocopherol (Miki, 1991) This same study also reports that astaxanthin shows strong activity against lipid peroxidation In addition, astaxanthin is reported to have no pro-oxidative properties: other carotenoids, such as β-carotene, lycopene, and zeaxanthin, under certain conditions, are considered to possess pro-oxidative properties (Martin et al., 1999) 18.5.4 HEMATOCOCCUS ALGAE H pluvialis, Flotow, Volvocales, Chlorophyceae, is a unicellular freshwater green microalga In response to environmental conditions, the green flagellated cells (vegetative cells) gradually transform into cyst cells without flagellae (the aplanospores), accompanied by a marked accumulation of astaxanthin, resulting in the formation of red-colored cells (Margalith, 1999) The size of a vegetative cell is less than 10 μm in diameter, although it gradually increases to over 40–50 μm after transforming into cyst cells In oxygenic photosynthetic organisms, carotenoids play important roles in the light-harvesting complex and in the protection of photosynthetic machinery, by dissipating excess light energy (Frank and Cogdell, 1996) These types of carotenoids are referred to as primary carotenoids and are essential in metabolism (Krishna and Mohanty, 1998) These carotenoids are localized in thylakoid membranes of the chloroplast In contrast, secondary carotenoids such as astaxanthin are not functionally obligatory for photosynthesis Astaxanthin in H pluvialis accumulates in cytoplasmic lipid globules of the cell: accumulation occurs in response to environmental stimuli, such as high light intensity and oligotrophic conditions In H pluvialis, it is considered that astaxanthin acts as a sunshade (Hagen et al., 1994), to provide protection from photodamage (Hagen et al., 1993), or to minimize oxidation of storage lipids (Sun et al., 1998) © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 333 as fish before Japan became westernized Thus, as a functional food, natto has been perceived as attractive to consumers who expect it to help them maintain a healthy body and to prevent various diseases Until recently, scientific evidence to substantiate the functions of natto has not been available, for example, its use as a folk remedy for heart and vascular diseases, to relieve fatigue, and as an antiberiberi agent (National Federation of Cooperatives on Natto, 1977; Sumi et al., 1987) In 1987 an enzyme contained in natto was found to have distinct fibrinolytic activity and was named nattokinase (NK) (Sumi et al., 1987) Fibrinolysis is the process by which fibrin clots or coagulated blood products are broken down Although the molecular mechanism of fibrinolysis is not expanded in this chapter, details can be found elsewhere (Cesarman-Maus and Hajjar, 2005) Such a process plays a crucial role in response to the formation of thrombus after an injury to a blood vessel Thrombus in the blood vessel probably causes vascular occlusions, leading to vascular diseases The intake of natto has been known to have a role in the prevention of such diseases for many years The finding of fibrinolytic activity in natto has accelerated studies on NK’s mechanistic action as well as on the safety and efficacy of the enzyme being supplemented for its health benefits In this chapter, we describe the evolution of this fibrinolytic enzyme known as NK into a dietary supplement or so-called healthy food (SCHF) thought to contribute to the prevention of thrombotic diseases such as cardiovascular and cerebrovascular diseases The term SCHF is used to define a scientifically substantiated ingredient with proven safety and efficacy (Ohama et al., 2006) Furthermore, we attempt to update the latest studies on NK, providing new insight about its biological activities in addition to the fibrinolytic activity of NK 19.2 NK AS A FIBRINOLYTIC ENZYME NK is scientifically known as Subtilisin NAT NK was produced by fermentation of B subtilis var natto NK is a serine protease, classified as a member of the Subtilisin family It has a molecular weight of 27,728 which is composed of 275 amino acid residues (Fujita et al., 1993) and its primary structure is shown in Figure 19.2 The nucleotide sequence of the NK (Subtilisin NAT) gene exhibits a high homology with other Subtilisin enzymes: it is 99.5% homologous to Subtilisin E and 99.3% homologous to Subtilisin Amylosacchariticus (Nakamura, 1992) H2N – Ala –Gln – Ser – Val – Pro – Tyr – Gly – Ile – Ser – Gln – Ile – Lys– Ala – Pro – Ala – Leu – His – Ser – Gln – Gly – Tyr – Thr – Gly – Ser – Asn – Val – Lys – Val – Ala – Val – Ile – Asp* – Ser – Gly – Ile – Asp – Ser – Ser – His – Pro – Asp – Leu – Asn –Val – Arg – Gly – Gly – Ala – Ser – Phe – Val – Pro – Ser – Glu – Thr – Asn – Pro – Tyr – Gln – Asp – Gly – Ser – Ser – His* – Gly – Thr – His – Val – Ala – Gly – Thr – Ile – Ala – Ala – Leu – Asn – Asn – Ser – Ile – Gly – Val – Leu – Gly – Val – Ala – Pro – Ser –Ala – Ser – Leu – Tyr – Ala – Val – Lys – Val – Leu – Asp – Ser – Thr – Gly – Ser – Gly – Gln – Tyr – Ser – Trp – Ile – Ile – Asn – Gly – Ile – Glu – Trp – Ala – Ile – Ser – Asn – Asn – Met – Asp – Val – Ile – Asn – Met – Ser – Leu – Gly – Gly – Pro – Th r – Gly – Ser – Thr – Ala – Leu – Lys – Thr – Val – Val – Asp – Lys – Ala – Val – Ser – Ser – Gly – Ile – Val – Val – Ala – Ala – Ala – Ala– Gly – Asn – Glu – Gly – Ser – Ser – Gly – Ser – Thr – Ser – Thr – Val – Gly – Thr – Pro – Ala – Lys – Tyr – Pro – Ser – Thr – Ile – Ala – Val – Gly – Ala – Val – Asn – Ser – Ser – Asn – Gln – Arg – Ala – Ser – Phe – Ser – Ser – Val – Gly – Ser – Glu – Leu – Asp – Val – Met – Ala – Pro – Gly – Val – Ser – Ile – Gln – Ser – Th r – Leu – Pro – Gly – Gly – Thr – Tyr – Gly – Ala – Tyr – Asn – Gly – Thr – Ser* – Met – Ala – Thr – Pro – His – Val – Ala – Gly – Ala – Ala – Ala – Leu – Ile – Leu – Ser – Lys – His – Pro – Thr – Trp – Thr – Asn – Ala – Gln – Val – Arg – Asp – Arg – Leu – Glu – Ser – Thr – Ala – Thr – Tyr – Leu – Gly – Asn – Ser – Phe – Tyr – Tyr – Gly – Lys – Gly – Leu – Ile – Asn – Val – Gln – Ala – Ala – Ala – Gln – COOH FIGURE 19.2 The primary structure of NK © 2010 Taylor and Francis Group, LLC *Active site 334 Biotechnology in Functional Foods and Nutraceuticals (a) (b) (c) 30 (d) (e) 1h 2h 5h FIGURE 19.3 Changes in fibrinolytic activity of NK with time as indicated using fibrin plates (a−e) to simulate artificial thrombus A potent fibrinolytic activity of NK is demonstrated using fibrin plates in Figure 19.3 The activity of NK was confirmed in an in vitro study (Sumi et al., 1987) A subsequent study using dogs with experimentally induced thrombosis further confirmed its fibrinolytic activity, when a mild enhancement of activity was observed after oral administration of NK (Sumi et al., 1990) Purified NK has been shown to degrade fibrin clots in two different ways One way is to act directly on the substrate or fibrin with a potent fibrinolytic activity which exhibits a much stronger activity than that of plasmin in clot lysis assays (Fujita et al., 1993, 1995b) The other mechanism is to degrade fibrin indirectly by affecting plasminogen activator inhibitor type (PAI-1), which is the primary inhibitor of tissue-type plasminogen activator (Urano et al., 2001) The elucidated activities of NK thus far are summarized in Figure 19.4 Importantly, NK does not suppress the formation of fibrin from fibrinogen; therefore, NK does not inhibit the formation of blood clots These NK Nattokinase Attack Degradation Attack Fibrin degradation products (FDP) Plasmin Fibrinogen Increase Plasminogen activator inhibitor type (PAI-1) Tissue plasminogen activator (t-PA) Activation (in vitro) Thrombin Degradation Fibrin thrombus Urokinase Plasminogen Pro-urokinase FIGURE 19.4 The proposed mechanism of NK on fibrinolysis Arrows show the sites where NK works NK as a serine protease exhibits multifunctional activities © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 335 activities differ from other fibrinolytic enzymes such as streptokinase and urokinase Moreover, NK is orally ingestible and still maintains its enzymatic activities, while the other fibrinolytic enzymes must be used intravenously in order to be active 19.3 PRODUCTION OF NK Natto is prepared by fermenting boiled soybeans with B subtilis var natto The soybeans are usually fermented at 40°C for 14–18 h until they convert into dark brown beans with the presence of sticky, viscous, string-like material In addition, natto possesses a notorious sour aroma and nutty flavor In this fermentation process, B subtilis var natto produces nutrients such as vitamin B2, amino acids, and dietary fiber as well as functional materials including NK, vitamin K2, poly-γglutamic acid (PGA), isoflavone, and polyamine Japan Bio Science Laboratory Co Ltd (JBSL) has a proprietary product containing a selected and patented strain of B subtilis var natto These bacteria were isolated from commercialized microorganism B subtilis var natto by JBSL The NK production method was found to yield NK in a culture medium primarily containing corn starch as the carbon source, defatted soybean as the nitrogen source, and inorganic salts such as calcium carbonate All culture medium nutrients are food or food additives approved by the Japanese government for human consumption NSK-SD is a raw material of the dietary supplements produced by JBSL The production methods of NSK-SD are as follows The first process of NSK-SD production is fermentation Providing proper aeration and agitation is an important process in large-scale fermentation NK is produced under an optimal temperature of around 35°C for the culture period of 2–3 days This cultured medium usually contains NK activity of about 1000–2000 FU/mL; a definition of the NK activity unit is given in the next section Additionally, vitamin K2 with concentrations ranging from 10 to 100 μg/g dry weight is contained in this cultured medium Although vitamin K2 is useful as a blood coagulant, it is an unfavorable by-product for the supplementation of NK since one of the major health benefits of NK is its antithrombotic effect; thus, vitamin K2 is eliminated subsequently The second process of NSK-SD production is filtration and removal of the vitamin K Chitosan dissolved in acetic acid solution as the high molecular weight aggregator and diatom earth as the filter aid are added to the cultured medium Filtration is performed under pressure to give a clear filtrate The chitosan–acetic acid solution absorbs the solids, bacterial cells, and hydrophobic materials including vitamin K2 in the cultured medium Consequently, after chitosan treatment 99.0– 99.9% of the vitamin K2 in the filtrate is removed This solution is then filtered by 0.2 μm pore size membrane The third process of NSK-SD production is the concentration and removal of natto flavor The filtrate is further concentrated by an ultrafiltration membrane unit The majority of compounds having a molecular weight of 10,000 (MW) or less can be removed by this procedure The obtained concentrate of the culture medium is then filtered by 0.2 μm pore size membrane filters At this point, the concentration of vitamin K2 in the solution is less than 0.01 μg/g dry weight The final process of NSK-SD production is powdering Appropriate additives such as watersoluble dietary fiber are added to the concentrate to produce powder upon spray-drying The production flow chart of NK is outlined in Figure 19.5 The finished product of NSK-SD is a white (milk-white) colored powder with little or no odor, having an enzymatic activity unit of more than 20,000 FU/g The recommended dosage is 2000 FU/ day All vitamin K2, which potentially increases blood coagulation, has been removed NSK-SD is produced from nongenetically modified soybeans and a selected, patented strain of B subtilis var natto NSK-SD is stable in a pH of 5.5–10.0 at 25°C for 24 h NSK-SD is stable at 50°C for h Optimal fibrinolytic activity takes place at around 65°C at pH 10.5 Also, NSK-SD is under pressure up to 2000 kg/cm2 and can therefore be pressed to prepare tablets NSK-II, the brand name for JBSL’s soft gel capsule product, retains 75–85% of the activity at a pH of 2.0, mimicking gastric fluid, for 30 at 37°C © 2010 Taylor and Francis Group, LLC 336 Biotechnology in Functional Foods and Nutraceuticals (a) (b) (c) (d) U F (e) (f ) (g) (h) Additive FIGURE 19.5 The production flow of NSK-SD using B subtilis var natto (a) Main culture, (b) filtration, (c) sterile filtration, (d) ultra filtration (UF), (e) sterile filtration, (f) membrane filtration, (g) standardizing, and (h) spray drying As dietary supplement, NK is generally used in soft gel dosage form Employing the assay method described below, the activity of commercially available natto products are compared and shown in Figure 19.6 Vitamin K2 concentration in NK is assessed by high-performance liquid chromatography (HPLC) NSK-SD is found to contain no vitamin K2, while the contents of vitamin K2 in other NK products ranged from 0.49 to 2.64 μg/g (Table 19.1) A B C D E F G H I J K L M N O P Q R S T U Ave 1590 1000 1610 930 1770 1550 3290 1270 2050 1170 1150 1480 2240 1670 1130 2040 620 2250 1180 1230 2100 1590 FIGURE 19.6 500 1000 1500 2000 2500 3000 A comparison of the NK activities of commercially available natto per pack © 2010 Taylor and Francis Group, LLC 3500 Production of Nattokinase as a Fibrinolytic Enzyme 337 TABLE 19.1 A Comparison of Vitamin K2 Concentrations in NK Products in the Marketplace in Japan and the United States Product Name NattoGoldTM Nattokinase NSP-2TM Ultra Nattokinase NSP-2TM NSK-SDTM Activity (FU/g) Vitamin K2 (μg/g) 15,600 11,400 19,000 24,900 0.49 1.93 2.64 Not detected 19.4 ASSAY METHOD It is essential to establish an assay method for the measurement of NK activity in order to ensure the presence of the expected enzymatic unit, as well as to guarantee the quality and safety of the product NSK-SD as a SCHF can be formulated in a wide variety of dosage forms such as soft gel capsule, hard shell capsule, tablet, paste, granule, and so on (Ohama et al., 2006) An accurate and standardized measurement of NK activity is crucial for determining the appropriate dose for daily oral ingestion The present assay method is officially validated by the Japan Health Food and Nutrition Association (JHFA) in Tokyo, Japan and the Japan Natto Kinase Association (JNKA) in Tokyo, Japan for any NK activities in SCHF products 19.4.1 DEFINITION OF ACTIVITY UNIT NK activity is determined by optically measuring the quantity of acid-soluble low-molecular products, which increases with the hydrolysis of the peptide linkages when NK acts on fibrin as a substrate One unit (1 FU) is defined as the amount of the enzyme that increases the absorbance of the sample at 275 nm by 0.01 per under the conditions described below 19.4.2 ASSAY METHOD OF NK A mixture of 1.4 mL of 50 mmol/L borate buffer (pH 8.5, containing NaCl) and 0.4 mL of 0.72% fibrinogen in buffer is preincubated in a water bath at 37.0 ± 0.3°C for 0.1 mL of thrombin, dissolved in buffer and adjusted to 20 U/mL, is added to the mixture and agitated by a vortextype mixer After reacting for 10 min, 0.1 mL of the sample solution is added and mixed for s at 37.0 ± 0.3°C After initiating the reaction, agitations at 20 or 40 are performed for s After reacting for 60 min, 2.0 mL of trichloroacetic acid (0.2 mol/L) solution is added to terminate the reaction The sample solution is further mixed and incubated for 20 at 37.0 ± 0.3°C The mixture is transferred into a micro test tube and centrifugation is performed at 15,000 × g for Finally, 1.0 mL of supernatant is delivered into a cuvette using a Pasteur pipette and the absorbance (AT) at 275 nm is recorded The following equation was used to calculate the NK activity per weight (FU/g): (AT − AB) _ × D, NK activity (FU/g) = × × _ 0.01 60 0.1 where AT is the absorbance of the sample, AB is the absorbance of blank (same as the sample except that it was added to the mixture after terminating the reaction), and D is the dilution rate of the sample Standardized NK 1.000 g is accurately weighed and diluent is added to prepare NK activity per volume to be 1.00 FU/mL The standard sample should be assayed simultaneously at every test batch to confirm the accuracy of the test result © 2010 Taylor and Francis Group, LLC 338 19.5 Biotechnology in Functional Foods and Nutraceuticals BIOAVAILABILITY OF NK The bioavailability of NK was demonstrated in a study using rats by measuring the transportability of NK across the intestinal tract A dose of 80 mg NK/kg was delivered intraduodenally to the rats Blood samples were drawn at predetermined intervals NK activity was detected in the plasma at and h after administration Furthermore, NK activity at 30 after administration was measured by monitoring the presence of degraded products of fibrinogen in the plasma Coagulation time, determined as plasma recalcification time, was prolonged when compared to baseline at and h following the administration of NK (Fujita et al., 1995a) 19.6 SAFETY NK is an enzyme which has been present in a commonly consumed Japanese food, natto, for centuries without any adverse effects (National Federation of Cooperatives on Natto, 1977; Sumi et al., 1987) The safety of NSK-FD, NSK-SD, or NSK II has been evaluated by JBSL in a wide range of studies using both animals and humans NSK-FD and NSK-SD are raw dietary supplement materials produced by JBSL NSK-FD is a freeze-dried powder with a natto flavor NSK II is a soft gel capsule product produced by JBSL 19.6.1 ANIMAL STUDIES Safety studies using animals include (1) a single oral dose toxicity study, (2) an oral repeated-dose 28-day toxicity study, and (3) a 13-week oral toxicity study 19.6.1.1 Single Oral Dose Toxicity Study NSK-FD with an activity of about 13,000 FU/g was tested orally by the administration of a single dose to Sprague-Dawley rats A group of 10 rats (five males and five females) received 2000 mg (26,000 FU)/kg body weight, and the other group received the placebo The animals were observed for 14 days and subjected to a necropsy at the end of the observation period The study was performed in compliance with the Ministry of Health and Welfare (MHW) Guidelines (1997) The trial of a single oral dose of the NSK-FD showed no deaths for the study periods while diarrhea was observed in two male rats and soft stools in three male rats and all female rats tested at one day after dosing The body weight gain of treated rats was not found to be adversely affected No gross pathological alterations were evident at terminal necropsy in any of the rats Based on these results, the minimal lethal dose (LD50) of NKS-FD after single oral administration in male and female Sprague-Dawley rats was found to be greater than the limit dose level of 2000 mg/kg body weight (BILIS, 1999) 19.6.1.2 Oral Repeated-Dose 28-Day Toxicity Study A repeat-dose study for 28 days was conducted with Sprague-Dawley rats A dose of 167 mg/kg/day NSK-SD (3700 FU/kg body weight/day) was orally administered to six male and six female rats The other group with the same number of animals was given a placebo The potency of NSK-SD was calculated as being equivalent to 100 times the common intake from commercially available packs of natto (50 g) by a 60 kg human Rats were examined for clinical signs, body weight, food consumption, urinalysis, and ophthalmological health The study methodology was in accordance with MHW guidelines (1993) At the end of 28 days the rats were bled for hematological and chemical analysis of blood and then euthanized for necropsy and histopathological examination The study results demonstrated that no toxic effects were found in the oral administration of NSK-SD (Kobuchisawa Laboratories, 2002) 19.6.1.3 A 13-Week Oral Toxicity Study A repeat dose of 90 days (13 weeks) was also conducted using Sprague-Dawley rats to assess the toxicity of doses at 100, 300, and 1000 mg/kg/day NSK-SD (21,900 FU/g) A placebo group was © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 339 included for comparison Four groups of animals were tested; each group consisted of 24 animals, 12 males and 12 females The study methodology was in accordance with the guidelines established by the MHW (1997) The results of the study showed no deaths of tested animals, no NK-related alterations in clinical signs, no changes in body weight or food consumption, no ophthalmological abnormalities, and no adverse effects on urinalysis, including water consumption Results also indicated that hematology as well as blood chemistry and pathology were normal at the end of the study (Bozo Research Center, 2004) 19.6.2 PATHOGENICITY STUDY OF NSK-SD PRODUCING BACTERIA The safety of the NSK-SD producing bacteria, isolated from commercially available natto bacteria (B subtilis var natto) was tested using 5-week-old mice (ICR-strain) A single oral inoculation of control or 7.55 × 108 colony forming unit (CFU) was given to a group of 10 mice Each group consisted of five males and five females The mice were observed for 14 days after inoculation Results demonstrated no mortality and no abnormalities in general health or body weight No treatment-related abnormalities were observed in the histopathology examinations during autopsy and no bacteria were found in any of the tissues examined during autopsy It was concluded that the bacteria used in the production of NSK-SD had no potential for infectivity, pathogenicity, or toxicity (Gifu Research Laboratories, 2003) 19.6.3 MUTAGENICITY It was demonstrated that NSK-SD was nonmutagenic in the reverse-mutation assay (Ames test) against five strains of bacteria and also in a chromosomal aberration study conducted in CHL/IU cells 19.6.3.1 Reverse-Mutation Assay The mutagenicity of NSK-SD (20,000 FU/g) was tested in five strains of bacteria: Salmonella typhimurium TA98, TA1537, TA100, TA1535, and Escherichia coli WP2uvrA NSK-SD was tested at six dose levels ranging from 15.5 to 5000 mcg/plate Negative and positive controls were included Positive controls included those without metabolic activation The results of a dose study as revealed by revertant colony counts of the test substance groups with and without metabolic activation were less than times the negative control values for all strains tested NSK-SD is not considered to be mutagenic (Kobuchizawa Laboratories, 2003a) 19.6.3.2 Chromosomal Aberration Test Tests for cell growth inhibition and chromosomal aberration were conducted with CHL/IU cells from the lung of a female Chinese hamster NSK-SD (20,000 FU/g) inhibited cell growth sharply at 0.156 mg/mL and higher concentrations The chromosomal aberration test was performed at concentrations lower than those which caused cell growth inhibition NSK-SD was incubated with the cells for h (short term) or 25 h (long term) with and without metabolic activation The period of 25 h was selected as it was 1.5 times the cell cycle for CHL/IU cells The short-term chromosomal aberration test was conducted without metabolic activation at three doses (0.110, 0.078, 0.055 mg/mL) The long-term test was conducted using the latter concentrations Both positive and negative controls were included The results of the experiments indicated that chromosomal aberration occurred at less than 5% in all doses tested without exhibiting dose-dependency It was concluded that NK did not produce chromosomal aberrations in CHL/IU cells at any concentration (Kobuchizawa Laboratories, 2003b) 19.6.4 HUMAN STUDIES The safety of NSK II was performed in a randomized, double-blind human clinical study with 31 healthy male and female subjects (20–64 years old; body mass index (BMI) between 18 © 2010 Taylor and Francis Group, LLC 340 Biotechnology in Functional Foods and Nutraceuticals and 28) Nine subjects (five males and four females) took a placebo and 22 subjects (10 males and 12 females) took three NSK II soft gel capsules (2000 FU/3 capsules) per day for weeks, followed by a 2-week observation period All subjects visited the clinic at the beginning of the study and then and weeks after initial treatment During visits to the clinic, interviews were conducted along with measurements of body weight, blood pressure, and pulse rate In addition, blood was taken and urine collected Subjective symptoms were recorded daily No significant adverse effects were reported for either group Mild adverse events reported for the placebo group were diarrhea (four cases) and back pain (one case) Mild adverse effects found in the treatment group included diarrhea (three cases) and common cold (two cases) The other effects observed were constipation, pimples, stomach pains, menstrual cramps, and headache Body weight slightly increased in both the placebo and treatment groups, which was considered to be clinically not significant There were also minor changes in hematological profi les in both groups that were not deemed clinically significant No effect was noted on blood pressure or pulse rate No significant changes in urinary analysis were observed It was confi rmed that oral daily administration of three capsules (630 mg) of NSK II for weeks was safe (Ogasawara et al., 2006) In recent years, consumers taking medications such as warfarin as an anticoagulant have asked questions about drug–food interactions Hence, another study was performed to assess the safety of the administration of NSK II to patients taking physician-prescribed warfarin This was a double-blind, placebo-controlled study with 60 adult cardiovascular patients (male and female) Treatment groups were administered two capsules of NSK II (1700 FU) as low dose and four capsules of NSK II (3400 FU) as high dose per day after breakfast for 26 weeks No adverse effects were observed for either group as a result of taking a combination of the two agents; therefore, concomitant intake of NSK II and warfarin may not cause any food–drug interaction (Ninomiya et al., 2008) Furthermore, an open label study evaluated the safety of NSK-FD as a concomitant oral fibrinolytic agent for those who had experienced strokes The study included 12 patients with an average age of 53.3 years who were in hospital in a conscious state with acute mild to moderate ischemic stroke of noncardiac origin All patients were administered heparin s.c (7600 IU/day) and antiplatelet drugs (low-dose aspirin 150 mg to 325 mg/day Clopidogrel) along with NSK-FD (6000 FU/day, three doses for 2000 FU) for days The patients were then monitored for months (90 days) The results exhibited no mortality during the course of the study No incidents of hemorrhagic transformation of the infarct as confi rmed by CT scan were reported in any of the patients The conditions of the subjects were evaluated using three internationally recognized scales: National Institute of Health Stroke Scale, Modified Rankin Scale, and Barthel Index According to these scales, five patients had an overall favorable response Coagulation and fibrinolytic assays were performed on days 1, 2, and Significant changes compared to day were as follows: bleeding time increased on day 7, clotting time increased on days and 7, and D-dimer levels decreased on days and There were three adverse events that might have been attributed to NSK-FD: (1) prolonged activated partial thromboplastin time, (2) moderate hematemesis, and (3) an abnormal liver function test All of these events were temporary Thus, the fi ndings in the study suggest that NK may be safely administered to patients with stroke as an adjunct to standard medical treatments (Japan Bio Science Laboratory, 2004; Shah et al., 2004) 19.7 EFFICACY In vitro experiments as well as animal and human studies prove that NK does indeed have fibrinolytic activity Results of such studies also suggest that serine protease may control high blood pressure, reduce red blood cell (RBC) rouleaux formation, decrease blood viscosity, and inhibit platelet aggregation © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 341 19.7.1 IN VITRO ASSESSMENT Initially, fibrinolytic activity was observed when the vegetable cheese natto was applied directly to fibrin The fibrinolytic activity was about 40 CU (plasma units)/g wet weight and the isolated protease was named NK (Sumi et al., 1987) The fibrin degradation unit is defined as fibrinolytic activity of 40 CU (equivalent to 30 FU) Experiments using a clot lysis assay (cross-linked fibrin) revealed that purified NK was 4- to 5-fold more potent than the fibrinolytic activity of plasmin NK cleaved fibrinogen and fibrin, producing degraded products similar to those of plasmin When the kinetics of NK and plasmin were measured, NK was 3-fold less active in the cleavage of fibrinogen as a substrate compared to plasmin (Kcat/Km) but 6-fold more efficient in the cleavage of crosslinked fibrin (Sumi et al., 1990) Preparations of pure NK and NSK-SD (bulk powder plus capsule contents) were tested in a series of in vitro experiments in human plasma Test concentrations (0.2–1.6 FU/mL) were calculated as twice the plasma concentration of the highest recommended dose (4000 FU) assuming 100% bioavailability in a 5-L average blood volume In this system, the functional activity of fibrinogen to form fibrin in response to thrombin was not altered by concentrations of 0.2–0.8 FU/mL of NK Only at the highest concentrations of 0.8 and 1.6 FU/mL did NK reduce the activity of fibrinogen This finding suggested that NK should not affect the body’s ability to respond to tissue wounding, when taken at recommended doses (Ero and Lewis, 2008) Unlike urokinase, NK does not promote fibrinolysis by directly stimulating plasminogen activator activity (Sumi et al., 1990) Instead, it was reported to degrade the PAI-1 PAI-1 is the primary inhibitor of the tissue-type plasminogen activator (t-PA) NK cleaves active recombinant PAI-1 into low-molecular-weight fragments at concentrations of 0.02–1.00 nmol/L NK reduces the activity of the inhibitor while enhancing t-PA-induced lysis of fibrin clots in a dose-dependent manner (0.06–1 nmol/L) (Fujita et al., 1995) In contrast to the above study, an in vitro test in human plasma reported that NK (0.8 and 1.6 FU/mL) slightly increased the presence of PAI-1 (Ero and Lewis, 2008) The effects of NK on RBC aggregation and blood viscosity were also measured in an in vitro experiment Blood samples incubated with NK at final concentrations of 15.6, 31.3, 62.5, and 125.0 activity units/mL resulted in 21.9%, 25.9%, 49.7%, and 62.0% inhibition of RBC aggregation respectively as compared to the control NK reduced blood viscosity at lower shear rates but no changes in viscosity at high shear rates were observed (Pais et al., 2006) 19.7.2 ANIMAL STUDIES The fibrinolytic activity of NK was tested in dogs using an experimental thrombosis model by infusing bovine fibrinogen and thrombin into the animals Three dogs were treated with NK and six dogs were given a placebo as control Four capsules of NK (250 mg/capsule: 2.13 CU/mg equivalent to 1600 FU) or placebo were orally administered Angiograms were obtained before the induction of the thrombus and from 2.5 to 24.0 h afterwards In the control group, there was no sign of lysis 18 h after induction of thrombosis In contrast, the dogs treated with NK had a complete restoration of blood circulation within h (Fujita et al., 1993) The fibrinolytic activity of NK was also examined in a rat model, in which thrombus was formed in the common carotid artery by damaging the endothelial cells of the vessel wall with acetic acid In this model, urokinase or t-PA given intravenously at a constant rate of 20 restored blood flow (45%) over 60 There was no restoration of blood flow with saline NK was tested in this model in doses of 0.02, 0.04, and 0.12 μmol/kg (intravenous) and its activity was compared to plasmin and elastase NK caused a dose-related recovery of blood flow (18%, 42%, and 62%) after 60 When the activity of NK and plasmin was compared on a molar basis, NK was 4-fold more efficient than plasmin There was no recovery with elastase Degradation of cross-linked fibrin was determined by the presence of D-dimer γ–γ chain remnants in the plasma D-dimer remnants were detected in the blood after treatment with NK as well as urokinase and t-PA The feasibility of using NK © 2010 Taylor and Francis Group, LLC 342 Biotechnology in Functional Foods and Nutraceuticals therapeutically for fibrinolysis would depend on its ability to digest fibrin without acting on fibrinogen Values for residual plasma fibrinogen following administration of a dose of NK were reduced by one-third to the approximate activity level of plasmin and the residual fibrinogen level was 53% This is a greater amount of residual fibrinogen than the 33% remaining after treatment with plasmin at a comparative level The results imply that NK may be safer than plasmin at an appropriate dose level (Sumi et al., 1990) Thickening of vascular intima is thought to be part of the progression of arteriosclerotic plaques which can lead to heart attack and stroke The ability of NK to inhibit the progression of intimal thickening was tested in a rat model In this model, endothelial damage to the femoral artery was induced by intravenous injection of Rose Bengal followed by irradiation with transluminal green light Twenty-one days after endothelial injury, significant intimal thickening was observed Administration of NK (50 or 100 CU/animal, equivalent to 38 and 75 FU/animal) began weeks before the endothelial injury and then continued for another weeks NK reduced the development of intimal thickening from an area of 1.28 ± 1.14 mm2 in the control group to 0.79 ± 0.60 mm2 and 0.71 ± 0.27 mm2 in the low- and high-dose groups The difference between the thickening in the control group and the high-dose group was significant (p < 0.05) When the intima/media ratios were compared for the three groups, both treatment groups were significantly different from the control group (p < 0.05) There was no difference between the control and treatment animals in the time taken to develop occlusion following injury However, differences were observed in the morphology of the mural thrombi In the control group the center of the vessel reopened with mural thrombi attached to the vessel walls In the NK groups, thrombi near the vessel walls showed lysis and most thrombi were detached from the vessel wall surface The control group had thrombi attachment lengths measuring 858 ± 430 mm at h after injury NK reduced the attachment length in a dose-dependent manner with the measurement of 173 ± 105 mm for the high-dose group, a significant difference (p < 0.05) as compared to that of the control Bleeding times for the three groups did not differ (Suzuki et al., 2003) NK was previously demonstrated to decrease blood pressure in animal models using Wistar male rats weighing 400–450 g The rats were intraperitoneally injected with 0.5 mL of a lyophilized natto extract dissolved in 80% ethanol which was equivalent to 25 mg natto or about 0.8 FU total or FU/ kg body weight Blood pressure was measured using the tail artery The average systolic blood pressure of six rats significantly (p < 0.05) decreased and h after administration of the natto extract by 12.6% and 13.2%, respectively The systolic blood pressure decreased from 166 ± 14 mm Hg at baseline to 144 ± 27 mm Hg after h (Maruyama and Sumi, 1998) 19.7.3 HUMAN CLINICAL STUDIES Various clinical studies demonstrated that NK or NSK-II possessed activities such as (1) fibrinolysis, (2) reduction of hypertension, (3) decrease of blood viscosity, and (4) platelet-aggregatory inhibition 19.7.3.1 Fibrinolytic Effect Preliminary evidence that NK would have a fibrinolytic effect on human subjects was reported in 1990 (Maruyama and Sumi, 1998) Twelve health volunteers (21–55 years old) were given a single dose of 200 g natto (equivalent to about 6000 FU) or a control of boiled soybeans in a crossover single-dose designed study at a 2-week interval Blood was collected 2–24 h after ingestion Euglobin (clot), lysis time (ELT) significantly decreased 2, 4, and h after intake of natto compared to the soybean control In another experiment the volunteers were given two enteric-coated capsules containing NK (650 mg/capsule; 2.13 CU/mg) times a day following meals (equivalent to 3000 FU/ day) for days Blood was collected each day Euglobulin fibrinolytic activity (EFA) gradually but not significantly increased over the course of days The degraded products from fibrin and fibrinogen (FDP) in the serum were not measured The FDP levels in the serum spiked on the first day and © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 343 then slowly decreased over the 8-day period The levels were significantly different from baseline on days through (Fujita et al., 1993) In a different study, a single oral dose of 30 g lyophilized natto (equivalent to 200 g original wet weight which is equivalent to 6000 FU) was given to five volunteers (51–86 years old) Blood samples were taken 2–24 h after intake Fibrinolysis was evaluated 4–8 h after intake EFA significantly increased after h and FDP measurements remarkably increased and h after administration EFA increased from 1.9 ± 2.7 mm2 at baseline to 4.5 ± 3.3 mm2 after h, 1.33 ± 7.2 mm2 after h, and 8.7 ± 7.4 mm2 after h The FDP levels at baseline, h, and h were 0.75 ± 0.52, 5.50 ± 2.74, and 2.75 ± 1.37 mcg/mL, respectively The FDP was further decreased following additional intakes on days and (Sumi et al., 1996; Sumi and Maruyama, 1998) A double-blind, placebo-controlled study with 30 human subjects (male and female; average age 59) explored the administration of NK to patients taking prescribed warfarin for possible food–drug interaction The theory behind the combination of the two agents was that the addition of NK might help stabilize the fibrinolytic effect of warfarin The treatment group was given two capsules of NSK II (1700 FU) per day after breakfast for 26 weeks As a result, there were significantly decreased rates of change in prothrombin and prothrombin-INR compared to placebo (p < 0.05) Treatment was particularly effective for those over 60 years of age Activated partial thromboplastin time and prothrombin time were closed to reference values compared to the placebo group after months (p < 0.05) In addition, lower rates of change were noted for activated partial thromboplastin and prothrombin, and prothrombin-INR times (Ninomiya and Yamada, 2008) 19.7.3.2 Reduction of Hypertension Hypertension is a global public health concern, affecting about 50 million individuals in the United States and one billion individuals worldwide (Kearney et al., 2005; Chobanian et al., 2003) High blood pressure is postulated as the most crucial risk factor for cardiovascular disease In Japan, the disease is the third highest mortality cause of the 2000 s (Ohama et al., 2006) Moreover, hypertension is an important predictor of stroke, acute myocardial infarction, and end-stage renal disease (Iseki et al., 2000) It has been reported that the occurrence of cardiovascular disease in Asia is relatively low, attributable to traditional food from Asia (Artaud-Wild et al., 1993; Keys et al., 1984) In fact, it is traditionally known that natto in the diet tends to lower blood pressure (National Federation of Cooperatives on Natto, 1977) A previous study of natto and NK for the control of high blood pressure suggested that such foods might exert their beneficial effect through the inhibition of the angiotensin converting enzyme (ACE) (Kim and Yamamoto, 1992) However a recent clinical study found no difference in blood levels of ACE following treatment with NK but did report a decrease in rennin activity (Kim et al., 2008) This study suggested that the mechanism whereby NK decreased blood pressure might be attributable to the inhibition of rennin activity as further described below Briefly, a randomized, double-blind, placebo-controlled study was conducted with 73 hypertensive subjects (20–80 years old) with initial systolic blood pressures between 130–159 mm Hg The subjects received one capsule of NSK-II (2000 FU/capsule) per day or a placebo for weeks After weeks of treatment there were significant decreases in systolic and diastolic blood pressure compared to placebo Both treatment and placebo groups had some reduction in blood pressure, with the net decreases for the treatment group being 5.5 mm Hg in systolic blood pressure and 2.8 mm Hg in diastolic blood pressure There was also a net decrease in plasma rennin activity (1.17 ng/mL/h) in the treatment group compared to the control (p < 0.05) However, there was no significant difference in ACE levels between the two groups In an open label clinical study, 30 g of lyophilized extract (80% ethanol; equivalent to 200 g natto, about 6400 FU) were orally administered for four consecutive days to human subjects who had high blood pressure In four of the five subjects the systolic and diastolic blood pressure decreased when measured in the supine position The systolic average values decreased by 10.9% © 2010 Taylor and Francis Group, LLC 344 Biotechnology in Functional Foods and Nutraceuticals from 173.8 ± 20.5 to 154.8 ± 12.6 mm Hg The diastolic blood pressure decreased by 9.9% from 101.0 ± 11.4 to 91.2 ± 6.6 mm Hg (Maruyama and Sumi, 1998) A randomized, placebo-controlled, crossover study was conducted with 20 male and female subjects (18–75 years of age) with a variety of disease states such as essential hypertension, hypercoagulable states, autoimmune diseases, and diabetes Half of the subjects in the study received 4000 FU (2000 FU twice a day of NSK) and the other half received placebo After weeks the groups crossed over and received the alternate intervention There was a significant decrease in systolic blood pressure compared to baseline for the NSK group (p = 0.039) and no significant change in diastolic blood pressure as compared to baseline The placebo treatment did not cause any change in systolic or diastolic pressure (Krishman Medical Association S.C, 2003) 19.7.3.3 Decrease of Blood Viscosity The effects of NK on blood flow were studied in a placebo-controlled crossover study with 15 healthy subjects aged 30–49 years (seven men and eight women) The volunteers were given three capsules of NSK (2000 FU/capsule or total of 6000 FU) in a single dose (Group A) or a placebo (Group P) There was a 2-week wash-out period before switching treatments Blood flow was measured using the PeriScan PIM II method In Group A, there was a significant increase in blood flow in the right and left middle fingers 80, 120, and 180 after intake of NK (p < 0.01) Compared to Group P (placebo) there was a significant effect 180 after intake (Group P 0.10 ± 011 V compared to Group A 0.42 ± 0.08 V; p = 0.034) Group A also had an increase in blood flow in the back of the right and left hands at 40, 80, 120, and 180 compared to baseline (p < 0.01) When the volunteers were subdivided according to the BMI, those with a BMI over 23 treated with NK had a statistically significant increase blood flow compared those on placebo (p = 0.046) (Pais et al., 2006) 19.7.3.4 Antiplatelet Activity A series of experiments to assess the antiplatelet effects of NK was performed in human studies An open label pilot study was first conducted by supplementing NK in soft gel capsules (4000 FU, equivalent to the NK activity of two packs of commercially available natto) to healthy human volunteers (Takaoka et al., 2003) The assay used was an aggregometer with a light-scattering method (Moriyama et al., 2003; Iizuka et al., 2007; Moriyama et al., 2009), which helped to determine the antiplatelet activity of NSK II based on the extent of platelet aggregatory inhibition and also the distribution of platelet aggregates based on the developmental formation of platelet aggregates with time and three sizes An additional study using NK demonstrated that administration of NSK II could inhibit spontaneously occurring platelet aggregation (Takaoka et al., 2004) A further study led to the establishment of a possible mechanistic action of NK on platelet-aggregatory inhibition, suggesting that fibrin degradation products might have played a pivotal role in this inhibition (Takaoka et al., 2005) A previous study reported that urokinase could inhibit platelet aggregation through mechanisms independent of the generation of plasmin but rather through mechanisms which might be associated with the fibrinogen of a fragment as a responsible inhibitor of platelet aggregation (Torr-Brown and Sobel, 1996) These studies appear to demonstrate the antiplatelet effects of NSK II and provide support for the observation that NK prevents the generation of platelet microaggregates in the early phase of platelet activation and the formation of thrombi which lead to vascular occlusion Thus, oral administration of NK soft gel is a good supplement for lifestyle-related diseases such as cardiovascular diseases 19.8 FUTURE PROSPECTIVES During the production process of NK useful by-products are also yielded in the culture media, including vitamin K2 and PGA Recently, vitamin K2 has been successfully recovered from the cultured media © 2010 Taylor and Francis Group, LLC Production of Nattokinase as a Fibrinolytic Enzyme 19.8.1 345 VITAMIN K2 Vitamin K2 is a fat-soluble vitamin which acts as a coenzyme and participates in the synthesis of a number of proteins involved in blood clotting and bone metabolism It is possible to recover vitamin K2, previously removed by extraction from the filtration residue in the chitosan treatment, with an organic solvent such as ethanol or less polar solvents and then to concentrate the extract Molecular distillation or steam distillation is employed The recovered vitamin K2 can be used in medical treatments or in the prevention of osteoporosis Although additional refinements could reduce the recovery cost of this vitamin K2, such products are already available in the marketplace Vitamin K2 is an ingredient in the foods with specified health claim (FOSHU) category in Japan, which allows the claim that it “helps absorb calcium into the bone” (Yamaguchi et al., 1999) 19.8.2 POLY-γ-GLUTAMIC ACID PGA is a water-soluble and biodegradable substance that comprises d- and l-glutamic acid polymerized through γ-glutamyl bonds Furthermore, PGA (consisting of 30–5000 glutamic acids) is responsible in part for the slippery characteristics of natto (Figure 19.7) In 2003, PGA was approved as an active ingredient of functional food under the FOSHU system with a health claim that it “helps absorption of calcium.” In light of its FOSHU approval, a largescale fermentation of PGA was attempted in order to search for suitable conditions for commercial PGA production (Ogawa et al., 1997) Incidentally, during the production of NK at its present industrial scale, a substantial amount of PGA has been detected in the culture media, which is eliminated as a by-product Thus, the introduction of an efficient recovery methodology is important to reclaim PGA during NK production 19.8.3 FOOD WITH SPECIFIED HEALTH CLAIM In 1991, the MHW, now the Ministry of Health, Labor and Welfare (MHLW), introduced the FOSHU system Details are discussed by Ohama et al (2006) FOSHU was based on the concept of “functional foods” which benefit the structure and function of the human body, as a result of research studies on the health benefits of foods since 1984 The Ministry of Education organized national research and development projects to evaluate the functionalities of various foods Researchers from diverse scientific fields defined new functions of foods by incorporating previously recognized functions of nutrition, sensory/satisfaction, and the physiological effects of ingredients in foods A representative FOSHU product based on material derived from traditional Japanese food is the fermented bonito or “Katsuobushi” peptide which has a health claim of “lowering blood pressure” (Fujita et al., 2001) Natto and its NK ingredient is another possible candidate for FOSHU, also categorized under the health claim of “lowering blood pressure.” CO CH2 CH2 NH CH – COO n FIGURE 19.7 The structure of PGA which is found in the stick-threads of natto © 2010 Taylor and Francis Group, LLC 346 Biotechnology in Functional Foods and Nutraceuticals 19.9 CONCLUSION Natto, a traditional Japanese food, is a source of functional ingredients such as the fibrinolytic enzyme known as NK, and vitamin K2 The large-scale production of functional foods has made it possible to commercialize them as ingredients for dietary supplements NK, in particular, plays an important role in antithrombosis, which may potentially help to prevent vascular disorders such as cardiovascular disease The present NK production methodology successfully eliminates vitamin K2 as a coagulation agent, which is considered as a contaminant in the final NK product NK has been demonstrated to be safe, and proven to show not only fibrinolytic activity but also antihypertensive, antiplatelet, and other blood circulatory beneficial activities to help prevent vascular diseases NK could thus be used as a dietary supplement replacement for aspirin or nonsteroidal anti-inflammatory drugs for the prevention of thrombi formation without any side effects The incorporation of additional steps in the NK production process can isolate vitamin K2 and possibly PGA for their utilization in other health benefits ACKNOWLEDGMENT One of the authors (Hiroyoshi Moriyama) is supported by funds from MTI, Inc (Tokyo, Japan) and the Japan Bio Science Laboratory Co., Ltd (Osaka, Japan) REFERENCES Artaud-Wild, S.M., Connor, S.L., Sexton, G., and Connor, W.E., 1993 Differences in coronary mortality can be explained by differences in cholesterol and saturated fat intakes in 40 countries but not in France and Finland A paradox Circulation 88: 2771–2779 BILIS, Environmental Biological Life Science Research Center, Inc., Shiga, Japan Final report: Singe oral dose toxicity study of BIOZYME NSK-FD POWDER in rats February 26, 1999 Bozo Research Center, Inc., Tokyo, Japan Final report: A 13-week oral toxicity study of nattokinase DS (NSK-SD) in rats B-5170, June 16, 2004 Cesarman-Maus, G and Hajjar, K.A., 2005 Molecular mechanisms of fibrinolysis Brit J Haematol 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