In the creation of an effi cient biofi xation system based on microalgae mass culture, Yamaha Motor began developing new conceptual photobioreactors using fl uid dynamics technology that
Trang 1for Microalgae Cultivation and Its Application in
Functional Foods and the Nutraceutical Industry
Akira Satoh, Masaharu Ishikura, Nagisa Murakami, Kai Zhang, and Daisuke Sasaki
18.1 INTRODUCTION
18.1.1 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
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 Benefi ts 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
Trang 2motorcycles 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 Y AMAHA MOTOR
In 1997, a meeting was held in Kyoto, Japan, to discuss the international convention on the zation of greenhouse gas concentration in the atmosphere At the meeting, many advanced coun-tries 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
stabili-on the road to researching and developing carbstabili-on dioxide reductistabili-on and fi xatistabili-on technology In
emission engines and fuel cells Yamaha Motor became interested in the biofi xation of CO2 using algal photosynthesis, which has a CO2-fi xing capability far superior to that of higher plants In the creation of an effi cient biofi xation system based on microalgae mass culture, Yamaha Motor began developing new conceptual photobioreactors using fl uid dynamics technology that was previously utilized in the fi eld of marine products (see Section 18.3 for details) Throughout the research, they accumulated specifi c 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 develop-ment 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
FOODS AND THE NUTRACEUTICAL INDUSTRY
Microalgae, including cyanobacteria, are microscopic photoautotrophs in which inorganic pounds and sunlight energy are converted into biomass Applied research on algal culture and bio-mass began in the late 1940s (Burlew, 1953) This research included alternative and unconventional protein sources, photosynthetic gas exchange for space travel, aquaculture feed, waste water treat-ment, renewable energy sources, biological fi xation of greenhouse CO2, and production of recombi-nant 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 technol-ogy 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 prod-ucts 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;
com-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
fl os-aquae, mainly cultured in China, India, USA, and Japan, with an annual production of 3000
Trang 3and 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
dif-fi culties 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, opti-mum 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, productiv-ity, quality, and seasonal effects) Before discussing the commercial production of microalgae at Yamaha Motor, details of the technology and problems are briefl y 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 tion risk, that is, diffi culties in maintaining a monoalgal culture (species control) and prevention
contamina-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 diffi culty of supplying an adequate amount of light to each algal cell Light irradiation reach-ing 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 produc-tion 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 diffi cult to control in open-air systems
Microalgal culture technology has developed a closed system using a photobioreactor to come the problems discussed above, especially contamination risk and light limitation, and conse-quently to achieve higher cell density In typical photobioreactors, a series of transparent tubes, fl at 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
over-of tube or thickness over-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 diffi culties 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 suffi ciently high light and perature control are required, since light intensity and temperature are the most critical factors
tem-affecting the growth of H pluvialis and astaxanthin accumulation (Margalith, 1999) In addition,
Trang 4sterility 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
containing H pluvialis algal biomass, namely the “YAMAHA High-effi ciency Photobioreactor.”
The development of this high-effi ciency photobioreactor is described in the next section
PRODUCTION OF MICROALGAE AT YAMAHA MOTOR
As described in Section 18.1.2, Yamaha Motor became engaged in the development of outdoor tobioreactors for the biofi xation of CO2 using algal photosynthesis In order to achieve high-density algal cultures with high photosynthetic activity, we at Yamaha Motor focused on the “fl ashing light effect,” also called “intermittent illumination,” which had been reported to be a means of utilizing
pho-a lpho-arger frpho-action of the sunlight shining on pho-a given pho-arepho-a (Emerson pho-and Arnold, 1932; Weller pho-and Franck, 1941; Rieke and Gaffron, 1942; Tamiya and Chiba, 1949; Kok, 1953) The “fl ashing 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 fl uid 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 fl uid 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.
Trang 5a valuable algal strain widely used as a feed in marine hatcheries A biomass productivity of
photobioreactor could be increased up to approximately 200 L In addition, the simultaneous use
of artifi cial light and sunlight to increase algal productivity was made possible by inserting fl cent 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 diffi culties in cleanup and sterilization The pipe-type photobioreactor is therefore applicable for small-scale on-site production of live microalgae
uores-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 fl at-plate-type reactor for this project, since previous research had shown this type
of reactor to have excellent light illumination effi ciency, leading to superior algal growth (Zhang
et al., 2001) Our fi rst 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 infl uenced by light availability and temperature (see Section 18.2) In order to establish a stable industrial manufacturing process, we moved to indoor cultivation using artifi cial lights, and this resulted in the development of the “YAMAHA High-effi ciency 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) certifi cation Details of our manufacturing process (GMP approved) are described in the next section
FIGURE 18.2 Computational fl uid dynamics analysis of the pipe-type photobioreactor.
Trang 618.4 ASTAXANTHIN RAW MATERIAL MANUFACTURING PROCESS
18.4.1 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 fi rst 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-effi ciency Photobioreactors” is the main production facility (Figure 18.4) This facility is operated in an
FIGURE 18.3 An outdoor vertical fl at-plate-type photobioreactor at Yamaha Motor, used in the cultivation
of H pluvialis.
FIGURE 18.4 The indoor cultivation system known as the “YAMAHA High-effi ciency Photobioreactor”
in the Fukuroi Factory II.
Trang 7unattended manner at night, enabling highly controlled batch cultures under continuous tion, 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 m2, with 3300 m2 total fl oor 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.
illumina-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.” Briefl y, 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 mini-mize 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 environ-ment under sanitary quality control Great care is also taken in the system design and operations at
Astaxanthin oil
Shipping inspection
Packaging (b)
(a)
Concentration
Extraction
Shipping inspection
Packaging
Drying
Separation
Cultivation
FIGURE 18.5 A production fl ow scheme for astaxanthin raw material production (a) and subsequent
astaxanthin-containing H pluvialis oil (b).
Trang 8subsequent 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 manu-facturers, 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 certifi cation by the Japanese Health Food Standards Association (JIHFS) Our quality assur-ance system had thus established a reliable GMP system, certifi ed by an outside organization Our GMP is composed of three basic principles: keeping mistakes to a minimum level; preventing pol-lution 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
1 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
2 A traceability system using serial numbers for each product lot
3 An education and training system, and skills improvement for all staff and workers at the plant
4 Execution of a rigorous plant self-check system, based on our GMP, at the end of every run
5 Clean-room installation, complete control of air-conditioning facilities and purifi cation control in all production processes
6 Manufacturing environment maintenance to prevent cross-contamination and foreign body mixing
7 Installation of appropriate inspection equipment for both the process control and product standard test
8 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 fulfi lls 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
Trang 918.5 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 car-otenoid group, with oxygen-containing functional groups, and also possesses hydroxyl and oxo functional groups In nature, astaxanthin is found in its esterifi ed 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 noids are known as provitamin A as they can be cleaved at the central C15=C15′ double bond and
carote-converted into vitamin A in vivo (Goodman and Huang, 1965; Olson and Hayashi, 1965) In fi sh, 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 metrically at the C9 position and is therefore regarded as a non-provitamin A carotenoid (Kistler
asym-et al., 2002)
Xanthophyll esters are thought to be hydrolyzed prior to absorption (Zaripheh, 2002) Only esterifi ed astaxanthin is detected in serum after the ingestion of esterifi ed astaxanthins (Coral-Hinostroza 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, reduc-ing their size, and xanthophylls reach the liver as chylomicra remnants In the liver, they are incor-porated 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
non-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.
FIGURE 18.6 Yamaha Motor’s GMP-grade astaxanthin oil product.
Trang 10Due to their high antioxidant properties and other functions (see Section 18.6), many containing nutraceuticals with potent effects on human health are coming onto the market.
astaxanthin-18.5.2 STRUCTURE AND SOURCE OF ASTAXANTHIN
The astaxanthin molecule has a symmetric confi guration and two chiral centers; two carbon atoms adjacent to hydroxyl functional groups are chiral There are three enantiomeric isomers of astax-
anthin, (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 fi eld
of aquaculture as a reviver and is not used as an ingredient in human neutraceuticals A green
microalga (H pluvialis), a red yeast (Phaffi a rhodozyma), and crustacean by-products are
com-mercially 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 esterifi ed with fatty acid to form mono- or diesters (Johnson and An, 1991) In contrast, Phaffi a rhodozyma is reported
to synthesize the (3R, 3 ′R) isomer (Torissen et al., 1989), which is mainly unesterifi ed (Andrewes and Starr, 1976) Haematococcus alga is considered to be the most effi cient 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 proper-ties (Martin et al., 1999)
18.5.4 H EMATOCOCCUS ALGAE
H pluvialis, Flotow, Volvocales, Chlorophyceae, is a unicellular freshwater green microalga
In response to environmental conditions, the green fl agellated cells (vegetative cells) gradually transform into cyst cells without fl agellae (the aplanospores), accompanied by a marked accu-mulation 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 caro-tenoids 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
pho-tosynthesis 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
olig-otrophic 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 tion of storage lipids (Sun et al., 1998)
Trang 11oxida-18.6 BENEFITS OF ASTAXANTHIN FOR HUMAN HEALTH MANAGEMENT
As a company which supplies functional food ingredients, it is important that we investigate the effectiveness of the ingredient we produce It is generally accepted that oxidative stress is involved not only in the normal aging process but also in the pathogenesis of many acute, chronic, and age-related diseases, including infl ammation, cancer, cardiovascular disease, neurodegenerative dis-ease, lung disease, and eye disease Natural antioxidants are scavengers of reactive oxygen species including free radicals, and therefore have broad implications in antiaging and human health man-agement As described in the previous section, astaxanthin has a strong antioxidative activity and hence various physiological effi cacies In the next section, we present our clinical evidence for the
effi cacy of our astaxanthin-rich H pluvialis oil on oxidative stress-related diseases/aging, especially
atopic dermatitis, brain health, and metabolic syndrome
18.6.1 ASTAXANTHIN AND ATOPIC DERMATITIS
There are many lines of evidence suggesting that allergic disorders, such as atopic dermatitis, asthma, and rhinitis, are mediated by oxidative stress (Bowler and Crapo, 2002; Okayama, 2005)
We observed that the administration of astaxanthin signifi cantly suppressed ear swelling in NC/Nga mice which had been previously sensitized by intradermal injections of mite antigen into the ear pinnae, inducing atopic dermatitis-like lesions (Iio et al., 2009) We further investigated the effect of astaxanthin intake on atopic dermatitis by a double-blind, randomized, placebo-controlled clinical trial (Satoh et al., 2009a)
In this study, patients (aged 19–51 years) with mild to moderate atopic dermatitis ingested
astaxanthin-rich H pluvialis oil, equivalent to 12 mg of astaxanthin dialcohol (Ax group: six men
and eight women) or corn oil (placebo group: six men and seven women) contained in soft capsules, once daily for 4 weeks Before and after administration, evaluations were made on severity (Scoring atopic dermatitis; SCORAD), pruritus (visual analog scale; VAS), quality of life (Skindex-16 and state trait anxiety inventory; STAI), immune function (Th1/Th2 and blood catecholamines), and antioxidative status (urine 8-OHdG and isoprostane) No signifi cant difference in severity and pru-ritus was apparent between the Ax and placebo groups However, the Ax group showed signifi cant amelioration of symptoms evaluated by Skindex-16 and on anxiety state evaluated by STAI The ameliorating effect on anxiety by astaxanthin was supported by the level of the stress hormone, dopamine, which was signifi cantly decreased in the Ax group The level of urine 8-OHdG, which
is reported to be high in patients with atopic dermatitis (Tsuboi et al., 1998), decreased signifi cantly
in the Ax group, indicating that astaxanthin intake has an antioxidative effi cacy Furthermore, the study revealed that there was a statistically signifi cant shift in the Th1/Th2 balance toward Th1 in the Ax group Atopic dermatitis is characterized by Th2-dominated allergic skin infl ammation It is known that higher anxiety in patients enhances the Th2-type response, due to dysregulation of the neuro immune system, leading to worsening of the allergic symptoms (Hashizume and Takigawa, 2006) The fi ndings of this study, which showed that patients in the Ax group showed reduced anxi-ety accompanied by a shift in the Th1/Th2 balance toward Th1, strongly suggests that astaxanthin
is a promising food factor in the management of atopic dermatitis
18.6.2 ASTAXANTHIN AND BRAIN HEALTH
Brain aging, either natural or in neurodegenerative disorders, is also closely associated with tive stress, subsequent damage to cellular components (DNA oxidation/mutation, protein modifi ca-tion/aggregation, and lipid peroxidation), infl ammation, and other factors, including excess calorie intake, insulin resistance, and mitochondrial dysfunction leading to neuron death (Matison et al., 2002; Schon and Manfredi, 2003) In connection with the effect of astaxanthin on the factors involved
oxida-in braoxida-in agoxida-ing, we have observed the followoxida-ing fi ndoxida-ings: (1) astaxanthoxida-in protects neuroblastoma
Trang 12cells from oxidative-stress induced apoptosis in vitro (Ikeda et al., 2008; Tsuji et al., 2008); (2)
in dopaminergic neuron-specifi c manganese superoxide dismutase (MnSOD)-defi cient mice which display Parkinsonian symptoms, oral administration of astaxanthin resulted in improvement of body weight loss and behavior function and hence an increase in life span (Tsuji et al., 2007); (3) astax-anthin improves mitochondrial function by maintaining a high mitochondrial membrane potential, stimulating respiration, maintaining mitochondria in a reduced state even under oxidative chal-lenge (Wolf et al., 2009) In addition, a memory-improving effect of astaxanthin in mice has been reported by other research groups (Hussein et al., 2005; Zhang, 2007) However, there had been no studies investigating the effect of astaxanthin on the higher cognitive function of the human brain
We therefore conducted a preliminary clinical evaluation, an open-label trial using 10 otherwise healthy male subjects (50–69 years of age) who complained of age-related forgetfulness (Satoh
et al., 2009b) Age-associated memory impairment is a common condition characterized by very mild symptoms of cognitive decline that occur as part of the normal aging process (NYU Medical
our study, subjects ingested astaxanthin-rich H pluvialis oil, equivalent to 12 mg of astaxanthin
dial-cohol, contained in soft capsules, once daily for 12 weeks Cognitive function was evaluated before administration (at baseline) and again every six weeks during the study, using either the CogHealth tool (CogState; Melbourne, Australia) or the event-related P300 recognition response elicited by
an auditory task CogHealth is a cognitive function test specifi cally designed to detect changes in healthy or mildly impaired subjects at an early date (Collie et al., 2003) The trial revealed signifi -cant reduction in response time on all tasks in CogHealth: psychomotor speed, impulse control, working memory, episodic learning, and attention, after 12 weeks intake of astaxanthin The accu-racy on the “working memory” task in CogHealth was also signifi cantly improved after 12 weeks of treatment The P300 peak amplitude tended to increase after 12 weeks astaxanthin administration, indicating that astaxanthin might increase patient information processing capacity and selective attention All tasks investigated by CogHealth and P300 are indispensable for a skilful perfor-mance in daily life activities such as driving, which is a complex form of activity involving specifi c cognitive and psychomotor functions Age-related decreases in these tasks are therefore a serious problem, causing an increased number of traffi c accidents and deaths in older people Double-blind, randomized, placebo-controlled clinical trials investigating the effect of astaxanthin on CogHealth tasks and driving performance are currently in progress
18.6.3 ASTAXANTHIN AND METABOLIC SYNDROME
Metabolic syndrome is characterized by a clustering of metabolic risk factors for cardiovascular disease in one person The risk factors include abdominal obesity, insulin resistance, elevated blood glucose, atherogenic dyslipidemia (high tryglycerides, low HDL cholesterol, and high LDL choles-terol), and hypertension (Cornier et al., 2008; Grundy, 2008) Metabolic syndrome is closely associ-ated with oxidative stress and adipose tissue infl ammation, and the improving effects of astaxanthin
on metabolic syndrome have been suggested in several animal models (Uchiyama et al., 2002; Naito
et al., 2004, Hussein et al., 2005; Ikeuchi et al., 2007; Watanabe et al., 2007; Akagiri et al., 2008) and in a clinical study (Satoh et al., 2009b)
We attempted to confi rm the clinical effi cacy of astaxanthin in an open-label uncontrolled study using volunteers at risk of metabolic syndrome (Uchiyama and Okada, 2008) In this trial, a total of
17 volunteers between 22 and 65 years of age (13 men and 4 women) at risk of developing metabolic
syndrome were administered with astaxanthin-rich H pluvialis oil, equivalent to 8 mg of
astaxan-thin dialcohol, twice daily for 12 weeks A signifi cant increase in adiponectin levels and a signifi cant decrease in tumor necrosis factor (TNF)-α levels were observed In addition, a signifi cant decrease
in glycohemoglobin (HbA1c), a diagnostic marker of diabetes, was observed Adiponectin has an antidiabetic effect; it decreases blood glucose and is therefore regarded as a good indicator of meta-bolic syndrome (Matsushita et al., 2006), while TNF-α increases insulin resistance It is generally
Trang 13accepted that the reduced secretion of adiponectin and increased secretion of TNF-α in tional adipose tissue (infl amed adipose tissue with enlarged adipocytes and impaired preadipocyte differentiation) increase insulin resistance in the pathogenesis of metabolic syndrome (Gustafson
dysfunc-et al., 2007) Our results showed that astaxanthin intake increased adiponectin levels and decreased TNF-α levels, which strongly suggests that astaxanthin has a positive effect on the proinfl ammatory state of dysfunctional adipose tissue and hence on elevated blood glucose This is confi rmed by a signifi cant decrease in glycohemoglobin (HbA1c) in humans at risk of metabolic syndrome
While the clinical effi cacy was in the process of evaluation by administrating astaxanthin as
an encapsulated oil-form in the studies above, we investigated the effect of astaxanthin on body fat in human subjects by using an astaxanthin-containing beverage as a test material We prepared the test beverage using water-mixable astaxanthin oil The study was a randomized, double-blind, placebo-controlled, parallel-group comparison trial, performed to conform with the Helsinki dec-laration A total of 36 volunteers, aged between 20 and 65 years (41.0 ± 7.3 on average), with a high body mass index (BMI) of 25 or more, were administered 50 mL of the beverage containing 0 mg (placebo group) or 6 mg of astaxanthin dialcohol (astaxanthin group), once daily for 12 weeks Body fat was evaluated by measuring abdominal visceral fat areas (VFA), subcutaneous fat areas (SFA), and total fat areas (TFA) using a computerized tomography (CT) scan, before (baseline) and after 8 and 12 weeks of dosing The statistical differences in VFA, SFA, and TFA between baseline
and postdosing values were determined by paired t-tests, while those between the placebo group and astaxanthin group were tested by unpaired t-tests, using SPSS for Windows (ver 13.0J; SPSS Inc., Chicago, Illinois, USA) Differences with p < 0.05 were considered signifi cant The time-course changes in VFA, SFA, and TFA are shown in Figure 18.7 The astaxanthin group showed a marked reduction in TFA in comparison with the placebo group, with signifi cant differences after eight weeks dosing VFA in the astaxanthin group was signifi cantly reduced after eight weeks when compared with the placebo; no signifi cant difference was observed after 12 weeks, possibly due to fat homeostasis A marked reduction in SFA was also observed in the astaxanthin group after 8 and
12 weeks when compared with the placebo In addition, no adverse effects, laboratory ties, or adverse events attributed to administration of the test material were apparent among the vital signs, laboratory analysis (hematology, hepatic, and renal function tests), and subject interviews
FIGURE 18.7 Time course changes in TFA (a), VFA (b), and SFA (c) in the astaxanthin group (closed
squares) and placebo group (open triangles) The differences in TFA, VFA, and SFA between respective line values and after treatment values are expressed as Δcm 2 The number of subjects was 26 and 10 in the
base-astaxanthin and placebo group, respectively *p < 0.05.
Trang 14These results suggest astaxanthin could be safely administered as a beverage for use in body fat management Further clinical studies remain to be performed to confi rm our fi ndings.
In this chapter, we discussed our indoor photobioreactor, a highly controlled microalgal cultivation technology, with high sterility, light illumination effi ciency, and therefore high and stable biomass productivity on a commercial scale Although microalgae have been recognized as promising organ-isms to provide sources of natural products in functional foods in the nutraceutical and pharmaceuti-cal industry, only a few algal strains and their products have been successfully marketed so far, as described in Section 18.2 Our microalgal cultivation technology, using an indoor photobioreactor, is considered to be competitive in the production of high-value bioactive metabolites with low cellular content The use of sterilized, high-density algal suspensions can be advantageous in downstream processing such as concentration and extraction processes, particularly for neutraceuticals and phar-maceuticals obtained from microalgae which are diffi cult to culture outdoors Another possible appli-cation of our technology seems to be the cultivation of genetically engineered microalgae that will most likely be banned from outdoor cultivation systems Transgenic algal strains that express good concentrations of several recombinant therapeutic proteins have already been successfully generated (Mayfi eld et al., 2007; Fletcher et al., 2007; Siripornadulsil et al., 2007) Further improvements of our cultivation technology, as well as innovations of other important technologies such as solar electricity and low-cost reactor materials, are needed to develop more competitive and economically feasible algal biomass production systems Together with fi nding useful algal strains and their metabolites, the innovations of our cultivation technology and other cultivation-related technologies will bring advancement and expansion to microalgal business opportunities in the human health industry
ACKNOWLEDGMENT
The authors wish to express their sincere gratitude to Dr Shigetoh Miyachi, Professor Emeritus, University of Tokyo, for his kind supervision and contributions to the Yamaha Motor Life Science Institute
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