Aphanizomenon flos-aquae as a Source of Dietary

Chapter 10

Blue-Green Alga Aphanizomenonflos-aquaeas a Source of Dietary Polyunsaturated Fatty Acids and a Hypocholesterolemic Agent in Rats Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 12, 2018 | https://pubs.acs.org Publication Date: March 27, 2001 | doi: 10.1021/bk-2001-0788.ch010

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Christian Drapeau , Rafail I. Kushak , Elizabeth M. V a n Cott , and Harland H. Winter 2

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Cell Tech, 1300 Main Street, Klamath Falls, OR 97601 Pediatric GI/Nutrition, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, V B K 107, Boston, MA 02114-2698 Current address: Research and Development, Desert Lake Technologies, 12750 Keno-Worden Road, Keno, OR 97627 3

The blue-green alga Aphanizomenonflos-aquaeis shown to be a good source of omega-3 polyunsaturated fatty acids in rats. It was able to partially reverse the effects on digestive functions induced by a deficiency in polyunsaturated fatty acids. Aphanizomenonflos-aquaeis also reported to significantly decrease total cholesterol and triacylglycerol levels.

Algae has been used worldwide as a food source or as a remedy for various physical ailments for thousands of years (7,2). In coastal regions of the Far East, notably in Japan, there is evidence that algae were used as a food source 6000 BC, and there are records of many species of seaweed used as food around 900AD (3). Many reports at the time of the Spanish conquest reveal that the natives of Lake Tezcoco, near the city of Tenochtitlan (today's Mexico city), collected blue-green algae from the waters of the lake to make sun-dried cakes called Tecuitlati, or "excrement of stone" as they believed stones were the source of algae (4,5). Still today in Africa, local tribes harvest blue-green algae growing in Lake Chad which is used to make hard cakes called Dihé (4). Similar to the inhabitants of Tenochtitlan, patches of floating microalgae are collected and sun-dried in shallow holes dug in the sand along the shore of the lake.

© 2001 American Chemical Society

Shahidi and Finley; Omega-3 Fatty Acids ACS Symposium Series; American Chemical Society: Washington, DC, 2001.

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Downloaded by UNIV OF CALIFORNIA SANTA BARBARA on March 12, 2018 | https://pubs.acs.org Publication Date: March 27, 2001 | doi: 10.1021/bk-2001-0788.ch010

126 For the past seventeen years Aphanizomenon jlos-aquae (Aph.flos-aquae),a naturally-occurring blue-green alga growing in Upper Klamath Lake, Oregon, has also been harvested and sold as a high protein nutritional source. Nearly one million consumers have been eating Aph. jlos-aquae over the past few years, and many anecdotal reports suggest thatAph.flos-aquaemight have health-promoting properties. Recently, it was shown that consumption of Aph.flos-aquaetriggers within two hours the migration of natural killer cells from the blood to the tissues, as well as a significant increase in the expression of adhesion molecules on the surface of natural killer cells (6). Such an effect was later confirmed and shown to be much more pronounced in regular consumers than in people exposed to Aph. flos-aquae for the first time (7). Aph.flos-aquaewas also shown to be useful as an adjunct to the treatment of mild traumatic brain injury, likely by facilitating neuroplasticity (8). Finally, a retrospective epidemiological study suggested that Aph.flos-aquaemay be beneficial in the treatment of various conditions such as attention deficit disorders,fibromyalgia,hypertension and chronic fatigue (P). Currently, the market for Aph.flos-aquaeas a health food supplement is close to $100 million in sales with an annual production exceeding 100 metric tons of dried algae. The present contribution would provide a brief review of the techniques of harvesting and processing of naturally-growing .4/?/?.flos-aquaeand reports the results pertaining to the effect of Aph.flos-aquaeon blood lipids.

Klamath Lake Ecology and Aph.flos-aquaeBloom 2

Upper Klamath Lake is the largest freshwater lake (324 km ) in Oregon and drains a watershed of9800 km . This shallow lake (ave. depth = 2.4 m) is flanked by the Cascade mountains to the west and the Great Basin to the east. The lake has two major tributaries, the Williamson and Wood Rivers, as well as many smaller springs and stream inflows. Historically, the lake has always been extremely productive and has supported not only a tremendous biomass of algae, but also fish, waterfowl, and predatory bird species. When ice was first collected from the lake (1906) it was reported to be green with algae (10). Lake suckers were so common that people used pitchforks to harvest them. Ospreys were reported in densities of up to 10 nests per square mile. Today the Klamath Basin is still home to the largest wintering congregation of bald eagles in the lower 48 states, and is the largest stopover for waterfowl in the Pacific flyway. Records of the dynamics of algal blooms in Klamath Lake have been maintained over the past 8 years and have established Aph. flos-aquae as the predominant algal species growing in the lake (11,12). Typically, Aph. flos-aquae starts blooming in early June with an initial biomass of 3-15 mg/L and remains the 2


127 dominant species until November, with a biomass sometimes reaching more than 100mg/L.


Harvesting and Processing of Aph flos-aquae One of the main harvesting facilities is located along an aqueduct system, approximately 14 kilometers from the southern end of Klamath Lake, at the site of a previous low head hydroelectric turbine facility with a 3m drop. This feature is utilized to feed water onto screens by gravity. The flow at this location averages 1.8 million liters per minute and is entirely processed for algae removal by dispersing the water over a total of 48 screens. This is the site of initial dewatering. A water spray assembly is located atop and across each screen and is manually operated to move the collected algae to a trough where it is pumped to a secondary vibrating filter screen. This screen is used to remove any unwanted macroscopic debris. The algae is then pumped to a series of slow speed horizontal centrifuges for the final removal of small extraneous material. This processing stage utilizes the fact that A flos-aquae has a specific gravity almost that of water, allowing for the separation of sand, silt, and any other light debris. The liquid algae concentrate is then pumped to a vertical centrifuge that applies high G-force to separate algal cells and colonies from water, removing virtually 90% of the remaining water. Once concentrated, the algae is immediately chilled to 5°C and stored in a chilled and agitated tank before being pumped to flake freezers to be instantaneously frozen. When needed, the frozen algae is shipped to an external freeze drying facility to be freeze dried into an algal powder containing 3-5% water content. This final product is processed into consumable capsules or tablets as needed. The other main harvesting process takes place directly in the algal blooms in the deep waters of Upper Klamath Lake, toward the Northern end of the Lake. Harvesting directly in the lake allows for a harvesting season that endures until late November. In brief, small harvesting platforms make passages in newly formed algal blooms that gather in large, thick patches at the surface. These harvesters are equipped with rotating screens that lift the algae from the water surface and transfer it on a screened conveyor system to perform the initial dewatering step. The algae is then transported to a 800 m processing barge located in close proximity to the blooms. On this platform the algae is slowly pumped through a tube and shell heat exchanger that brings the concentrated algae to a temperature of 5° C. The algae is then stored in refrigerated tanks. At this point, the algae isfilteredthrough a centrifugal sieve to remove debris and certain species of algae such as Microcystis. The algae is then passed through a concentrating system to further concentrate the liquid algae. The concentrated 2


128 algae is then transported to shore in a refrigerated tank and then trucked to a drying facility to be driedfresh.The powder is finally processed into consumables.


Ability of Aph.flos-aquaeto Provide Essential Lipids Over the past 15 years, many empirical reports have been made on the beneficial effect of Aph.flos-aquaeon conditions such as depression, attention deficit disorders, epilepsy, skin diseases, rheumatism, Crohn's disease and other inflammatory disorders. The occurrence of many of these conditions have been associated with a deficiency in polyunsaturated fatty acids (PUFA), mostly omega3 fatty acids. In brief, omega-3 fatty acids have been shown to improve arthritic and other inflammatory conditions (13). Epidemiological studies suggest that decreased omega-3 fatty acid consumption correlates with increasing rates of depression (14). Attention deficit disorder has been associated with a decrease in blood PUFA (15). Skin conditions such as eczema have been associated with altered blood levels of PUFA and oral PUFA has been shown to improve such conditions (16). Omega-3 fatty acids prevent platelet aggregation (17,18), arrhythmia and ventricular fibrillation (19,20), and protect myocardial cells against hypoxiareoxygenation-induced injury following ischemic heart disease (21). Eicosapentaenoic acid (EPA) was also shown to prevent epileptic seizures in a rat model of epileptogenesis (22,23). Finally, omega-3 PUFA have been shown to lower cholesterol, possibly by stimulating its excretion into bile (24,25). PUFA constitute 30% to 50% of the lipid content of dried Aph. flos-aquae (5% to 9% of total dry weight), with the main contributor being alpha- linolenic acid. It was hypothesized that the PUFA content of Aph. flos-aquae might be mediating some of the reported health benefits. Experiments were designed to assess the bioavailability of the PUFA contained in Aph. flos-aquae. Here we report the results of experiments using rats, in which Aph. flos-aquae proved to be not only a good source of PUFA, but also lowered blood cholesterol and triglyceride levels.

Methods Animals. Thirty-two adult male Sprague-Dawley rats were randomly distributed into 4 groups. Animals were placed into individual wire cages, and maintained at 22°C with a 12-hour light-dark cycle. Food and water were supplied ad libitum. Animals were fed for 32 days with the following semipurified test diets (Table I) based on the American Institute of Nutrition (AIN-76) standards: 1) Standard diet


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Table L Fatty acid profiles of selected oils. Fatly Acid (%) Caprylic acid Capric acid Laurie acid Myristic acid Palmitic acid Palmitoleic acid Heptadecanoic acid Stearic acid Oleic acid Linoleic acid (LA) Linolenic acid (LNA) Arachidic acid Arachidonic acid (AA) Eicosapentaenoic acid (EPA) Behenic acid

Soybean 0 0 0 0 14.69 0 0 5.4 26.8 44.4 8 0.35 0 0 0.33

Coconut 9.7 7.5 42.1 22.4 18.2

Polyunsaturated (PUFA) Monounsaturated (MUFA) Saturated (SFA)

52.4 26.8 20.77

0

Alga

9.1 36.6 11.9 0.89 2.7 6.7 7.4 22.3

0.14 0.65 0.08

100.04

30.43 18.6 49.29

SOURCE: Fromref. 28.

containing 5% soybean oil (SBO); 2) PUFA deficient diet containing 5% coconut oil (PUFA-D); 3) PUFA-D diet containing 10% algae (0.50% algal lipids) (Algl0%); and 4) PUFA-D diet containing 15% algae (0.75% algal lipids) (Algl5%). The algal material used in this study was supplied by Cell Tech (Klamath Falls, OR) and contained 6.29% lipids. Feed was provided by Purina Test Diets (Richmond, IN). The weight of the animals was monitored prior to and during the 32 day feeding trial. After the feeding trial, the animals were fasted overnight and euthanased by carbon dioxide inhalation. Viscera (liver, kidney, small and large intestine, pancreas, cecum and spleen) were collected and frozen in liquid nitrogen and kept at -80° C for further biochemical testing. Plasma was collected by heart puncture in a tube containing 100 μΐ 0.5 M ethylenediaminetetraacetic acid (EDTA; pH 8.0), spun at 3,000 g for 15 minutes, and stored at -80°C.



130 Fatty Acid Analysis. Blood plasma fatty acid analysis was performed using direct transesterification method (26,27). Fatty acid identification was performed using external and internal standards on a Hewlett-Packard 5890 series II model gas chromatograph-mass spectrometer GC-MS with a Hewlett-Packard 5971 mass spectrometer (Hewlett-Packard, Wilmington, DE). Soybean and coconut oils were methylated before analysis. The algal material was soaked in methanol, extracted and then methylated before analysis. Plasma triglycerides and cholesterol were measured using Boehringer Mannheim kits on the automated clinical chemistry analyzer Roche BHO/H917.

Statistical Analysis. For the various parameters, statistical difference between groups was determined using unpaired Student's Τ test. Differences in fatty acid profiles were determined using univariate and multivariate repeated measures analysis. For both analyses, differences of p

Aphanizomenon flos-aquae as a Source of Dietary

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