Klamath Lake Aphanizomenon Flos-Aquae: Wild-Harvesting, Extracts and Benefits

Abstract

This chapter examines Aphanizomenon flos-aquae (AFA) from Oregon’s Klamath Lake, emphasizing its nutritional richness and health benefits. Thriving in a unique volcanic ecosystem, this wild-harvested cyanobacterium is a powerhouse of nutrients, making it a prime focus in the health supplement domain. The chapter highlights AFA’s comprehensive nutritional profile, packed with proteins, essential amino acids, vitamins, minerals, and bioactive compounds. Special attention is given to AphaMax® and Klamin®, two AFA extracts with significant nutraceutical potential. AphaMax®, rich in AFA-phycocyanins, shows strong antioxidant, anti-inflammatory, wound-healing and anti-cancer properties. Klamin®, containing β-phenylethylamine (PEA), is notable for its mental health benefits, particularly in alleviating depression and anxiety, and shows promise in ADHD treatment and neurodegenerative disease management. In essence, the chapter underscores the importance of AFA from Klamath Lake as a key natural resource in the nutritional supplement industry, owing also to its potent health-promoting extracts.

1. Introduction

Cyanobacteria, also commonly known as blue-green algae, constitute a diverse group of ancient photoautotrophic prokaryotes [1]. These photosynthetic life forms played a pivotal role in Earth’s evolutionary history during the oxygenic revolution around 2.5 billion years ago, contributing significantly to the release of oxygen into the atmosphere [2]. Displaying a remarkable diversity of morphological forms, cyanobacteria range from unicellular entities, like Synechococcus sp., to intricate filaments, exemplified by Anabaena sp., and colonial structures, found in Microcystis sp. [3]. Furthermore, these microorganisms have adapted to a wide array of habitats, from freshwater bodies, like Planktothrix sp., to oceans, like Trichodesmium sp., to terrestrial and desert-like ones, as observed with Chroococcidiopsis sp [3].

Beyond their ecological significance, cyanobacteria have been studied for various applications, such as for the production of biofuel or for wastewater treatment [4, 5]. However, it is in the realm of nutritional and nutraceutical supplements that cyanobacteria truly shine. Athrospira sp., colloquially known as Spirulina, has gained prominence as a nutritional powerhouse and has become a staple in the health supplement industry [6]. Its nutritional density and ability to be cultivated in open ponds/photobioreactors make it an attractive choice for those seeking dietary enhancements [6]. Beyond Spirulina, a singular Aphanizomenon flos-aquae (AFA) strain, Klamath AFA, has carved its niche in the supplement industry, owing to its superior nutritional and nutraceutical benefits, and it is directly harvested wild from Upper Klamath Lake (UKL), in Oregon, U.S.A [7].

Klamath AFA is a nitrogen-fixing obligate phototroph composed of cylindrical cells that self-assemble into filaments (Figure 1) [7]. It finds its taxonomical place within the order of the Nostocales and is the only Nostocales strain, alongside a few Nostoc sp., like N. commune and N. flaggeliforme, to be regularly consumed as a dietary supplement [8, 9]. AFA is well known for forming fascicles, filaments that self-aggregate together into leaf-like structures which can be observed with the naked eye [7]. Furthermore, unlike Spirulina, AFA features specialized vegetative cells, known as heterocysts, which house the active nitrogenase enzyme responsible for converting atmospheric nitrogen (N²) into ammonia [10]. Additionally, AFA forms akinetes, dormant cells that serve as spore-like structures, allowing the cyanobacterium to withstand unfavorable conditions and germinate into new vegetative cells when environmental conditions become favorable again [11].

Figure 1.

Microscope image of a Klamath AFA fascicle, illustrating AFA filaments, each of which is composed by cylindrical vegetative cells. The white arrow indicates a heterocyst. Magnification: 40×.

AFA thrives in nutrient-rich lakes or ponds, which contribute is its ability to form blooms, dense mats of biomass that blanket the surface of lakes. Particularly renowned are the Klamath AFA blooms that grace UKL in Oregon, USA [7]. It is this biomass that is then harvested, processed and distributed as a nutritional supplement across the planet. UKL is the only lake in the world to be harvested for AFA and AFA is the only commercially distributed cyanobacterial supplement to be harvested from the wild at an industrial scale [12].

AFA boasts a comprehensive nutritional profile: it is rich in proteins, up to 70% of total biomass, containing high amounts of all essential amino acids [9]. It contains all the vitamins, many of which at high concentration. It’s a good source of polyunsaturated fatty acids (PUFAs), circa 75% of which are made of anti-inflammatory Omega-3 s [13]. Finally, thanks to the mineral richness of Klamath Lake’s sediment, AFA provides 73 bioavailable minerals, including the complete spectrum of trace minerals [12]. The distinct biochemical profile of this substance is characterized by the presence of bioactive molecules. These include a spectrum of carotenoids, including the main xanthophylls, and an abundant concentration of chlorophyll [12]. Additionally, it contains a unique form of AFA-phycocyanins, notable for their potent antioxidant, anti-inflammatory, and anti-cancer properties, as well as their efficacy in promoting wound healing [14]. Furthermore, this composition encompasses phenylethylamine, a compound recognized for its role as a neuromodulator and its capacity to modulate the immune system [15]. Two important AFA extracts, known respectively as AphaMax^® and Klamin^®, concentrate such molecules, and have shown, in many clinical studies, to produce beneficial effects on a number of diseases.

2. Industrial importance

Circa 120 distinct AFA strains have been confirmed globally [12]. However, the strain consumed as a nutritional supplement is found thriving in Klamath Lake, Oregon, USA. This particular strain is identified by its specific name, Aphanizomenon flos-aquae Ralfs ex Born. & Flah. Var. flos aquae [7]. Klamath AFA nutritional supplements have emerged as a prominent player in the health supplement industry over the past 35 years [7]. Since then, the AFA nutritional supplement market is estimated to have reached an overall value of around $100 million a year, indicating a robust consumer interest in AFA’s unique benefits [12]. Furthermore, while the majority of Klamath AFA’s market is predominantly limited to its use as a nutritional supplement, it is now also expanding into novel areas as well, such as the cosmetic and beauty industry. As the AFA industry grows and the availability of its biomass made easier, AFA-based products are expected to enter numerous other markets as well, such as the animal feed, aquaculture and biofertiliser ones [12]. The latter holds particular promise because AFA stands as the only microalgae or cyanobacterium harvested at scale with the unique capability of nitrogen fixation.

2.1 Harvesting and processing

Klamath AFA wild harvesting occurs in Klamath Lake, Oregon, USA, and takes advantage of the unique ecological conditions of this natural habitat (Figure 2) [7]. Typically 500–1000 t are harvested per year [12]. The harvesting process is strategically conducted during the AFA bloom period, typically occurring in early summer, specifically between the end of June and the beginning of July, and then again in late summer and early fall, between September and November, when environmental factors such as temperature and nutrient availability are optimal [12]. Harvesting begins with the identification of specific areas within the lake where AFA concentrations are high. The desired concentrations range between 5 and 7% solids. Harvesters typically use specialized boats equipped with fine mesh nets or other non-intrusive filtration systems to gently collect the AFA from the lake’s surface [7].

Figure 2.

Klamath Lake, its bloom and the associated harvesting barges. The top image illustrates Klamath Lake with Mount Shasta in the background. The bottom left image is an example of a Klamath AFA bloom, while bottom right of an AFA harvester.

Once harvested, the AFA biomass is transported to processing facilities near the lake. In the processing phase, the collected AFA undergoes meticulous cleaning processes to remove any impurities or debris, ensuring the purity of the final product. Subsequently, the AFA is dewatered in a two-step process: firstly, the majority of the water is removed via a non-specific centrifugation process, and subsequently, it is carefully dried, often utilizing methods like air drying or low temperature drying to preserve its nutritional profile [7]. It is at this moment that the high-value AFA-phycocyanin and phenylethylamine compounds are concentrated. High-grade phycocyanin is concentrated through a water-based filtering centrifugation process (Patent: EP2032122A2); while PEA and synergic molecules, such as mycosporine-like aminoacids, are concentrated through water ultrafiltration (Patent: EP2046354B1). The obtained AFA biomass and extracts are then commonly transformed into various consumer-friendly forms, such as powders, capsules, or tablets, making it suitable for use as a nutritional supplement [12].

2.2 Wild growth properties

AFA exhibits prolific growth and blooms in Klamath Lake due to a combination of specific ecological factors that create an optimal environment for its thriving population. Klamath Lake measures approximately 52 × 12 km with an approximate surface area of 250 km² [16]. Nevertheless, it is a fairly shallow lake, with a mean average depth of 2.4 m. Situated at an altitude of 1300 m, Klamath Lake is bordered by the Cascade Mountains to the west and lies adjacent to a desert area, the Great Basin, to the east [7, 16]. Furthermore, the pristine waters of Klamath Lake are home to a number of different animal species, from the common Sucker fish to Pelicans and Bald Eagles. Currently, the Klamath Basin serves as the primary wintering destination for the most substantial gathering of bald eagles in the contiguous 48 states. Moreover, it functions as the largest rest area for waterfowl along the Pacific flyway [7].

Klamath Lake consistently witnesses numerous AFA blooms each year, attributed primarily to its geological origins as a volcanic basin [17]. The lake’s geological history traces back 7700 years to the eruption of Mount Mazama, which left substantial mineral sediments at the lake’s bottom. Following the explosion, Mount Mazama’s crater, now known as Crater Lake, was formed and continues to supply Klamath Lake with water [7]. The mineral-rich composition of Klamath Lake, including elements like Iron, Magnesium, Manganese, Molybdenum, Boron, and Zinc, is a fundamental factor contributing to the formation of AFA blooms [18]. These minerals play vital roles in supporting AFA’s biological activities; for instance, Molybdenum is crucial for the development and function of the nitrogenase enzyme specific to heterocysts, essential for AFA’s nitrogen-fixation capability [19].

The three most important nutrients for AFA’s growth, however, are Carbon, Nitrogen and Phosphorous sources [18]. The former is provided by the lake itself due to the occurrence of methane springs across the lake and, of course, of decomposing organic matter. Carbon is expected to compose circa 50% of AFA’s total biomass [20]. AFA’s consumption of dissolved CO₂ is also reflected in the rise of lake pH from around 7.5 at the start of a bloom to around 9–10 towards the end [18]. CO₂ + water is known to form carbonic acid, thereby lowering the overall pH. Its removal, therefore, leads to the opposite. Nitrogen availability, instead, is not of real concern when it comes to the formation of AFA blooms as the cyanobacterium possesses the ability to fix nitrogen directly from the air and convert it into bioavailable forms, like nitrates or nitrites [21]. This ability favors its growth over other organisms, which cannot (see below), allowing it to completely dominate the lake under low-nitrogen conditions [22]. Nevertheless, nitrogen-fixation is costly, from an energetic point of view, and the bioavailability of nitrates/nitrites/ammonia certainly enhances its overall growth rate [23]. The latter nutrient, phosphorous, is most likely the determining factor underlying the amount and frequency of AFA blooms, as it would not be able to grow without it. It comes to no surprise that there is an increase in both nitrogen and phosphorous levels in the spring, right before AFA starts to bloom [17, 18].

Another critical reason for the formation of AFA blooms is that Klamath Lake receives 300 days of sunshine per year, a key factor for the growth of a photosynthetic organism [7]. AFA actually possess the capacity to control its buoyancy and height within the water column by generating bubbling vesicles that allow it to float up and down [24]. The consequence is of this is each cell is able to optimize the amount of light needed. Furthermore, Klamath Lake also offers optimal temperatures for AFA growth and life cycle. During its blooms, water temperatures will range anywhere between 18 and 25°C [18]. Laboratory studies have found that AFA’s optimal growth temperatures for growth range between 20 and 28°C, with a faster growth rate observed between 25 and 28°C [25]. Furthermore, AFA’s akinites are able to survive at the lake’s sediment during the winter, which can see Upper Klamath Lake freeze over [26].

2.3 Contamination and toxicity

The occurrence of M. aeruginosa within the lake has heavily impacted the Klamath AFA harvesting business, especially after the government of Oregon introduced the limit of 1 μg of microcystins per gram of cyanobacteria [7].

Microcystins are potent cyclic peptides that, when consumed in elevated quantities, disrupt protein synthesis within liver cells, leading to cellular death and, potentially, organ failure [27]. As a result, Klamath Lake microcystin outbreaks have triggered public health advisories, warning against water contact and consumption. The World Health Organization (WHO) has established a suggested drinking water guideline value of 1 μg/L [28]. Similarly, due to the possible contamination of these toxins within AFA blooms, the government of Oregon has imposed an incredibly stringent limit of 1 μg/g of microcystins in Klamath AFA supplements [29]. As a result, quality control of AFA blooms for microcystin presence has become an important concern. This has not only caused harvesting companies to offhold any AFA harvest during the occurrence of M. aeruginosa, but has also led to microcystin AFA biomass testing throughout the harvesting and processing stages. This has increased overall costs and any biomass testing higher than 1 μg/g is, for the most part, unusable [12]. Furthermore, microcystin contamination and the associated public concern has also impacted the reputation of AFA-based products. Several studies have in fact questioned the safety of AFA nutritional supplements, based upon this 1 μg/g requirement [30, 31]. However, there is abundant data to show that the imposed threshold is wrong and should be revisited.

The government of Oregon decided upon this limit by directly translating the WHO limit (1 μg/L) of microcystin in drinking water to Klamath AFA biomass. As pointed out by a recent article, there are deep flaws both in this decision, and in the WHO standard [29]. Firstly, the WHO limit is too stringent as it is based upon Fawell et al.’s study which looked at the toxicity (liver damage) of purified microcystins to mice via gavage administration [32]. The methodology employed is not representative of real-world human exposure to these cyanotoxins. Moreover, purified microcystins are not found in nature. Furthermore, the gavage model bypasses the complex interplay between microcystins and the digestive system’s protective barriers [29]. While gavage delivers the toxin directly to the gastrointestinal tract, it bypasses the initial enzymatic and acidic breakdown in the stomach, potentially overestimating the bioavailability and subsequent hepatotoxicity observed in real-world scenarios—like that of natural cyanobacterial blooms [29]. Stomach acids and intestinal enzymes inactivate the toxins, almost completely reducing their potency [33]. Unfortunately, most animal studies on microcystin toxicity have been done through the gavage or intraperitoneally injection, and very few reproducing, normal oral ingestion. Studies comparing oral and intraperitoneal administration of microcystins show substantial differences in toxicity [32, 33]. For instance, the other two studies considered by the WHO panel, both of which performed through normal ingestion, generated, for a human being of 60 kg, a safe chronic limit of 45 μg/day and 8.4 μg/day, respectively [34, 35]. The EPA, in fact, recommends, for water consumption, a safety limit of 8 μg/L [36].

This begs for a cautious interpretation of microcystin-related research. Exaggerating the hepatotoxic risks based solely on intravenous studies might create unnecessary public fear and potentially misdirect valuable resources. It is also important to notice that there has been only one animal study investigating the potential toxicity of microcystins within Klamath AFA biomass: mice were administered a diet of AFA whole-cell biomass containing 333 μg/g of microcystins over a duration of six months. Subsequent evaluations revealed optimal health conditions, including liver health. The researchers, after implementing all pertinent safety thresholds, deduced that a daily intake of 10 μg/g of microcystins from AFA cyanobacterial supplements would be considered safe for an individual weighing 60 kg [28]. Finally and importantly, no human toxicity has ever been reported, even though this biomass has been consumed for decades.

3. Nutraceutical properties

In virtue of its unique ecological setting, wild-harvested Klamath AFA emerges as an exceptionally nutrient-dense food source, surpassing many other foods and superfoods in absolute nutritional richness [12]. Its remarkable nutritional profile encompasses all 14 essential vitamins, featuring high concentrations of pro-vitamin A carotenes, substantial levels of B vitamins, crucial for homocysteine regulation, and vitamin K, pivotal for bone health, dental well-being, and blood coagulation [12, 13]. Klamath AFA stands out with a mineral content of 73 minerals and trace elements, including noteworthy amounts of iron, natural fluorine, and vanadium, essential for insulin metabolism and addressing metabolic syndrome [12]. Furthermore, it serves as a notable source of Omega-3 fatty acids, with a high quantity of alfa-linoleic acid [13]. It boasts an extensive array of carotenoids, encompassing significant xanthophylls like lutein, canthaxanthin, and lycopene [12]. Recent revelations highlight Klamath AFA’s richness in polyphenols, featuring a diverse and potent assortment of nutraceutical molecules, including a high quantity of chlorophyll [12, 13, 37]. See Table 1 for a summary of AFA’s nutritional profile.

Properties	Amount/types	Description
Amino Acids	All 20 amino acids	Essential for life as they are the building blocks of proteins
Protein	~65% of dry mass	Necessary for energy metabolism, all cellular processes and tissue homeostasis
Vitamins	All 14-vitamins: A, K, B1, B3, B5, B9 and B12 are at RDA-relevant amounts	These are necessary nutrients that perform a myriad of essential tasks, from wound healing to bolstering of the immune system
Minerals	All 73 minerals and trace minerals – vanadium, iron, fluoride, iodine, molybdenum, at RDA-relevant amounts
Chlorophyll	~4% of dry mass	Linked to anti-inflammatory and anti-cancerous effects
Carotenoids	High concentration of canthaxanthin, lutein, and lycopene, plus astaxanthin and zeaxanthin	Lutein possesses eye protection and age-related macular degeneration prevention properties; canthaxanthin is a powerful antioxidant; lycopene is a neuroprotectant
Polyphenols	Caffeic, vanillic and hydroxytyrosol acid	These ameliorate GI tract issues and help prevent the onset of certain metabolic, cardiovascular, and neurodegenerative diseases.
MAA’S	High concentrations of porphyra and shinorine	Linked with antioxidant, immunomodulatory, and anticoagulant activities
PUFA’S	~12–15 mg/g of an omega-3 fatty acid, alfa-linoleic acid (ALA).	ALA reduces the onset likelihood of cardiovascular diseases, IBS, rheumatoid arthritis, and neurodegenerative pathologies. Furthermore, they decrease cholesterol (low-density lipoproteins)
AFA-PC	~60–100 mg/g of biomass	See Section 3.1
PEA	~3 mg/g of biomass	See Section 3.2

Table 1.

Summary of Klamath AFA’s nutritional profile.

3.1 C-phycocyanin and phycoerythrocyanin

The exceptional anti-inflammatory and antioxidant properties associated with AFA and cyanobacteria-based supplements predominantly arise from the intricate composition of pigments, specifically chlorophyll, C-phycocyanin (C-PC), and phycoerythrocyanin (PEC) [38]. Despite comprising around 4% of AFA’s total biomass and impacting its taste, chlorophyll proves beneficial in reducing inflammation, aiding in weight loss, and preventing cancer [12, 39]. In the realm of cyanobacteria, AFA, much like Spirulina, encompasses both C-phycocyanin (C-PC) and allophycocyanin (AP). Although a 2004 study suggests AFA’s phycocyanin content at 15% of the total biomass, recent estimates propose a range of 6–10% [14]. The therapeutic efficacy of C-PC is attributed to their bioactive chromophore, phycocyanobilins (PCB) [40]. Animal studies showcase the effectiveness of C-PC in reducing in vivo edema induced by oxidizing factors and inhibiting liver lipid oxidation caused by hepatotoxic chemicals [41]. As powerful anti-inflammatory agents, C-PC inhibit molecules like NO and, notably, COX-2, serving as selective COX-2 inhibitors without the side effects observed in common NSAIDs [12, 42]. Their reversible antagonism on platelets preserves platelet survival [43]. Furthermore, in-depth in vitro studies validate the potential of C-PC in inhibiting cancer cell proliferation across various tumor cell lines [44]. A comprehensive review affirms their role in cancer treatment, influencing cell cycle arrest, activating apoptotic pathways, and modulating cancer-promoting and fighting molecules [45, 46]. C-PC’s diverse effects extend to cardiovascular improvements, wound healing, and immune enhancement, including the normalization of cholesterol levels, platelet aggregation inhibition, cardioprotective roles, fibrinolytic activity, fibroblast release stimulation for wound healing, and immune system support (Figure 3) [12, 43].

Figure 3.

Summary of AphaMax® and Klamin® nutraceutical properties.

AFA, unlike Spirulina, also expresses the light-harvesting phycoerythrocyanin (PEC) pigment within its phycobilisome. PEC displays a unique chromophore attachment compared to C-PC, one phycoviolobilin (purple color) chromophore instead of a phycocyanobilin (blue color) [47, 48]. More specifically, both C-PC and PEC are both heterodimeric proteins comprising of monomers that are made of two distinct subunits, α and β. Each αβ monomer typically binds three chromophores. In the case of C-PC each αβ monomer binds three phycocyanobilins, while for PEC it binds two phycocyanobilins and one phycoviolobilin [47, 48, 49]. Importantly, the exact nutraceutical properties of phycoviolobilin are yet to be studied. Crucially, the Klamath AFA phycocyanin extract, known as AphaMax®, combines both C-PC and PEC, as the industrial extraction methodology employed cannot discern between the two due to their overall similarity [42]. Recent research has shown that the AFA-phycocyanin extract, AphaMax®, exhibits superior antioxidant and anti-inflammatory responses compared to Spirulina’s C-PCs, potentially suggesting that AFA C-PC’s activity is enhanced by PEC [12, 14]. For example, in-depth in vitro studies on lipid oxidation showcase AphaMax®‘s ability to achieve a 50% inhibition of malonyldialdehyde (MDA), a late by-product of lipid peroxidation, with a dosage 75x lower than Spirulina’s PCs (0.14 nM vs. 11.35 μM) and a 90% inhibition 200x lower (1 μM vs. 200 μM) [14]. In terms of inflammation, an unpublished in vitro study on COX-2 enzyme acitivity inhibition, reveals that, at human intake dosages (250 mg), AphaMax® inhibits COX-2 activity by 65%, while Spirulina C-PCs by 40%. Overall, there is a clear need to investigate the nutraceutical properties of PEC to better assess the potential nutraceutical impact of AphaMax®.

Numerous other studies have also highlighted AphaMax® anti-inflammatory antioxidation, anti-cancer properties. AphaMax® has the highest oxygen radical absorbance capacity (ORAC) value among all purified molecules, about 300x higher than even quercetin and epigallocatechin [14, 43]. Comparative studies show that while quercetin, at 10 μM, reduces erythrocyte damage by benzoic acid by 25% AphaMax® at 100 nM yields a 95% reduction against copper chloride, a mild oxidative agent like benzoic acid [50]. Furthermore, it should be noted that ORAC tests are limited in their ability to evaluate the full antioxidant spectrum. However, an in vivo human study has demonstrated that long-term AphaMax® administration significantly reduces MDA levels, averaging a 37% reduction within 1–2 months [43]. In terms of cancer, a study testing the efficacy of AphaMax® to inhibit prostate and thyroid cancer cells, showed the ability of the AFA PC & PEC extract to inhibit 95% of cancer cell growth with just 100 nM [51]. In comparison, quercetin and gallic acid only inhibited the proliferation of MCF-7 human breast cancer cells by circa 66% at a concentration of 500 μM, a concentration approximately 5000x higher than the one of AphaMax® [52]. Similarly, at the same concentration of AFA (100 nM), the cannabinoid JWH-33, known for its potent anti-cancer properties, inhibited lung cancer cell proliferation by circa 75%, compared to up to 98% inhibition achieved by AphaMax®. This distinction is significant, as achieving higher levels of inhibition is notably challenging: JWH-33 attains a comparable 95–98% inhibition rate as AFA-PCs, but requires a concentration 1000x higher – 100 μM [53]. In terms of inflammation, a 2006 study investigated effects of AphaMax® in mice. In the experiment, one group of mice received an injection of capsaicin directly into the stomach, leading to a marked increase in inflammation, as measured by Evans Blue extravasation. In a second group, pre-treatment with AFA-PCs extract significantly inhibited inflammation, with an approximate reduction of 95%. A further test involving capsaicin injection in the urinary tract resulted in over 100% inhibition of urinary inflammation [54] (Figure 4). This outcome not only demonstrates the potent anti-inflammatory properties of AphaMax® but also their effectiveness at the systemic level after traversing the gastrointestinal tract [54].

Figure 4.

Bar charts highlighting AphaMax®‘s ability (800 mg/kg) to inhibit -induced inflammation as measured by Evans Blue extravasation in mice. Left: Stomach capsaicin injection; right: Urinary bladder capsaicin injection.

AphaMax® has demonstrated also efficacy as a dermatological therapeutic agent in a clinical trial involving human subjects [55]. This study included 10 patients diagnosed with varying stages of psoriasis, who had previously shown no improvement with standard or biologic treatments. Participants were administered three doses of an AphaMax® product daily over a period of three months. Post-treatment assessment revealed substantial remission in 90% of the participants (9 out of 10), with the remaining individual exhibiting significant symptomatic improvement [55]. Additionally, the pharmacological impact of AphaMax® was evaluated in an experimental model of colitis induced by 2,4-dinitrobenzenesulfonic acid (DNBS) in rats [56]. Varied dosages of AphaMax® (20, 50, or 100 mg/kg/day) were administered. The results indicated a notable reduction in histological damage to the colon (Figure 5). Furthermore, there was a decrease in myeloperoxidase activity, inhibition of NF-κB activation, and reduced expression of inducible nitric oxide synthase and COX-2. These changes suggest an improvement in the aberrant immune response associated with colonic inflammation. Additionally, the treatment led to a decrease in the inflammatory interleukins IL-1β and IL-6 expression. Finally, AphaMax® exhibited antioxidant properties, evidenced by decreased levels of reactive oxygen species (ROS) and nitrites [56].

Figure 5.

Effects of Aphamax® on DNBS-induced histological damage in rats. Photomicrographs of the colon stained with hematoxylin & eosin from (a) Sham (control) rats; (b) Sham (control) rats treated with Aphamax® (100 mg/kg); and (c) DNBS rats showing colonic damage and the infiltration of inflammatory cells in mucosa and submucosa. (d), (e), and (f) show DNBS rats treated with Aphamax® at a concentration of 20, 50 and 100 mg/kg, respectively, showing progressive reduction of inflammatory cell infiltration and fewer inflammatory cells close to the mucosal layer. (Scale bar = 100 μm, magnification 20, red arrows = colonic damage, black arrows = inflammatory infiltrate) [56].

3.2 β-Phenylethalamine

A distinguishing feature of AFA is its capacity to produce the endogenous phenolic compound β-phenylethalamine (PEA), setting it apart from other microalgae and cyanobacteria. PEA stands out for its role in neurotransmission, coupled with energizing, anti-anxiety, anti-depressant, and hunger-suppressing properties (Figure 3) [12]. This phenolic compound is notably produced during exercise and experiences of “love.” It is an agonist to a widely-spread receptor within the body, known as the trace amine-associated receptor (hTAAR) [57]. This is found in the gut, on immune cells and in neuronal synapses. Its activation in the brain, for example, is associated with the release and inhibition of reuptake of biogenic amines such as norepinephrine, dopamine, and serotonin. The resultant increase in catecholamine concentrations can lead to elevated endorphin levels, making PEA an indirect natural painkiller, and an increase in testosterone, contributing to heightened libido. Notably, PEA exhibits rapid and profound effects on mental clarity and alertness without side effects or tolerance [57]. However, the challenge lies in the rapid degradation of purified PEA once ingested: it is a well-known fact that monoamines are rapidly degraded by MAO-B enzymes already in the gut. For this reason, an extract concentrating PEA together with selective MAO-B inhibitors, namely AFA-phycocyanins, Mycosporine-like amino acids (MAAs), and phytochrome C, has been developed. This extract is known as Klamin® [58]. The three molecules are the most potent among all natural substances, and most of all they are reversible inhibitors, blocking the MAO-B activity only temporarily, thus producing no side-effects. This combination facilitates the absorption of a significant portion of PEA through the gut and the blood–brain barrier [15] (Table 2).

MAO-B inhibitors	IC50	Ki	Inhibition mode
AFA-phytochrome	0.02	0.01	Mixed
Deprenyl	0.28	0.04	Irreversible
AphaMax®	1.44	0.14	Mixed
AFA MAAs	1.30	0.58	Competitive
Emodin	35.40	15.10	Mixed
Paeonol	42.50	38.20	Competitive
Epicatechine	58.90	21.00	Mixed
Piperine	91.30	79.9	Competitive

Table 2.

Table describing the IC₅₀, Ki and inhibition mode of MAO-B inhibitors concentrated within the Klamin® compared with other synthetic and natural MAO-B inhibitors.

IC₅₀: Half-maximal inhibitory concentration; Ki: dissociation constant [15].

PEA’s crucial attribute lies in its promotion of brain tissue regeneration, as it is able to stimulate the production of erythropoietin (EPO) and its receptor (EPOR). Endogenous erythropoietin (EPO) within the brain acts as a fundamental regulator of neural stem cells, which are totipotent and pivotal for the generation of all neural tissues and neurotransmitters [59]. This positions EPO as a crucial factor in the potential repair and regeneration of brain and nervous system tissues. Notably, the observed changes extend beyond mere functional alterations in EPO dynamics. There is evidence of structural brain changes, notably an increase in EPO receptors [59]. This increase suggests not just a functional modification but also a physical transformation and regeneration of the brain tissues themselves. Such findings hold significant implications for a range of neurodegenerative conditions, including Multiple Sclerosis and Amyotrophic Lateral Sclerosis (ALS). The increased EPO activity and receptor expression could potentially offer new avenues for therapeutic interventions aimed at mitigating the progression of these diseases, emphasizing the role of EPO in neural repair and regeneration mechanisms [12].

One study specifically looked at Klamin® effects on EPO brain levels [58]. Two mice groups, one of group suffering from accelerated senescence (AS) and one not (A), were evaluated on learning ability through the Morris Test (Figure 6). After Klamin® administration (100 mg/kg of body weight), the AS mice were able to complete the test 15 s faster than ordinary, from 25 s to 10 s. The normal group of mice (A), instead, lowered the time to complete the test by 4 s, from 9 s to only 5 seconds [58] (Figure 6A). Subsequently, the brain of the mice was analyzed, and the following results were found: a) a strong decrease of brain oxidation (less MDA) and an increase in brain antioxidants (thiols); b) a strong increase in cerebral erythropoietin (EPO) (+500%), as well as in EPO receptors (+300%) [58] (Figure 6B). This ability to moderate and mobilize stem cells was already found by a study from Jensen et al., where it was shown that an AFA extract increased the release of stem cells from the bone marrow, triggering the mobilization of CD34+ CD133+ and CD34+ CD133− cells in vivo, associated with repairing of the central nervous system, heart, and other tissues [60].

Figure 6.

(A) Illustration of the Morris Water Maze Test, which looks at the learning ability of a mouse to reach the platform (gray circle) and leave the water, and the effect of Klamin® on the mouse speed to reach the platform. (made with biorender). B) Bar chart demonstrating the 500% increase in brain EPO levels and the 300% increase in EPOR following Klamin® administration (100 mg/kg of body weight) in both AS and A mice groups. EPO & EPOR expression were measure via western blot band intensity, with β-actin expression used as the control. Readapted from [58].

The AFA PEA extract, due to its ability to increase brain catecholamine levels, has also been investigated for its impact on mental health, including depression, anxiety, ADHD, and autism. Research indicates significant improvements in depression, anxiety, self-esteem, and overall well-being in individuals with depression, including post-menopause and cancer-induced depression [12]. In a study conducted by the Department of Gynecology at the University Hospital of Modena in Italy, a study was carried out involving 40 menopausal women, divided into two groups: 20 receiving Klamin® and 20 in a placebo control group. These participants were selected based on their exhibition of typical psychosomatic symptoms associated with menopause [61]. The intervention group was administered a daily dose of 1 gram of Klamin® for a duration of two months. Post-treatment evaluation using specific psychiatric scales, namely the Kellner-Sheffield Scale and the Zung Self-Rating Scale, revealed a statistically significant improvement in the levels of depression, anxiety, and self-esteem among the women who received Klamin® [61, 62].

Furthermore, Klamin® was also shown to have important beneficial effects on the mood and well-being of terminally ill patients. At the Ovada Oncology Center (Italy), 18 terminally ill cancer patients, being treated only with palliative care, took approximately 1 g of Klamin® for 2 months [63]. Statistically significant improvements were observed in the areas of anxiety, fatigue and depression, confirming that Klamin® is able to balance even apparently conflicting states such as anxiety and depression and to sustain the ability of the body to produce energy [63]. Similarly, Klamin® also had a positive effect on children with ADHD. A recent study looked at 30 children diagnosed with ADHD, and the associated impact of Klamin® administration, at dosages ranging from 0.25–1.20 g (according to weight). The observed improvements were noteworthy, and the areas affected were as follows: 1) the overall condition of the child; 2) the levels of attention and hyperactivity; 3) in executive functions; 4) in the quickness and precision [64]. The researchers also found significant improvements in the 25% of children who were also affected by autistic symptoms [63].

In addition, Klamin® has been shown to have a positive impact on neurodegenerative illnesses, most likely due to its effect on EPO brain levels [12]. Neural stem cell proliferation homeostasis has implications for memory improvement and the reduction of beta-amyloid plaques associated with neurodegenerative diseases, like Alzheimer’s. A recent Alzheimer study by Nuzzo et al. demonstrated Klamin®‘s ability to prevent the accumulation of the beta-amyloid substance, while inactivating its toxicity [65]. In this study, we administered the oxidizing agent tert-butyl hydroperoxide (TBH) into the mitochondria of live neuronal cells. This intervention resulted in a marked increase in the production of reactive oxygen species (ROS) within the cells, compared to the control group. However, the simultaneous introduction of 0.8 μg of Klamin® alongside TBH effectively inhibited the TBH-induced overproduction of ROS in the mitochondria [65]. Furthermore, the study explored the implications of Klamin® in the context of Alzheimer’s disease, particularly its interaction with beta-amyloid, a substance closely associated with the disease’s pathogenesis. Human neuronal cultures were stimulated to produce beta-amyloid, and the effect of Klamin® addition was observed. Remarkably, the presence of Klamin® led to a 63% reduction in the production of beta-amyloid compared to the control group [65]. Additionally, the beta-amyloid aggregates that were still formed in the presence of Klamin® were significantly smaller in size and exhibited a substantial loss of toxicity. This result is particularly significant given the role of beta-amyloid aggregates in Alzheimer’s disease progression [65].

Klamin®‘s nutraceutical properties have also been tested on obesity and its associated metabolic imbalances, which have been linked to neurodegenerative conditions, including Alzheimer’s disease. To investigate this connection, a study was conducted on mice using KlamExtra®, a novel product combining Klamin® and Aphamax® extracts [66]. The mice were divided into three groups: one group was fed a standard diet (Lean group), another received a high-fat diet (HFD), and the third group was given a high-fat diet supplemented with the AFA product (HFD + AFA) for a duration of 28 weeks. The study focused on several key aspects: metabolic parameters, brain insulin resistance, the expression of apoptosis (cell death) biomarkers, the modulation of astrocytes and microglia activation markers (key components of brain inflammation), and the accumulation of beta-amyloid plaques, which are characteristic of Alzheimer’s disease (Figure 7) [66]. These factors were analyzed and compared across the brains of the different mouse groups. Results indicated that the AFA product, KlamExtra®, mitigated neurodegenerative effects induced by the high-fat diet. This included a reduction in insulin resistance and a decrease in neuronal loss. Additionally, AFA supplementation was found to enhance the expression of synaptic proteins and significantly reduce the activation of astrocytes and microglia - a typical response to high-fat diet-induced stress. Moreover, the accumulation of beta-amyloid plaques, often associated with Alzheimer’s disease, was also reduced in the mice receiving the AFA supplement [66]. These findings suggest that KlamExtra® has potential therapeutic effects in addressing neurodegeneration linked to obesity and metabolic dysfunctions.

Figure 7.

Thioflavin T staining of beta-amyloid aggregates on cerebral cortex section of lean, HFD and HFD + AFA mice. Thioflavin T-positive amyloid deposits are prominent in cortex areas of HFD mouse compared with those from lean and HFD + AFA.

Finally, towards the start of the century, PEA was shown to possess immune enhancement properties. in their 2000 study, Jensen et al. discovered that consuming 1.5 g of AFA biomass leads to a broad enhancement of immune surveillance, without directly stimulating the immune system [67]. This enhancement is characterized by a rapid increase in the movement of immune cells, such as monocytes and lymphocytes, from bodily tissues into the bloodstream. Specifically, there is a notable mobilization of CD3+, CD4+, CD8+ T cells, and CD19+ B cells. Notably, individuals who regularly consume AFA biomass exhibit a 40% increase in natural killer (NK) cell recruitment within 4–6 hours post-ingestion [67]. The study attributes this immune modulation to various low-molecular compounds present in the AFA cyanobacteria, with PEA likely being a key contributor. PEA acts as an agonist to TAAR, which are found on monocytes, B cells, T cells, and NK cells. The stimulation of these cells by PEA is thought to be a crucial factor in their mobilization and the resultant enhanced immune surveillance observed following AFA biomass ingestion [68].

4. Conclusion

AFA, sourced from Klamath Lake, Oregon, is an example of nutritional excellence and industrial relevance in the health supplement sector. This wild-harvested cyanobacterium, flourishing in the lake’s unique volcanic ecosystem, boasts a rich nutritional profile, ranging from a high protein content, up to 70%, to an elevated concentration of Omega-3 s PUFAs. The nutraceutical value of AFA is epitomized by its specialized extracts, AphaMax® and Klamin®. AphaMax® is enriched with C-PC and PEC and confers notable anti-inflammatory benefits, due to its ability to reversibly inhibit the inflammatory COX-2 enzyme, while also having important antioxidant, anti-cancer and dermatological properties. On the other hand, Klamin®, containing β-phenylethylamine (PEA), has shown significant potential in improving mental health. It is particularly effective in alleviating symptoms of depression and anxiety, as shown in post-menopausal women and cancer patients, due to PEA’s ability to increase brain catecholamine concentrations. Additionally, its promising results in managing ADHD and its potential in treating neurodegenerative diseases such as Alzheimer’s further underscore its therapeutic versatility. In conclusion, AFA from Klamath Lake emerges as a powerhouse of health benefits, especially through its extracts AphaMax® and Klamin®. Its impressive nutritional profile and the health-promoting properties of its extracts solidify its standing as an invaluable component in the realm of nutritional supplements.

Conflict of interest

Scoglio, S. & Scoglio G. D. own and manage a Klamath AFA harvesting and nutritional supplement company.