Edible algae allergenicity – a short report

The use of seaweed and algal derived products in the food industry has grown rapidly in recent times. Major areas of expansion have been in Western countries where algae derived commodities are being utilised as edible foods or sources of high value ingredients. However, studies focused on potential allergenicity attributed to these food items, prevalence of allergenicity, and public health awareness are limited. Therefore, the current research summarises the existing literature focused on algal induced allergy in humans. Of the available literature, a total of 937 titles were identified, and 33 articles underwent subsequent full-text screening. Most research focused on prevalence and were derived from studies conducted in Europe (58%), North America and Canada (33%), and the remainder Australia and South Korea (9%). No studies addressed the need for public education or labelling of algal products. Our review reports that the available evidence identified points to algal derived products as being potential sources of allergens in the human food chain. Several components have been characterised that are shown to induce allergic responses in humans. Few studies have assessed the prevalence of algal allergenicity in the general population and as such further research is warranted given the increased usage of these products in the food industry.


Introduction
Several hundred edible seaweed and microalgal species are recognised, with many being widely used in the food industry as foods, additives, extracts, and sources of functional ingredients (Pereira 2011;Cai et al. 2021). Such is the demand for algal derived products, that the global algal market has seen significant growth viz. an estimated value of US$20.16 billion in 2021, looking to grow at a compound annual growth rate (CAGR) of 10.9% from 2022 to 2031; with food being the predominant application at 37.0% and pharma and nutraceuticals at 8.1% (Transparency Market Research 2022). Red seaweeds (Rhodophyta) and brown seaweeds (Phaeophyceae) are the most prominent species used (FAO 2018(FAO , 2021. Similarly, microalgae production reached 56,456 tonnes in 2019, and this figure was largely dominated by Spirulina (Arthrospira spp) (totalling, 56,208 tonnes), and to a lesser extent other green microalgae species (totalling, 248 tonnes), (FAO 2021). Demands have spawned a rapid growth in algal commodity markets, particularly in Western regions, with reported growth of seaweed-flavoured foods and drinks in many European countries (Mintel 2016). Given the increased use and the interest in the further usage of algal products as sustainable new food sources in the food production chain (FAO 2022), research has become more focussed on the composition and nutritional quality of proteins, polysaccharides, lipids, vitamins, minerals, and various antioxidants present in the tissues of these commodity food sources (Dawczynski et al. 2007; reviewed in Wells et al. 2017). In addition, researchers have highlighted potential concerns relating to the safety of algal based foods products (Banach et al. 2020a, b). These concerns relate to the bioaccumulation of heavy metal ions in tissues (Hwang et al. 2010;Smith et al. 2010;Taylor and Jackson 2016;Circuncisão et al. 2018), excess iodine intake in consumers (Crawford et al. 2010;Bouga and Combet 2015), the accumulation of various toxicants such as polychlorinated biphenyls (PCBs), dioxins, and microplastics (Hanaoka et al. 2001;García-Rodríguez et al. 2012; García-Salgado et al 2012; Gutow et al. 2016), radioactive materials (Goddard and Jupp 2001) and potential allergenicity to algal components (Cherry et al. 2019).
Food allergenicity, follows the induction of an immune response after exposure to an ingested item, via dermal contact or through inhalation of food allergen particulates. The National Institute of Allergy and Infectious Diseases (NIAID/NIH) recognises food allergy as key to public health initiatives and is of clinical significance to the general population. Current estimates show that globally approximately 8% of children and 3-10% adults are affected by food allergy (Zarkadas et al. 1999;Sicherer 2011;Gupta et al. 2018Gupta et al. , 2019Messina and Venter 2020), associated with a spectrum of food items including various nuts, shellfish, fish, egg, milk, wheat, soy, some fruits and vegetables, and various seeds (Codex Alimentarius 2020;FAO and WHO 2022). Various proteinaceous allergens have been identified in algal food sources and have been documented within the Allergome database (allergome.org), though some of these have not yet been biochemically characterised and require further study (listed in Table S1, supplementary materials). Therefore, given the increased growth in the macro-and microalgal production systems, and the promotion of algal based products in the food industry, it seems timely to report on current data on the allergenicity to algal products encountered in the human food chain.

Materials and methods
The current scoping review was conducted to assess the state of primary research into the allergenicity of edible algal sources emerging in food systems, and employed the framework set out by Arksey and O'Malley (2005) and Levac et al (2010). A scoping review differs from a systematic review in relation to the research question posed, as scoping reviews favour broader and more open-ended questions (Arksey and O'Malley 2005). Objectives and methods were identified, and the respective search terms and inclusion criteria adapted during the search process as the scope of the literature evolved.

Review questions
This scoping review sought to answer the following questions: 1) What algal food allergens have been introduced into food systems? 2) What research has been conducted on these algal food allergens? 3) What are the known health effects on humans? 4) How does the safety of these algal food allergens need to be addressed? 5) What are the priorities for future research into algal food allergens?

Identifying relevant studies
Relevant studies were determined following a trial of possible search terms, this to gain an overview of the literature, and to define key concepts. Initially, the search terms 'Allergy AND Algae' were used in a search of PubMed. The titles and abstracts of the articles were scanned, and recent reviews were read in full (Banach et al. 2020a, b;Borsani et al. 2021;Hadi and Brightwell 2021), allowing the identification of key concepts and areas of relevant research. These concepts were used to develop a search matrix for a systematic like literature search (Table 1), and to refine inclusion and exclusion criteria as recommended by Levacet al. (2010). For the scoping review, all searches were conducted using two databases PubMed and Web of Science, and identical search terms were used. The most recent searches were completed on 28 April 2022. After screening study titles and abstracts, the reference lists of relevant articles were searched to identify additional studies to be incorporated. The literature search uncovered a wide range of uses for algal sources in food systems (Wells et al. 2017;Ścieszka and Klewicka 2019;Francezon et al. 2021;Mendes et al.   . This review only focused on those health outcomes associated with the ingestion of algal food products (and potential exposure to allergens), rather than allergic reactions caused by inhalation or skin contact. Key metrics linked to the year of publication, geographic location of study, and population age/demographic were not restricted. As such this review includes primary research in humans in which algal allergens were ingested or administered during allergenicity testing. Studies were excluded if they were not primary peer reviewed studies, not written in English, studies were based on animal or cell culture models, the research was not focused on algal food allergy, or were studies duplicated results.

Study screening
The process of article screening is summarised in the flow diagram in Fig. 1. Following database searching, title and abstract screening were carried out using the defined inclusion and exclusion criteria stated above. Once found, included articles were read in full and the data extracted. Microsoft Excel was used to collate relevant information following assessment of the available data. These metrics including the location, study population, study design/purpose, definition of food allergy/food hypersensitivity, algal food allergen, type of reaction, prevalence, and outcomes. Due to a scoping review aiming to quickly highlight the gaps and areas in a field of research, the quality of research is not considered a priority (Armstrong et al. 2011), therefore no systematic quality assurance process was applied to the search. Study characteristics and key available data were tabulated to enable easy comparison of the findings.

Eligibility and study characteristics
Nine hundred thirty seven research papers were identified. These were reduced to 33 papers via title and abstract screening. Further scrutiny via full text screening led to the removal of 20 papers. In addition, a further 700 research articles were excluded due to them not being related to algal food allergy in humans or focused on one of the following key areas namely, prevalence, burden, labelling information, or education strategies. A further 32 papers were excluded due to not being written in English. Subsequently, 191 articles were excluded on the basis of publication type, as these represented conference abstracts, review articles and letters to the editor. Following the removal of duplicate sources, 12 relevant articles were identified, and data extraction was performed to allow for the final analysis. The paper selection process is summarised in Fig. 1.
Of the 12 included articles selected, 33% were published between the years (1989 to 1999), 25% (2000 to 2010) and 42% (2011 to 2022), suggesting increased interest in research of food allergic reactions to algal foods and associated products. The studies identified were, 8 case studies, 2 studies of prevalence, 1 randomised control trial and a comparative study (Table 2). These reports were composed of research conducted in several geographic locations with the majority (58%) based in European countries. 58% of the selected papers are more than a decade old, published between 1989 to 2010. A further 3 studies were performed in United States of America or Canada (25%), 2 of which were published in the last decade (2012 to present, 2022). The 2 most recent studies were carried out in Australia and South Korea. Of the 12 identified articles, 6 focused exclusively on younger populations (ages ranging from 10 months -17 years) with the remainder looking at adults of ages ranging from 18 to 75 years. Assessment of the 12 research articles allowed for the identification of two key themes from the available papers focused on allergenicity to edible algal species and associated clinical outcomes of allergenicity. These themes will be addressed below.

Edible algae and allergenicity
Most research focused on microalgal species, notably, Spirulina (Arthrospira) and Chlorella (58%) and spanned cases ranging from simple allergic reaction through to anaphylaxis (summarised in Tables 2 and 3). Reports were limited to papers associated with allergic responses to macro-algae.
Of the available studies, red algae, Chondrus crispus and Palmaria palmata, and the algal derived component carrageenan (a polysaccharide), isolated from a number of sources including Chondrus crispus, Eucheuma and Gigartina seaweeds respectively, were noted. Results from these studies included information on case studies, population details, diagnostic tests used, and treatment regime as defined in each study.
In reference to macro-algae, several studies have reported on food allergy to seaweed. These studies are summarised in Tables 2 and 3. One randomised control trial was identified in the current analysis. This assessed seaweed derived carrageenan (Vojdani and Vojdani 2015) and comprised 288 healthy participants of different ethnicities and ages (18-65 yrs). Of these participants, 57 (20%) had positive immunoglobulin E (IgE)-specific immune reactivity to carrageenan. Seaweed associated allergenicity was found in 4 case reports (Tarlo et al. 1995;Lauerma et al. 2001;Kular et al. 2018;Thomas et al. 2019). The report by Kular et al. (2018) documents the first reported case of IgE-mediated reaction following carrageenan ingestion. In this case, carrageenan was obtained from Eucheuma, Chrondrus, and Gigartina seaweed. Similarly, in the case identified by Tarlo et al. (1995) there was an IgE-mediated reaction to carrageenan, though this was identified during a barium enema study. Lauermaet al. (2001) identified positive IgE binding and skin prick tests to agar-agar and seaweed extract, as they were components of a consumed antacid that resulted in an anaphylactic reaction. Thomas et al. (2019), indicated generalized urticaria, facial angioedema, lip and tongue tingling, throat tightening, and nose congestion following a positive skin prick tests to red seaweeds Porphyra, C. crispus, and P. palmata, whereas Ulva spp. and Undaria pinnatifida were negative. In this study, patient history and positive skin test results suggested an immunoglobulin E-mediated response (Thomas et al. 2019). As a likely mechanism, studies have highlighted the link between the chemical structure of carrageenan containing the oligosaccharide epitope galactose-α-1,3-galactose (alpha-gal) and the potential for this structure to produce IgE mediated reactions in humans (Tobacman 2015;McGuire et al. 2020;Borsani et al. 2021), which have also been documented in exposure to meat products from mammalian sources and tick bites (Steinke et al 2015;Wilson et al. 2019).
Furthermore, protein sequence similarities have subsequently been found for food allergens from the green seaweed Ulva with known allergens (Polikovsky et al. 2019). Indeed, dried Porphyra spp. (nori), reportedly contains components with immunoreactivity similar to that of known lobster allergens (Motoyama et al. 2007). It is widely known that crustaceans, such as shrimp, lobster, and crab, induce IgE mediated allergies. Therefore, the possibility that dried nori products could potentially cause severe allergic reactions in people sensitive to crustaceans cannot be ruled out and further work is needed (Bito et al. 2017). Similarly, Mildenberger et al. (2022) identified marine allergenic proteins from crustaceans (crustacean tropomyosin), molluscs (mollusc tropomyosin) and fish (fish parvalbumin) in the seaweed Saccharina latissima when produced using growth methods such as Integrated Multi-Trophic Aquaculture. Whether some crustacean associated allergens are due to direct contamination of seaweed products cannot be ruled out.
By far the most widely reported indication of allergy to edible algal products comes from reports linked to unicellular species of both Spirulina and Chlorella. These algae are widely used in the supplementation industry, with Spirulina (Arthrospira) and Chlorella comprising the largest proportion of seaweed-based supplements produced worldwide (5,000 and 2,000 t of dry matter per year respectively), with an estimated global production values of about US$40 million each, per year (Enzing et al. 2014). Of the included research, 2 studies documented algal allergens within Chlorella spp. identified at 13, 17, 19, 25-26, 46-50, 72 kDa (Tiberg and Einarsson 1989;Tiberg et al. 1990) and 1 study discovered the algal allergen C-phycocyanin beta subunit at 15-35 kDa, within Arthrospira platensis. Another study examining Spirulina (Arthrospira) conducted by Yu et al. (2002) has also identified a similar algal allergen of C-phycocyanin beta subunit in Arthrospira maxima using in silico methods.
This review identified 9 papers, each using skin prick tests in the assessment of allergy to microalgae (75%), and 7 articles (58%) documented positive IgE measures in participants in response to exposure. 7 (88%) of the included case reports highlighted allergenicity to skin prick tests in individuals challenged with algal sources (Tarlo et al. 1995 ;  Table 3 The identification of algal food allergens and the prevalence of adverse reactions to these products Author ( Lauerma et al. 2001;Yim et al. 2007;Petrus et al. 2010;Le et al. 2014;Kular et al. 2018;Thomas et al. 2019). These articles were conducted in males and females, reporting on the severity of reaction ranging from localised inflammation to anaphylaxis. Interestingly, no questionnaire-based screening was used in any studies to scope for known allergic reactions to algal foods in the selected populations. Common symptoms associated with allergenicity included oedema (33%), gastrointestinal symptoms (25%), urticaria or erythematous (rash) (42%), anaphylaxis (42%, 5 of 12 papers).

Discussion
It is widely reported that algal species are good sources of fibre, various vitamins, minerals, and that several algal derived bioactives have anti-inflammatory properties when tested in models of inflammation (Lee et al. 2017;Ismail et al. 2020;Alkhalaf 2021;Cuevas et al. 2021;Chen et al. 2022;Mihindukulasooriya et al. 2022). These beneficial properties have spawned growth in the algal food production industry and algal products are largely good for health. However, less widely reported is the potential for edible algae to induce allergenicity and studies in this area are sadly lacking. Several studies using rodent models show that some algal derived components like carrageenan can induce intestinal inflammation, and to significantly alter gastrointestinal tract microbiota composition (Benard et al. 2010;Chassaing et al. 2017). Some algal based constituents like carrageenan are widely used to induce the innate immune response in animal models of inflammation, and points to the possibility of immune modifying properties for some algal species or constituents (Wei et al. 2016;El-Dershaby et al. 2022;Yi et al. 2022). Indeed, several studies have reported inflammatory responses of human colonic epithelial cells to carrageenan (Borthakur et al. 2007;Bhattacharyya et al. 2008;Choi et al. 2012). And recent assessment of edible seaweed species in Denmark (two brown and one red), indicate immunogenicity and allergenicity in rat models, and the capacity to raise IgG1 and IgE levels to elicit an allergic reaction (Vega et al. 2021). Moreover, in the last five years, the first reported incidence of carrageenan induced allergy via ingestion has been published (Kular et al. 2018). In view of these studies and rapid growth in the edible algal industry, the current scoping review was conducted to summarize the available information relating to the incidence of edible algal allergenicity in the general population. It is hoped that this will prompt further interest in the field and may indicate the need for more research in this area.
Most studies identified were case reports, and were conducted in Europe, with additional research from Asia, and North America (summarised in Table 2). A broad literature search was adopted in this work to capture all available pieces of evidence linked to edible algae and allergy. Of the studies identified, only 12 were suitable for further analysis. The research by Vojdani and Vojdani (2015) gave an indication of the prevalence of algal allergenicity in the general population, identifying that the sera of 20% of the 288 individuals tested had IgE-specific reactivity to carrageenan. One other study by Tiberg et al. (1995), conducted in 94 Swedish children, showed no specific IgE to Chlorella. These two studies point to significant gaps in our knowledge, particularly in reference to the incidence of algal allergenicity in the general population, the differing and developing use of algae in the food industry, consumption rates and relative exposure to algal based products. Moreover, there is a significant gap in our understanding and characterisation of potential algal allergens in food commodities and the levels of contamination of algal products with marine derived allergens. Adventitious allergen cross-contact of algal products with seafood allergens has been considered when other marine-based products are being grown near seaweed such as when implementing integrated multi-trophic aquaculture (Banacha et al. 2020a;Mildenberger et al. 2022). In the United States of America, researchers have highlighted the potential of seaweed cultivated on longlines, to be contaminated by organisms including crustacean shellfish allergens (e.g. tropomyosin) (Concepcion et al. 2020). Further studies have identified that crustacea in the form of amphipods can be present in raw or dried seaweed products and have the potential to cause serious allergic reactions within the general public in those with crustacean allergy (Motoyama et al. 2007;Bito et al. 2017). The global prevalence of allergy to crustacea has been documented by the FAO and WHO (2022) as mixed due to some regions having > 1% (Laoaraya and Trakultivakorn 2012; Lyons et al. 2019;Li et al. 2020) and others having populations ranging from 0.5-1.0% (Ben-Shoshan et al. 2010;Lyons et al. 2020). Food allergy to crustacea is more prevalent in Thailand and Southeast Asia, Australia and areas of Europe, including Spain, where crustacean seafoods are consumed more widely (FAO and WHO 2022). Tropomyosin is considered the major crustacean food allergen followed by other clinically relevant allergens viz. arginine kinase and myosin light chain, these allergens demonstrate high levels of homology amongst crustacean shellfish species and are accountable for crossreactive allergies (Lopata et al. 2010;Hajeb and Selamat 2012;Ruethers et al. 2018).
In the United States, the European Union, and other countries, labelling of all ingredients, including major allergenic sources is required. In the European Union 14 major allergens are outlined by Annex II of the 'Council Regulation (EU) No.1169/2011 on the provision of food information to consumers' (2011): cereals containing gluten (wheat, rye, barley, oats, spelt, kamut or their hybridised strains), crustaceans, eggs, fish, peanuts, soybeans, milk, nuts (almonds (Amygdalus communis), hazelnuts (Corylus avellana), walnuts (Juglans regia), cashews (Anacardium occidentale), pecan nuts (Carya illinoinensis), Brazil nuts (Bertholletia excelsa), pistachio nuts (Pistacia vera), macadamia or Queensland nuts (Macadamia ternifolia)), celery, mustard, sesame seeds, sulphur dioxide/sulphites at concentrations > 10 mg kg −1 or 10 mg L −1 (total SO 2 ), lupin, molluscs and products thereof. Similarly, in the US eight major allergens are currently recognised under Food Allergen Labelling and Consumer Protection Act (FALCPA) (2004), namely: milk, eggs, fish, crustacean shellfish, tree nuts, peanuts, wheat and soybeans, with sesame being added as the 9 th major food allergen under the Food Allergy Safety, Treatment, Education, and Research (FASTER) Act (2021), effective 1 January 2023. Currently, no such requirement is needed for algal based components and no studies were identified that addressed labelling of algal products or ingredients. However, it is worth noting the novel food status of different algae species to be potentially used as food and food supplements, subject to the pre-market authorisation requirements of the 'Council Regulation (EU) 2015/2283 on novel foods' (2015) before they can be freely placed in the European market.
In recent times, the use of algal derived ingredients in the food production industry has increased as a result of the numerous reported health benefits like antioxidant, anti-inflammatory, anti-acne, anti-microbial and anti-aging properties (Montero et al. 2018;Hannan et al. 2020;Thiyagarasaiyar et al. 2020;Ashaolu et al. 2021). In addition, algae have many beneficial nutritional attributes being rich in proteins, carbohydrates, lipids and providing good sources of vitamins and minerals such as vitamin A, B1, B2, B6, B12, C, E, potassium, iron, magnesium, calcium and iodine (Wells et al. 2017;Koyande et al. 2019). Due to this, many algal sources are being developed to meet the growing demand for dietary proteins to sustain an increasing human population, and environmental impacts of conventional protein production routes (Bleakley and Hayes 2017). The rapid growth in commercial and wild algal farming sector was recently acknowledged by the Food and Agriculture Organisation of the United Nations and highlights the importance of this sector in modern food production systems (FAO 2021). While this is a positive shift in driving commodity food markets and future food sustainability, questions still remain as to the potential allergenicity of algal derived products, and this justifies the current research. As it currently stands, only a handful of studies have shown macroalgae to induce allergy in humans (Table 3). These studies centre on skin prick assessment of seaweed extracts in sensitive individuals. Moreover, in the last few years, allergy associated with seaweeds are becoming more widely reported, however, the potential significance of this is not known (Thomas et al. 2019). Several studies have identified potential allergens in seaweeds namely, phlorotannins , polysaccharides (Borthakur et al. 2012) and proteins (Polikovsky et al. 2019) respectively. These constituents having varying degrees of potency at inducing allergy. Whether these molecules drive allergenicity in humans requires further investigation. Research has also been conducted into the aquaculture and production of brown seaweeds Laminaria digitata and S. japonica and their applications within pharma-or nutraceuticals, due to bioactive molecules and health benefits (Vadalà and Palmieri 2015;Li et al. 2022), as well as functional foods for example flavouring materials or food additives such as thickening agents or to decrease lipid oxidation in cooked meat products (Shirosaki and Koyama 2011;Purcell-Meyerink et al. 2021). However, allergic reactions, including anaphylaxis, have been documented in humans during medical procedures inserting Laminaria spp. with positive skin testing and specific IgE (Kim et al. 2003;Sierra et al. 2015;McQuade et al. 2020). Due to this, further examination is needed into these relationships and the possibility that the introduction of this item into products for human consumption could trigger allergic reactions.
More is known regarding microalgae like Spirulina (Arthrospira) and Chlorella and these species were the most commonly reported to induce allergic reactions in humans. These microalgae are used in a variety of products ranging from supplements through to single-cell food protein resources currently in development. Therefore, cases may reflect increased usage of these products. Historically these unicellular algae have been used in a sustainable manner for human consumption (Mooney and Klamczynska 2017) since they contain high concentrations of protein (51%-70% of dry matter) and are rich in amino acids, vitamins, dietary fibre and a variety of antioxidants, bioactive materials and chlorophylls (Bernaerts et al. 2019). However, several immunological studies have shown Chlorella to contain constituents that bind to IgE antibodies (Tiberg and Einarsson 1989). In addition, in assays of relative allergen potency determined using radio-allergosorbent test (RAST) inhibition, significant variation is reported between strains of Chlorella (Tiberg et al. 1990). This indicates that natural variation in the levels and composition of allergens in Chlorella is common and this may translate to cases of allergenicity in the general population. More recently, Bianco et al. (2022) used in silico assessment of algal protein sequences of Spirulina (Arthrospira platensis) and Chlorella (Chlorella vulgaris) using the AllergenOnline (AO) (AllergenOnline.org) and Allergome databases (allergome.org) to identified six proteins of interest. These proteins were identified in spirulina and exhibit significant homology with several known food allergens including thioredoxins, superoxide dismutase, a glyceraldehyde-3-phosphate dehydrogenase, triosephosphate isomerase and C-phycocyanin beta subunit, respectively. Importantly, the first case report of spirulina induced anaphylaxis was associated with phycocyanin (Petrus et al. 2010). Potential allergens in chlorella are also reported and have been shown to share sequence homology to calmodulin (A0A2P6TFR8), troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Allergic properties of chlorella have been reported on several occasions (Pukhova et al. 1972;Stewart et al. 2006). Taken together, the current work highlights a significant lack of studies, particularly prospective studies focused on food allergens linked to edible algae. Given the current expansion in the algal commodities market, particularly those targeting the human food chain, more work is needed in this area. Of the available evidence, several case studies point to the possibility that algal based food could be a source of allergic reactions and is of public health interest.

Conclusion
Growth in the production of macro-and microalgae and the continual promotion of algal based products in food systems will support drives in sustainable food production systems. The available evidence points to the presence of allergens in edible algae, but what significance this has to the general population remains largely unknown. Allergenicity to edible algal species has been reported and induces several clinical outcomes ranging from urticaria and gastrointestinal symptoms to oedema and anaphylaxis. Therefore, further research is needed to assess the allergenicity of edible algae species. This will enable the provision of information for clinicians, industry, regulators and legislators to put in place controls to manage algal allergens and provide information to protect public health and educate consumers.
Author contributions CAJ and PR contributed to the conception, design and drafting of the review. CAJ carried out literature searching, data extraction and analysis. PR, SW and CAJ and PR reviewed and revised the manuscript.
Funding This research was funded by Campden BRI.
Data availability All data generated or analysed during this study are included in this published article and its supplementary information files.

Declarations
Competing interests The authors declare they have no competing interests.
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