Novel Food Information – Lyophilized Tetraselmis chui strain 8/6 as a novel food

Health Canada has notified Fitoplancton Marino, S.L. (Cádiz, Spain) that it has no objection to the food use of lyophilized Tetraselmis chui. The Department conducted a comprehensive assessment of this microalga according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

Background:

The following provides a summary of the notification from Fitoplancton Marino, S.L. and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Tetraselmis chui has been used for decades in aquaculture as a feed for crustaceans (e.g., shrimps and king prawns) mainly in Central American countries. Other Tetraselmis spp. are used in the culturing of mollusk larvae and adults (e.g., clams, oysters, and scallops) (FAO, 1996 ^{Footnote 1}).

The manufacturer, Fitoplancton Marino S.L., has been selling lyophilized T. chui since 2005 for fish larvae culture, mollusk culture, and crustacean culture. It has been sold as food in the United States since 2009. In 2014, the manufacturer received approved novel food status for their T. chui product in Europe and has subsequently been commercializing it for human consumption.

The proposed use of this lyophilized microalga is as a condiment (100%), added to salt (up to 1%), or to be added as an ingredient in other foods such as condiment sauces (~20%). The suggested intake of this product is based on its capacity to give a desired flavor to different foods.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada’s Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: the history of use of the organism, the safety of the source organism, the manufacturing process for this product, the nutritional composition of this product, and what the potential is for this product to present a toxic or allergenic concern. Fitoplancton Marino, S.L. has provided data to support that lyophilized T. chui is safe for use as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods). T. chui and foods containing this lyophilized microalga are considered novel foods under the following part of the definition of novel foods:

“a) a substance, including a microorganism, that does not have a history of safe use as a food;” 

2. Development of the product

Tetraselmis chuiis a unicellular microalga which can be found in marine environments. Its taxonomic classification is as follows: Plante, Chlorophyta, Prasinophyceae, Chlorodendrales, Chlorodendraceae, Tetraselmis. This microalgae was first isolated off the coast of Great Britain in the 1950s, although it was later isolated in different locations around the world including Spain (specifically Cádiz Bay).

The T. chui strain (i.e., 8/6) used in the production of the lyophilized product originated from the microalgal collection of the Andalusian Marine Sciences Institute (Lubián and Yúfera, 1989 ^{Footnote 2}). This strain has since been maintained in the manufacturer’s in-house collection where it is kept and preserved in optimal conditions to be used as inoculum for the production systems.

The identity of the T. chui strain has been confirmed in both the original collection reference and production samples. These samples were identified by the Molecular Biology Laboratory of the IFAPA Centre (Research and Training Institute for Agricultural and Fisheries) El Toruño, which is attached to the Ministry of Agriculture, Fisheries and Environment of the Junta de Andalucía.

The process by which lyophilized T. chui is obtained consists of four main sub-processes: culture, harvesting, lyophilization, and packaging. The production T. chui strain is cultured photoautotrophically in tubular photobioreactors. Described by the petitioner, tubular photobioreactors are systems consisting of a closed tank and a transparent tube circuit through which the microalgae culture circulates. The microalgae are cultured to obtain the desired cell density, followed by harvesting. Once harvested the cells are subjected to centrifugation to obtain a microalgal cell ‘paste’, which is subsequently lyophilized for preservation. Lyophilization produces microalgal ‘crackers’ that are disaggregated into a powder that is vacuum-packed into 250-g bags. The lyophilized biomass of T. chui is not viable in the finished product. According to the manufacturer the overall production process is similar to the production process of other microalgae that have already been authorized for human consumption (e.g., Chlorella).

3. Dietary Exposure

Based on the proposed uses, the petitioner’s estimated intakes of T. chui are 780 mg/day (12.97 mg/kg body weight (bw)/day) for adults and 761.5 mg/day (29.2 mg/kg bw/day) for children. With the higher intake of 780 mg/day, T. chui would provide approximately 0.312 g of protein, 0.273 g of carbohydrates, 0.078 g of fat, and 0.023 g of fibre per day based on the highest specification for these nutrients.

4. Microbiology

Microalgae have been cultured for decades (Borowitzka, 1999 ^{Footnote 3}). Out of approximately 10,000 species of microalgae described (Chaumont, 1993 ^{Footnote 4}; Radner, 1994 ^{Footnote 5}), only a few thousand are maintained in different collections located around the world. Among these few thousand, only a few hundred species are or have been studied to obtain different chemical products or applications and from these species only a few are cultured industrially (Oliazola, 2003 ^{Footnote 6}).

According to the petitioner, the most relevant microalgal species produced industrially are Chlorella, Haematococcus, Dunaliella, and Spirulina. These species have been used as food supplementation and additives. Other species that have been cultured for direct consumption in the aquaculture industry are Nannochloropsis, Tetraselmis, Isochrysis, Phaeodactylum, and Chaetoceros (Yamaguchi, 1997 ^{Footnote 7}; Borowitzka, 1997 ^{Footnote 8}; Apt and Behrens, 1999 ^{Footnote 9}; Muller-Feuga, 2000 ^{Footnote 10}).

The use of T. chui is widespread in the culturing of crustaceans (e.g., shrimps and king prawns), mainly in Central American countries. The development of crustacean larvae and their survival depend on the type, quality, and food ingested at various stages (Sánchez, 1986 ^{Footnote 11}; Treece and Fox, 1993 ^{Footnote 12}). It is in the first stage of development that the larvae are directly fed microalgae such as T. chui. The genus Tetraselmis is also used in the culturing of mollusc larvae and adults (e.g., clams, oysters, and scallops) (FAO, 1996). These filtering animals need microalgae throughout their life cycle.

According to the manufacturer, there is no literature on direct consumption of T. chui by humans, although companies exist that sell other species of microalgae for human consumption via the internet to North America (i.e., Canada and the United States). Therefore, the manufacturer provided information on the introduction of T. chui into the food chain through its consumption by fish, mollusc, and crustacean from aquaculture.

The manufacturer provided a detailed, stepwise description of the manufacturing process and the individual measures taken to ensure the microbiological safety of the finished product. The manufacturer declared that the lyophilized biomass of T. chui is not viable in the finished product.

The manufacturer has provided the following microbiological specifications for the finished T. chui product: an aerobic mesophilic count of less than 1´104 CFU/g, an enterobacteria count of less than 10 CFU/g, an absence of Salmonella spp. in 25 g, and an absence of Listeria monocytogenes in 25 g.

The manufacturer has provided Certificates of Analyses for 3 non-consecutive batches of T. chui. In addition to the above mentioned specifications, these batches were also analysed for the presence of other pathogenic microorganisms (e.g., Bacillus cereus, Staphylococcus aureus, Clostridium perfringens, etc.) ^{Footnote 13}.

The results of the batch analyses demonstrate that no pathogenic microorganisms were detected in each of the batches. Additionally, each batch conforms to the microbiological specifications as mentioned above, therefore demonstrating that this product can be safely produced in a consistent manner.

6. Nutrition

The manufacturer provided data on the nutritional composition of lyophilized T. chui, including macronutrients, amino acids, fatty acids, and minerals. As per the specifications, the protein, carbohydrate, total fat, and fibre content of T. chui ranges from 35-40%, 30-35%, 5-10%, and 2-3%, respectively. The composition of T. chui reported from three different batches was within the specifications provided.

Lyophilized T. chui will be available in the form of salts containing 1% T. chui, condiment sauces containing 20% T. chui, and the lyophilized powder itself which will be used as seasoning. T. chui is composed of amino acids, fatty acids, and minerals that are all expected to be commonly consumed in the typical diet of Canadians and metabolized through normal physiological pathways.

The amino acid analysis of T. chui showed that it contains all essential amino acids and the levels of amino acids are similar to levels reported in the literature for other T. chui strains. Overall, distribution of the amino acid profile is balanced and does not present any safety concerns from a nutritional perspective.

Approximately 47% of the fat content of T. chui is polyunsaturated fatty acids, which include 25% of linolenic acid, 10% of eicosapentaenoic acid (EPA), 7% of stearidonic acid, 3% of linoleic acid, and 0.5% of docosahexaenoic acid (DHA). The other major fatty acids in T. chui are palmitic acid and oleic acid providing approximately 27% and 15% of the fat content, respectively.

The estimated intakes (see Dietary Exposure) were used to determine the contribution from minerals in T. chui, which is not expected to be a major source of any of the minerals, with the exception of iron being the only mineral that could contribute up to 22% of the Recommended Dietary Allowance (RDA).

The proposed use of lyophilized T. chui is not expected to significantly alter the exposure to protein, individual amino acids, or other nutrients in the Canadian population.

7. Chemistry/Toxicology

The culture medium to grow the T. chui is seawater that is sterilized. Other substances that are used include sodium nitrate to maintain the concentration of nitrate, a nutrient, in the culture medium, and carbon dioxide as a carbon source. Once harvesting is finished the pipe is washed and then disinfected. None of these substances are regulated as food additives in this context of use. Their use, like that of any other substances used in food manufacture, must not result in a violation of Section 4 of the Food and Drugs Act.

The manufacturer provided specifications and data for cadmium, mercury, lead, and arsenic, which was based on a batch analysis of T. chui. The addition of T. chui as an ingredient in the foods specified by the petitioner at the proposed levels of use is not expected to significantly increase the total dietary exposure to these trace elements.

The manufacturer referenced several studies which showed that species in the Plante Kingdom, in which T. chuibelongs, are not amongst those known to produce phycotoxins. Nevertheless, the petitioner analysed three separate batches of the T. chui testing for the presence of phycotoxins. None of the phycotoxins were detected above their respective limits of detection (LODs). Even under the conservative assumption that these phycotoxins are present at their respective LODs, dietary exposure to these phycotoxins as a result of the consumption of T. chui in the proposed foods at the indicated levels of use would not be expected to represent a safety concern.

The manufacturer provided the results of analysis for more than 30 organochloride and organophosphide pesticides including some for which Health Canada’s Pest Management Regulatory Agency has developed maximum residue limits in certain foods. Since none of the pesticides were detected in the final product above their respective LODs, exposure to these substances from the consumption of T. chui is not expected to be of concern to human health.

The petitioner demonstrated that T. chuidoes not contain the marine toxins produced by some algae and microscopic plants which are associated with paralytic shellfish poisoning, amnesic shellfish poisoning, and diarrhetic shellfish poisoning. The expression of these toxins by T. chui is not anticipated as this microalga shares no evolutionary lineage with microalgae which produce any marine toxins ^{Footnote 14}. T. chui belongs to the Plantae Kingdom in the Prasinophyceae class. No microalgae in the plant kingdom are known to produce toxins.

An acute oral toxicity assay was conducted with lyophilized T. chui in rats (3 rats/sex). No treatment-related effects or mortalities occurred at the limit dose. The LD50 for lyophilized T. chui in rats of both sexes was determined to be greater than 2500 mg/kg bw.

A 90-day oral toxicity assay was also conducted with lyophilized T. chui in rats (10 rats/sex/dose), at dose levels 0, 625, 1667, or 2500 mg/kg bw/day which were administered orally. No treatment-related effects on mortality, body weight or weight gain, food or water consumption, hematology, clinical chemistry, organ weights, gross lesions, or histopathology were observed following dosing. The No Observed Adverse Effect Level (NOAEL) for lyophilized T. chui following the 90-day treatment in rats of both sexes was determined to be 2500 mg/kg bw/day.

Lyophilized T. chui was not mutagenic in a bacterial reverse mutation assay (Ames assay) at concentrations of up to 4 mg/plate.

Total dietary exposure was conservatively estimated by the petitioner to be 29.2 mg/kg bw/day in children and 12.7 mg/kg bw/day in adults. The Margin of Exposure (MOE) between the NOAEL from the 90-day study and the dietary exposure is over 85-fold in children, the most sensitive subpopulation, and nearly 200-fold in adults. The MOE is considered sufficient from a safety perspective.

The manufacturer stated that of the 70 foodstuffs which cause food allergies listed by the World Health Organization, there is no vegetable food of marine origin.

T. chui belongs to the Chlorophyta phylum, as does the microalga Chlorella. Published data on the allergenic potential of Chlorella species has not been observed even in atopic individuals. According to the petitioner, there are no reports of allergy following Chlorella consumption. These facts are considered to be generally supportive of the lack of concern for the potential allergenicity of T. chui.

T. chui was tested in a skin prick test or a skin patch test conducted in 100 human volunteers (50 healthy volunteers and 50 volunteers with histories of atopy or allergy). None of the volunteers showed sensitization or irritation in response to the microalgae.

The manufacturer performed analyses which revealed T. chui does not contain several common allergens such as soy, fish, or crustacean allergens, nor does it contain gluten. The level of sulphites was also analysed and reported to be 15 mg/kg. These sulphites are stated by the petitioner to be naturally present in the lyophilized microalgae ^{Footnote 15}.

Information indicates that there is no similarity or cross-reactivity between the proteins in the lyophilized T. chui and proteins found in common allergens like soy, fish, and crustaceans. Further, there are no reports in the literature associating T. chui with food allergy. The information is not conclusive on whether T. chui would cause allergenic reactions, but the available evidence presents no concern.

Conclusion:

Health Canada’s review of the information presented in support of the food use of lyophilized T. chui does not raise concerns related to food safety. It is the continuing responsibility of the manufacturers, sellers, and distributors of lyophilized T. chui to ensure that marketed products are in compliance with all applicable statutory and regulatory requirements, including labelling and advertising.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada.  This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate                                                         
Health Products and Food Branch                 
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
novelfoods-alimentsnouveaux@hc-sc.gc.ca