NOVEL FOOD INFORMATION - Whole Algal Flour to be used as a partial replacement for cream, milk, eggs/egg yolks and butter/shortening in a variety of foods

Health Canada has notified Solazyme Inc. that it has no objection to the sale of Whole Algal Flour to be used as a partial replacement for cream, milk, eggs/egg yolks and butter/shortening in a variety of foods. The Department conducted a comprehensive assessment of whole algal flour according to its Guidelines for the Safety Assessment of Novel Foods.

BACKGROUND:

1. Introduction

Solazyme Inc., has developed a Whole Algal Flour (WAF) for use as a partial replacement for cream, milk, eggs/egg yolk and butter/shortening in a variety of baked goods, beverages, dairy and egg products, sauces, gravies, margarines, salad dressings and soups.

Whole Algal Flour is a high lipid (40 -70% fat) algal flour consisting of the milled dried biomass of the microalgae Chlorella protothecoides strain S106. The microalgae are grown by the petitioner in large industrial fermenters under conditions which enrich the cells with high lipid content. The cells are then pasteurized and dry-milled to produce the final powder product.

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 reflect international guidance documents in this area. The assessment considered: the development of WAF; its history of use; the manufacturing and processing methods; its intended use; nutritional composition; microbiological and toxicological information; presence of potential allergens and estimation of its level of consumption by consumers.

The Food Directorate has a legislated responsibility for the 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). Information on the history of food use of Chlorella protothecoides strain S106 has not demonstrated that this species of microalga has a history of safe use as food. Therefore, Chlorella protothecoides strain S106 is considered a novel food under the following part of the definition for novel foods:

2. Product Development

Solazyme obtained C. protothecoides strain S106 from the culture collection maintained at the University of Texas. This strain is designated as UTEX 250 in the collection and was originally obtained from the culture collection of the Scottish Association of Marine Science, where it was deposited in the 1950's as an isolate of C. vulgaris. This original deposit was collected from fresh water in the Netherlands. Other deposits of this strain have been made in Germany and it is also known by the additional designation C. protothecoides var. communis. C. protothecoides Kruger is a synonym for Auxenochlorella protothecoides (Krüger) Kalina & Puncochárová (family Chlorellaceae), which is the name currently accepted taxonomically. All C. protothecoides strains held within the American Type Culture Collection (ATCC) are preserved under Biosafety level I conditions.

C. protothecoides is characterized as being tolerant to temperatures (28-34 ○C), acidic pH (3.5-4.0) and salt concentrations (3-4%). The petitioner notes that C. protothecoides is the only species known to de-green in the presence of glucose, meaning that it has decreased or no chloroplasts when grown in a high glucose, low nitrogen containing medium. This species has also been shown, under nitrogen depleted conditions, to synthesize and store oil.

WAF is manufactured by fermenting and harvesting cultures of C. protothecoides S106. A pure, clonally isolated culture is initially used to prepare a master seed bank from which working seed vials are prepared. Three samples from the master and each working seed bank are characterized by molecular genotyping to demonstrate that they are genetically identical.

For a production lot, a cryo-preserved working seed vial is thawed and the contents used to inoculate a flask culture, which is transferred into larger flasks at mid-log phase and then to standard, industrial seed fermenters. Throughout the fermentation process, pH, temperature, agitation and aeration rates are controlled, and glucose or sucrose and nutrients are added. Lipid production is induced by limiting inorganic nitrogen during the fermentation process.

Following fermentation, the cells are inactivated by pasteurization, and separated from the culture broth by centrifugation. The separated cells are washed with water to remove the medium and other non-biomass related material, concentrated and milled. After milling, the pH is adjusted to neutral and food-grade antioxidants are added.  In the diagram presented by the petitioner this pH adjusted biomass may then be subject to a second, optional pasteurisation step.

The resulting biomass is homogenized, dried and packaged. If needed, food grade flow agents may be added to assist processing through drying and packaging; at present the use of such agents is not part of the standard process. The final product, AlgaVia™, is packed in a bag-in-box arrangement in quantities of 15 – 25 kg.

3. Dietary Exposure

WAF is expected to replace a portion of the cream, milk, eggs/egg yolks and/or butter/shortening in the Canadian market for various conventional foods which lack a standard of identity.  WAF is not expected to change the overall consumption of these foods by the general population.

Estimated daily intakes based on data derived from the USDA's What We Eat in America (2003-2004) Continuing Survey of Food Intakes by individuals were provided. The mean dietary intake in adults (eater's only) consuming baked goods, beverages, dairy and egg products, sauces, gravies, margarines, salad dressings and soups as 4.7g/day (90^th percentile: 10.5 g/day). For the same group exposure was calculated at 78.3 mg/kg b.w./day (90^th percentile: 175 mg/kg b.w./day). The petitioner also provided mean exposure levels for children (eater's only) of 228.98 mg/kg b.w./day (90^th percentile: 457.96 mg/kg b.w./day).

On a body weight basis, children are the greatest consumers of the foods that could contain the WAF.  The 90^th percentile of consumers in this subpopulation could be exposed to ~ 0.5 g WAF/kg b.w./day.

4. Chemical Assessment 

The petitioner provided certificates of analysis from three different lots of WAF which included concentrations for arsenic, cadmium, lead, mercury and chromium. This information was used to estimate the concentrations of these contaminants in foods in which WAF is used as an ingredient.  The estimated concentrations are found to be within the range of background levels of these contaminants normally seen in foods on the Canadian market in which WAF is proposed for use as an ingredient

No chemical food safety concerns were identified with respect to the WAF itself, which is a discreet product that exists independent of any processing aids and additives that the manufacturer may choose to use in manufacturing WAF.  The petitioner was advised that the use of any additives in the manufacturing process must be compliant with food additive provisions, and that the use of processing aids, growth medium nutrients, and any other substances in the manufacturing process must not result in a violation of Section 4(a) of the Food and Drugs Act. 

5. Microbiological Assessment

C. protothecoides, a synonym for Auxenochlorella protothecoides, is distantly related to members of the genus Chlorella in the phylum Chlorophyta. Evidence provided by the petitioner indicates that while distantly related, C. protothecoides is more closely related to the type strain of C. vulgaris, the most commonly used Chlorella species, than some other strains currently classified as being a member of the C. vulgaris species. This strain, as well as many other strains, is still maintained as C. protothecoides and in the literature C. protothecoides and A. protothecoides are used interchangeably.  

Species of Chlorella, including C. protothecoides, are widespread in fresh and salt water, soil and air. A non-motile, unicellular organism, axenic cultures of this microalga are easily established and replication under optimum conditions tends to be rapid.

The petitioner noted that, while ubiquitous in nature, algal infections are rare in humans and other animals. A survey of the literature was provided and found only a single case of an opportunistic human infection, which was the result of the exposure of surgical wounds to river water and presented as localised lesions. No reports of human infection due to ingestion were identified. Furthermore, as the WAF is pasteurized at least once during manufacturing, inactivating C. protothecoides, it is unlikely that WAF will pose a risk to consumers.

Specifications for assuring microbial safety were provided by the petitioner. These specifications include testing for total plate count, coliforms, E. coli, Staphyloccoci, Salmonella, Pseudomonas aeruginosa, yeast and mold. All testing is conducted according to AOAC, USP or FDA-BAM methods. Certificates of analysis for three production lots demonstrated compliance with these specifications.

6. Nutritional Assessment

The petitioner provided data on the nutrient composition of WAF which included information on the nutritional composition, including macronutrients, amino acids, fatty acids, vitamins and minerals.  As the composition of Chlorella species is dependent upon the growing conditions during fermentation, the composition of each macronutrient in WAF varies based on the conditions.  However, the composition of WAF reported from different batches was always within the specifications provided by the petitioner. The fibre, protein, and total fat content of WAF ranges from 10-50%, 2-15%, 0-10%, and 40-70%, respectively.  WAF is high in oleic acid with typical content in the range of 60-72% of the total fat.

The complete fatty acid profile of WAF was compared to that of butter, cream, eggs, and common vegetable oils.  The fatty acids myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid and alpha-linolenic acid represent approximately 98.5% of the total fat content of WAF.  All fatty acids in WAF are present in the comparators, with the exception of two (C21:0 and C23:0), very long chain fatty acids with an odd number of carbons that are present in negligible quantities of 0.02% of the total fat.  The levels of all other fatty acids are within the ranges established for butter, cream, eggs, and common vegetable oils.

Based on the proposed food uses, the estimated mean and 90th percentile intakes, eaters only, of WAF were 4.7 g/day and 10.5 g/day per person, respectively.  With the 90th percentile dietary intake for all ages of 10.5 g/day, WAF would provide 7.35 g of fat, 1.6 g of protein, and 5.25 g of fibre per day based on the highest specification for these nutrients.  Furthermore, all vitamins and minerals in WAF would provide less than 8% of their respective % Daily Value.  Therefore, WAF is not expected to significantly contribute to the daily vitamin and mineral consumption.  As WAF is proposed as a partial replacement for cream, milk, egg/egg yolks, and/or butter/shortening, it is not expected to significantly impact the current exposure of the Canadian population to fatty acids, vitamin, minerals, proteins or fibre.

7. Toxicological Assessment

WAF is a mixture of lipids, proteins and nutrients from the milling of unicellular, eukaryotic green alga C. protothecoides strain S106.  The body's ability to digest these components is expected to be typical to that of nutrients from most food sources derived from plant materials. 

WAF does not contain algal toxins (e.g., amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, diarrhetic shellfish poisoning toxin, neurotoxic shellfish poisoning toxins and cyanobacterial toxins) as determined by high performance liquid chromatography (HPLC) with fluorescence or mass spectrometry detection.

Pheophorbide A is a plant pigment which has been reported to cause photosensitive dermatitis (i.e., skin inflammation that is aggravated by exposure to light) in people who consume natural health products containing Chlorella with high levels of pheophorbide.  Pheophorbide A levels in C. protothecoides S106 were below the limit of detection of HPLC (LOD = 0.5 ppm) and were below the limits prescribed by the Japanese Public Health Ministry (1.2 mg/g; enforced in 1981).  Phototoxicity is not considered a concern for people consuming WAF.

A 28-day subchronic oral toxicity study (OECD Test No. 407) was performed in rats (10 animals/sex/group) that were given WAF provided as 2.5, 5 or 10% of the diet (equivalent to 1794, 3667 and 7557 mg/kg b.w./day in males and 1867, 3918 and 8068 mg/kg b.w./day in females).  Animals in the control group were given animal feed supplemented with soybean oil.  No treatment related mortalities occurred in animals.  No clinical or physical changes in the rats were attributed to the test substance, considered adverse or considered toxicologically relevant.  A NOAEL of 7557 mg/kg b.w./day was proposed based on the highest dose tested in males.

A 90-day subchronic oral toxicity study (OECD Test No. 408) was performed in rats (10 animals/sex/group) that were provided the WAF at a constant dietary concentration of 25 000, 50 000 and 100 000 ppm in the diet (equivalent to 1248.6, 2478.1 and 4806.8 mg/kg b.w./day in males and 1413.3, 2739.4 and 5365.9 mg/kg b.w./day in females).  The control groups were provided the basal diet with similar levels of dietary fat from soybean oil and lard.  No treatment-related mortalities occurred during this study.  There were no clinical signs of toxicity, significant differences in body weight or body weight gain, or food efficiency changes associated with the administration of WAF.  The ophthalmoscopic examinations were normal in all WAF-treated animals. Some statistically significant changes occurred in food consumption, hematology, clinical chemistry and organ weight data in rats of the treatment group; however, they were not accompanied by concomitant changes in physiology, did not demonstrate a dose response relationship or were consistently observed in both genders.  As such, no adverse or toxicologically significant effects were associated with the WAF test substance.  The NOAEL was calculated to be 4806.8 mg/kg b.w./day, the highest dose tested in males.

The results of the Ames test (OECD Test No. 471) performed with the WAF were confounded by cytotoxicity and poor solubility of the test substance.  However, the WAF was not clastogenic in an in vivo mammalian bone marrow chromosomal aberration test (OECD Test No. 475) when orally administered to NMRI mice at 2 g WAF/kg b.w.  The weight of evidence (which includes the results of the 90- and 28-day subchronic oral toxicity studies) suggests that the WAF would not be considered genotoxic. 

Children are the greatest consumers of the foods that could contain the WAF.  The 90th percentile of consumers in this subpopulation could be exposed to 457.96 mg WAF/kg b.w./day.  The WAF was calculated to have a margin of exposure (MOE) that is 10-fold less than the NOAEL reported in the 90-day subchronic oral toxicity study (the highest dose tested in male rats).  The margin of exposure is considered sufficient from a safety perspective.

The petitioner provided additional test results from a repeat-insult patch test in which the WAF was spread as a dry solid over the skin of 28 male and 82 female volunteers (18 - 67 years of age).  The test material was held in place by a patch for 48 hours and then removed.  The skin was then assessed for irritation 48 to 72 hours after application and repeated nine times over the course of three weeks.  After 10-12 days without exposure to the WAF, the original test site and a new and untested test site was challenged with WAF for another 48 hours challenge phase.  The WAF was then removed and assessed again for irritation 48 to 96 hours post-application. Under the conditions set out in this test, the WAF did not induce contact sensitization and the authors concluded that there was little potential for the WAF to act as an allergen when consumed.  The study was later repeated with dried and milled C.protothecoides that had higher protein content (Whole Algal Protein or WAP).  The conclusions of the WAP study were consistent with those of the WAF study. 

WAF was approved in the United States in June, 2013, for the same proposed food uses (baked goods, beverages, dairy and egg products, sauces, gravies, margarines, salad dressings and soups).  Americans have been consuming the WAF in a subset of these foods for approximately one year without reported allergic reaction. 

There is no published information on the allergenicity of Chlorella protothecoides when it is consumed as a food.  However, Chlorella protothecoides is distantly related to Chlorella vulgaris, a product that is currently available in the market as a natural health product.  The published literature on consumed Chlorella products currently available in the market as natural health products suggests that Chlorella is a weak allergen that may produce dermal reactions in sensitive consumers.  Despite a long history of use as a food supplement, there is no evidence to suggest that Chlorella is a major allergen when ingested.

CONCLUSION:

Health Canada's review of the information presented in support of the food use of Whole Algal Flour as a partial replacement for cream, milk, eggs/egg yolks and butter/shortening in a variety of foods concluded that food products containing WAF do not raise concerns related to safety. It is the continuing responsibility of the manufacturers/sellers/distributors of WAF to ensure that marketed products are in compliance with all applicable statutory and regulatory requirements.

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.

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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