Novel Food Information - Acrylamide-reducing Baker’s Yeast

Health Canada has notified Renaissance BioScience Corporation that it has no objection to the food use of acrylamide-reducing baker's yeast, RBSC AR. The Department conducted a comprehensive assessment of this baker's yeast 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 Renaissance BioScience Corporation and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Renaissance BioScience Corporation has developed RBSC AR yeast to metabolize the acrylamide precursor asparagine in foods, thus reducing levels of acrylamide in foods. This Saccharomyces cerevisiae strain was developed using adaptive evolution to constitutively express a native ASP3 gene which codes for Asparaginase II.

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 (e.g., Codex Alimentarius). The assessment considered: how RBSC AR yeast was developed; how the composition and nutritional quality of RBSC AR yeast compared to non-modified varieties; and the potential for RBSC AR yeast to be toxic or cause allergic reactions. Renaissance BioScience Corporation has provided data that demonstrates that RBSC AR yeast is as safe and of the same nutritional quality as traditional yeast varieties used 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 the Food and Drug Regulations (Division 28). Foods derived from or which contain RBSC AR yeast are considered novel foods under the following part of the definition of novel foods: "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that

(iii) One or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism"

2. Development of the Modified Microorganism

Information was provided to describe the methods used to develop RBSC AR yeast and data that characterize the modification which results in increased ASP3 gene expression and therefore asparagine breakdown in otherwise repressive conditions.

RBSC AR yeast was developed by an adaptive evolution methodology that consists of successive or iterative adaptations of an organism based on novel growth conditions and/or environments. Random mutations are introduced, followed by characterization of a large pool of variants, and selection of individuals with desirable traits. This strategy employed on RBSC AR yeast involved selection of a commercial baker's yeast strain on D-asparagine and methylamine media with Ultra Violet (UV) light treatments in between. This strategy slowly increases selection pressure over time in order to build up genetic diversity allowing for full ASP3 gene expression.

3. Characterization of the Modified Microorganism

The host yeast strain is an industrial bread yeast strain of the domesticated S. cerevisiae, which is widely used in the baking industry. S. cerevisiae is found as a commensal organism on our bodies, is in the air we breathe, and grows naturally on many foods that are regularly consumed. Yeast is well understood given that it is a model organism for scientific research in numerous disciplines. Yeast is generally known to be non-pathogenic, but may cause infection in individuals with compromised immune systems. Commercial baker's yeast is used extensively in the baking and snack food industry for its ability to ferment dough, thereby leavening and conditioning the dough. Commercial baker's yeast is normally manufactured by growth on cane and/or beet molasses under highly aerobic conditions.

Conventional (wild type) baker's yeast is capable of degrading asparagine to use it as a nitrogen source for growth. However, this ability is only activated when the yeast are starved of other nitrogen sources such as ammonia, glutamate, and glutamine. The genes responsible for asparagine degradation are ASPl and ASP3, which encode an intracellular Asparaginase I and extracellular cell-wall associated Asparaginase II, respectively. ASP3 exists as a quadruple tandem repeat locus but is generally not constitutively expressed.

The application of adaptive evolution in the development of RBSC AR yeast was a step wise process. The commercial parent baker's yeast strain was continuously subcultured in the presence of media containing D-asparagine as the sole nitrogen source. This started with the wild-type strain being subcultured in minimal media, in which the wild-type exhibited very slow growth, resulting in cells with natural mutations and higher ASP3 expression to have a selective advantage. These yeast cells were then subjected to UV mutagenesis weekly for a period of time until the growth rate of the cells reached that of cells grown on non-selective/rich media. The subculture at this point in development was then continuously evolved in selective media now containing methylamine and subjected to UV mutagenesis weekly for a period of time until the growth rate of the cells in the presence of methylamine reaches the same as cells in the absence of methylamine. Individual colonies were plated on selective media containing methylamine. Colonies are selected for the ability to degrade L-asparagine under non-inducing conditions. The final concentrations of D-asparagine and methylamine used in the development were increased gradually in a step-wise manner in order to provide the selective conditions necessary to ensure full de-repression of ASP3 and to prevent the growth of non-adapted yeast.

4. Product Information

After the adaptive evolution was carried out, a series of tests were performed to characterize the RBSC AR yeast in relation to its parent strain. The identity of the RBSC AR yeast was confirmed as belonging to S. cerevisiae species using genetic identification to compare ribosomal DNA (rDNA) in RBSC AR yeast to conventional baker's yeast. 25S, 18S and internal transcribed spacer region of the parent baker's yeast strain and the RBSC AR yeast strain matched the reference S288C S. cerevisiae genome.

Inter-delta fingerprinting PCR was used to compare long terminal repetitive DNA sequences known as delta elements between RBSC AR yeast and the parent strain. An identical number, size distribution, and relative intensity of bands was observed for the parent and AR yeast strains.

A phenotypic profile index (API) which uses the presence or absence of growth on 20 different carbon sources to differentiate between species was also used to confirm identity. The API profile of the parent baker yeast, RBSC AR yeast, and a reference S cerevisiae strain all matched.

The growth rate of RBSC AR yeast was compared to the parent industrial baker's yeast strain and no differences were noted.

Asparaginase II is a cell wall associated asparaginase. Conventional baker's yeast is capable of degrading asparagine in order to use it as a nitrogen source only when the yeast are starved for other nitrogen sources such as ammonia, glutamate, and glutamine. There are two genes in yeast which are responsible for asparagine degradation, ASP1 and ASP3. ASP1 is known to act intracellularly and ASP3 acts extracellularly on the surrounding growth medium given its location in the cell wall. The presence of extracellular Asparaginase II is usually not very high due to nitrogen catabolite repression which causes yeast to select nitrogen sources in order of priority. Conventional baker's yeast is therefore not capable of breaking down asparagine to the degree of lowering acrylamide levels in food. RBSC AR yeast is able to constitutively degrade asparagine regardless of the growth conditions.

The evaluation of RBSC AR yeast did not raise any specific concerns regarding the method of development and it would be highly unlikely that the yeast was different from the parent strain in any way other than ASP3 expression.

5. Dietary Exposure

RBSC AR yeast is intended to be used as a replacement of conventional yeast in cooked and baked foods. The proposed level of use is comparable to that of conventional baker's yeast in breads and leavened snack foods (5000-50000 ppm of food). RBSC bakers yeast is not expected to change overall consumption of these foods by consumers.

6. Nutrition

Nutrient analysis of bread produced with RBSC AR yeast and conventional baker's yeast was provided to validate assertions of nutritional equivalence. The outcome of this analysis should be representative and sufficient for conclusion about other foods where RBSC AR yeast will be incorporated or used as a transient processing aid.

In the analysis of vitamins, minerals and macronutrients no statistically significant differences were observed between bread baked with conventional yeast and RBSC AR yeast for any nutrients analyzed, with the exception of maltose. This is not expected to be a nutritional concern as the level of total carbohydrates is not significantly different, which would include measures of maltose.

The levels of several nutrients were not analyzed, some of which yeast would be considered a source of, including potassium, folate, niacin, riboflavin, thiamin, pantothenic acid and phosphorus. However, the petitioner has indicated that industry accepted quality control product specifications for baker's yeast, published by Confederation of European Yeast Producers, will be met for each batch of RBSC AR yeast produced. This standard includes specifications for potassium, folic acid, niacin, riboflavin, and thiamine. Yeast currently being sold in Canada has levels of these nutrients that are within the ranges specified in this standard, according to the Canadian Nutrient File. As a result, it should not be expected that there would be any significant difference in these nutrient levels between RBSC AR yeast and conventional yeast; therefore, this is not expected to present any nutrition safety concerns.

Phosphorus and pantothenic acid were not analyzed in the nutrient analysis and are not included in this standard; therefore, the petitioner provided additional data showing levels of these nutrients in RBSC AR yeast and conventional yeast. The level of phosphorus was found to be 490 mg higher per 100 g of RBSC AR yeast than conventional yeast. Based on the exposure assessment provided by the petitioner this could result in an additional phosphorus intake of 50 -120 mg (5 - 11 % DV) per day at the 97.5th percentile of yeast intake. This is unlikely to pose a nutritional safety concern as high phosphorus intakes are not nearing the Tolerable Upper Intake Level for any age groups in Canada. The level of pantothenic acid was found to be 8 mg less per 100 g of RBSC AR yeast than conventional yeast, which could result in 0.81 -1.97 mg (12 -28% DV) per day less pantothenic acid at the 97.5th percentile of yeast intake. However, pantothenic acid deficiency is rare and has only been observed in individuals who were fed diets devoid of pantothenic acid or given a pantothenic acid metabolic antagonist, as reported by the Institute of Medicine. In addition, a wide variety of foods are reported to be major sources of pantothenic acid in addition to yeast, including chicken, beef, potatoes, oat cereals, tomato products, egg yolk, broccoli and whole grains. Therefore, it is unlikely the lower level of pantothenic acid in RBSC AR yeast will pose a nutritional safety concern.

In the amino acid analysis no statistically significant differences were observed between bread baked with conventional yeast and RBSC AR yeast for histidine, glycine, cysteine, lysine, methionine, and tryptophan. Asparagine was found to be significantly lower and aspartic acid was found to be significantly higher in bread baked with RBSC AR Yeast. However, these differences were expected, as RBSC AR yeast was evolved to convert L-asparagine into L-aspartic acid.

The levels of serine, arginine, glutamic acid, glutamine, threonine, alanine, proline, tyrosine, valine, isoleucine, leucine and phenylalanine were also found to be significantly different in bread baked with RBSC AR yeast compared to conventional yeast. However, for these amino acids the magnitude of difference in 100 g of bread made with RBSC AR yeast is very low and would not be expected to present any nutritional safety concerns. In addition, the level of ammonia was found to be significantly lower in bread baked with RBSC AR yeast compared to conventional yeast. However, there is no level of dietary requirement for ammonia; therefore, this is unlikely to present any nutritional safety concerns.

In addition, the phytase activity of the RBSC AR yeast was compared to that of the parental conventional yeast strain, as well as a number of other commercial baker's yeast strains. Data submitted by the petitioner showed that RBSC AR yeast exhibits comparable levels of phytase activity-as determined by growth on phytic acid media-relative to both the conventional parent yeast, as well as commercial baker's yeast strains. As a result, it is unlikely the phytase activity in RBSC AR yeast would be significantly different than conventional yeast and therefore, not expected to affect the bioavailability of minerals in foods to which RBSC AR yeast is added.

Acrylamide has known adverse effects. The petitioner has shown that adding or applying RBSC AR yeast to foods before heating/cooking reduces acrylamide content of the food by converting L-asparagine, the precursor for acrylamide, to L-aspartic acid by the Asparaginase II enzyme expressed in RBSC AR yeast. Furthermore, evidence suggests that alternative Maillard reaction products produced from aspartic acid are unlikely to pose a safety concern.

RBSC AR yeast has similar composition to conventional baker's yeast and no nutritional safety concerns have been identified.

7. Toxicology

RBSC AR baker's yeast was found to be generally equivalent to conventional baker's yeast and not a source of toxins. On this basis, RBSC AR baker's yeast is considered as a safe microbe for food use from a toxicological perspective.

Asparaginase II can be expressed at low levels in baker's yeast under conditions of food manufacturing. Consumers have had a history of low level oral exposure to Asparaginase II without apparent adverse effect.

Asparaginase II is an enzyme that is naturally expressed in baker's yeast under certain conditions. It shares 50% sequence identity with asparaginase I and approximately 43-46% sequence identity with asparaginases from other commonly consumed, food-grade microorganisms. All asparaginases share common functional groups that are necessary for their specific enzymatic activity; Asparaginase II is composed of some divergent amino acid sequences when compared to the other soluble asparaginases due to its hydrophobic domains which help it integrate into the plasma membrane.

The amino acid sequence of the Asparaginase II protein was compared to sequences of known toxins and antinutrients retrieved from the MvirDB database (29,576 toxin sequences). No significant matches were found between Asparaginase II and the toxins listed in these databases. It was concluded that Asparaginase II does not share significant sequence similarity to known toxins.

Based on the above, RBSC AR baker's yeast would not be expected to pose a toxicological safety concern to consumers.

8. Allergenicity

The amino acid sequence of Asparaginase II was compared with sequences of known allergens retrieved from the AllergenOnline database (1956 sequences). Asparaginase II did not share ≥ 35% amino acid identity over an 80 amino acid segment with any known allergen. Further, the eight amino acid segment analysis showed that Asparaginase II did not share sequence similarity with any allergen epitopes. Based on the results of the bioinformatics analysis, it was concluded that Asparaginase II did not match any known human allergens.

RBSC AR baker's yeast is expected to be used in foods where it is advantageous to reduce acrylamide production, such as baked goods where high-temperature cooking exceeds 120°C. The petitioner provided data showing that the RBSC AR baker's yeast Asparaginase II loses activity at temperatures exceeding 85°C. Therefore, Asparaginase II is denatured and potentially degraded in the consumed food, like other asparaginases, and is not expected to cause an allergic reaction.

The Asparaginase II protein was technically difficult to purify and sufficient amounts could not be produced to perform a digestion assay with the purified protein. Instead, experimental data was provided to show that total protein extracts from RBSC AR baker's yeast produced the same protein banding patterns as total proteins from a control baker's yeast after exposure to simulated gastric fluid and simulated intestinal fluid. The study results suggest that the proteins in RBSC AR baker's yeast will be generally digested in the stomach and intestines in a manner that is similar to other baker's yeast. It is expected that RBSC AR baker's yeast will not be any more allergenic than conventional baker's yeast.

The majority of reactions to baker's yeast are reported following inhalation of the yeast. Allergy to baker's yeast from oral exposure is a rare occurrence in the general population and those individuals who do have an allergy can experience generalized urticaria and asthma after consuming baker's yeast. It is not expected that RBSC AR baker's yeast would pose any new level of hazard.

Based on the available data, RBSC AR baker's yeast is considered as safe as conventional baker's yeast that is currently available in the Canadian market and would not be expected to pose a greater allergenic concern when consumed.

9. Microbiology

RBSC AR yeast will be produced under Current Good Manufacturing Practices (cGMP) and appropriate food grade starting materials will be used. Commercial batches of the RBSC AR yeast will meet all common industrially accepted food manufacturing standards for baker's yeast including but not limited to cell count, viability, microbial contamination and the presence of heavy metals. The industry accepted quality control and microbial product specifications for baker's yeast will be met for each batch of the RBSC AR yeast produced.

No antimicrobial compounds have been identified in S. cerevisiae besides the production of ethanol during fermentation. This activity requires live yeast cells actively fermenting to produce ethanol. RBSC AR yeast will be limited to heat killed yeast in cooked food products, so there should be no reasonable opportunity for ethanol production and antimicrobial activity.

Conclusion:

Health Canada's review of the information presented in support of the food use of RBSC AR yeast does not raise concerns related to food safety. Health Canada is of the opinion that food containing or derived from RBSC AR yeast is as safe and nutritious as food from current commercial baker's yeast varieties.

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.

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