Novel Food Information: Sourvisiae®

On this page

• Background
• Introduction
• Development of the Modified Microorganism
• Characterization of the Modified Microorganism
• Product Information
• Dietary Exposure
• Microbiology
• Nutrition
• Chemistry
• Toxicology
• Allergenicity
• Conclusion

Background

Health Canada has notified Mascoma LLC (a Lallemand company) that it has no objection to the food use of Sourvisiae®. The Department conducted a comprehensive assessment of this product 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 characteristics.

The following provides a summary of the notification from Mascoma LLC and the evaluation conducted by Health Canada. This document contains no confidential business information.

Introduction

Mascoma LLC has developed a a genetically modified (GM) Saccharomyces cerevisiae (brewer’s yeast) strain M16141. The commercial preparation of this strain will be sold as Sourvisiae®. This yeast strain was modified to express a lactate dehydrogenase (LDH) enzyme based on the wild type ldhA gene from Rhizopus oryzae.

Sourvisiae® is intended for use in beer fermentation to produce alcohol and impart a sour flavour via lactic acid production (catalysed by the expressed LDH enzyme). Sourvisiae® is intended to replace yeast from other available commercial sources in the fermentation of beer. Sourvisiae® is produced through fermentation of a pure culture under controlled conditions.

The safety assessment performed by Food and Nutrition 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: how Sourvisiae® was developed, its nutritional safety, and what the potential is for Sourvisiae® to present a toxic or allergenic concern. Mascoma LLC has provided data to support that Sourvisiae® is safe for use as food in Canada

The Food and Nutrition 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). Sourvisiae® is considered a novel food 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

1. the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism.”

Development of the Modified Microorganism

The modified yeast strain of Sourvisiae® (i.e., strain M16141) was developed through chromosomal integration of a linear recombinant DNA fragment containing an expression cassette for the LDH enzyme. The cassette was flanked by sequence homologous with the native S. cerevisiae targeted gene locus.

No DNA from the donor organism (i.e., R. oryzae) was used in the construction of the production microorganism, as the codon-optimised LDH coding sequence was generated synthetically.

The linear recombinant DNA fragment was transferred into the parental strain by electroporation, along with a co-transformation plasmid. The plasmid contains a hygromycin antibiotic resistance gene, which aided in the selection of successful transformants.

Successful transformants were recovered overnight in medium containing hygromycin and plated on selective media. The plasmid containing the hygromycin antibiotic resistance marker was subsequently removed from successful transformants through subsequent passages on non-selective media (i.e., media not containing hygromycin). A successful transformant containing the LDH expression cassette at the targeted locus and lacking the co-transformation plasmid was ultimately selected and designated as strain M16141.

To confirm that the modified strain M16141 was sensitive to hygromycin and insensitive to selective media, the parental strain was compared to the modified strain M16141 on various media. Both strains show similar growth on normal yeast extract peptone dextrose (YPD) and yeast nitrogen base (YNB) media, whereas the parental strain is unable to grow on selective media and the strain M16141 lacking growth on other relevant selective media. Both strains are sensitive to the hygromycin antibiotic.

Characterization of the Modified Microorganism

The genomes of strain M16141 and its parental strain were sequenced using whole genome sequencing to verify the genetic modifications introduced in the production microorganism and confirm the absence of non-S. cerevisiae DNA beyond the intended expression cassette purposefully introduced.

Short-read Illumina and long-read PacBio sequencing was used to confirm the chromosomal integration at the targeted locus, to screen the genome and verify that no ‘off-target’ integrations occurred, and that no plasmids were retained in the production microorganism.

To verify that the chromosomal integration at the targeted locus in strain M16141 was as designed, sequence comparison between the de novo genomes of strain M16141 and the parental strain, as well as the linear recombinant DNA fragment was performed.

A BLAST search with the nucleotide sequence of the LDH coding sequence from the linear recombinant DNA fragment against the strain M16141 de novo genome sequence identified a single copy of the LDH coding sequence in the production microorganism genome sequence.

To confirm that the LDH coding sequence had integrated at the desired location in the strain M16141 genome, replacing the targeted gene coding sequence, the R. oryzae LDH BLAST hit was extracted from the strain M16141 genome sequence with 5-kb flanks. The targeted gene coding sequence of the S228C S. cerevisiae reference genome was subsequently blasted against the genome of the parental strain to localize its copy of the targeted gene coding sequence (the parental strain’s targeted gene allele was similarly extracted with 5-kb flanks). A sequence alignment of both regions demonstrated that the targeted gene coding sequence in the strain M16141 had been replaced by the LDH coding sequence.

To determine whether the full expression cassette had integrated as designed, the nucleotide sequence of this cassette was aligned to the mapped integration site in M16141. This alignment showed no structural differences between the cassette sequence of the linear recombinant DNA fragment and the integration site in strain M16141, showing 99.1 % pairwise sequence identity. The observed discrepancies consist of variants in the upstream and downstream targeted gene flanks and in the cassette terminator sequence. No variants were observed in the cassette promoter sequence and the LDH coding sequence.

Additionally, it was verified whether other components of the linear recombinant DNA fragment had integrated elsewhere in the strain M16141 genome by blasting the fragment sequence against the genome sequence. This BLAST search showed only three hits: one for the full-length fragment sequence at the characterized integration site), and two hits for the promoter and terminator sequences, respectively, which were confirmed to represent these elements in the native copies of these S. cerevisiae genes. Based on these results, it is demonstrated that no components of the linear recombinant DNA fragment integrated elsewhere in the production microorganism genome.

Illumina reads of strain M16141 were imported into Geneious Prime 2020.2.1 (https://www.geneious.com/prime/), trimmed, and simultaneously aligned to the S. cerevisiae S288C reference genome and the sequences of the plasmid used for transformation. Read coverages for the reference genome and plasmid sequences were manually inspected to determine whether plasmid sequence had been retained in the production microorganism.

To confirm that the antibiotic marker genes from the co-transformation plasmid used in the construction of the production microorganism had not been retained (either as a plasmid or through chromosomal integration), the Illumina whole-genome sequence reads of strain M16141 were aligned in Geneious Prime to this plasmid as well as the S288C reference genome.

No full-length read coverage was observed for either the hygromycin or ampicillin resistance markers of the plasmid. For the ampicillin marker, read coverage was only observed for the first 36 nucleotides of the 861st ORF, with only 22 % sequence identity between this subregion of the marker sequence and the consensus sequence of the aligned Illumina reads. The petitioner stated that the read coverage in this region is likely an artifact caused by the nearby 2-micron ORI in the co-transformation plasmid (the S288C reference genome does not include a 2-micro plasmid sequence). These results indicate that both antibiotic marker genes are absent in strain M16141.

To screen the strain M16141 genome for DNA sequence not present in its wild-type parental strain, Illumina whole-genome sequence reads of strain M16141 and the parental strain were aligned to the strain M16141 de novo genome sequence. The median genome coverages for the strain M16141 and parental strain sequence reads were 224x and 100x, respectively. Subsequently, coverage gaps (nucleotide positions with a sequence read coverage of 0) were determined for both alignments, and coverage gaps unique to the alignment of the parental strain Illumina reads were inspected, as these could indicate the presence of newly introduced DNA.

The absence of additional coverage gaps demonstrates the genome-wide absence of non-S. cerevisiae DNA in the production microorganism (with the exception of the synthetic LDH coding sequence), with no other regions in the strain M16141 genome not already present in the parental strain genome.

Genotypic stability of the production microorganism was determined over 100 generations by serially passing the strain through 11 transfers in YPD medium. Genomic DNA was isolated from the 11th transfer of the production microorganism and PCR was performed to confirm stability of the modified integration site at the targeted locus. Similarly, the parental strain was also passed as a control. The results of all the genotype PCRs were as expected, indicating no loss in the genetic stability of the integration site over 100 generations of strain M16141.

Based on the safety of the host and donor organisms, the well-characterized genetic modifications introduced into the parental strain to create strain M16141, and the phenotypic characterization of the production microorganism compared to its parental strain (see Microbiology), it can be concluded that the production microorganism, S. cerevisiae strain M16141 of Sourvisiae® is safe for food use from a molecular perspective.

Product Information

Sourvisiae® is produced according to current good manufacturing practices (cGMP). A Hazard Analysis and Critical Control Points (HACCP) plan, which includes confirmation of microbiological purity, is employed during the entire manufacturing process. The production is conducted at a fermentation facility with established procedures and equipment suitable for large-scale contained production of a genetically modified S. cerevisiae strain.

Physical inspection and appropriate microbiological and fermentation analyses are conducted to confirm strain purity and functionality in application, ensuring that the yeast product meets the finished product specifications. These methods are based on generally available and accepted methods used to produce microbial production organisms and microbial enzymes^{Footnote 1}.

Dietary Exposure

Sourvisiae® is intended for use in beer fermentation to produce alcohol and impart a sour flavour via lactic acid production. Sourvisiae® is intended to replace yeast from other available commercial sources in the fermentation of beer. The petitioner does not anticipate a significant change in the food use of beer with the introduction of Sourvisiae® as a food ingredient.

Microbiology

To further support the safety of Sourvisiae®, the petitioner conducted pathogenicity characteristic testing on strain M16141 and its parental strain.

It has been reported by de Llanos et al. (2006)^{Footnote 2} that there are 3 properties of S. cerevisiae strains that can predict if the strain possesses pathogenic properties: 1) the ability to grow at 42 °C; 2) pseudohyphal growth on low ammonia dextrose media; and 3) phospholipase production.

Strain M16141and its parental strain were evaluated for these phenotypic virulence factors, along with two commercially available industrial S. cerevisiae strains (i.e., Industrial distillers S. cerevisiae strain FALI, producer: ABMauri; and an Industrial biofuel S. cerevisiae strain, producer: Mascoma). Additionally, the reported pathogenic yeast strain Issatchenkia orientalis (Candida krusei), which possesses all 3 characteristic properties, was used as a positive control^{Footnote 3}.

The assay results confirmed that neither strain M16141 nor its parental strain display pathogenic characteristics as described by de Llanos et al. (2006). In all of the studies, the pathogenic positive control performed as expected in the respective assays whereas all of the S. cerevisiae strains were negative as expected. The strains of interest do not have the ability to grow at temperatures above 40 °C, do not produce pseudohyphal growth on low nitrogen plates, and do not possess phospholipase activity.

The petitioner provided the batch analyses of 3 non-consecutive commercial batches of Sourvisiae®. The results of these batches demonstrate that the microbiological specifications outlined for the finished Sourvisiae® preparation are consistently met in a production run. All analytic methods are (or are comparable to) internationally recognized methods and validated.

Based on the pathogenicity characteristic testing of the production microorganism and its parental strain, and the microbiological specification results of 3 non-consecutive batches, the Bureau of Microbial Hazards (BMH) has not identified any microbiological food safety concerns with the proposed uses of Sourvisiae®.

Nutrition

Beer is not consumed for its nutritional value. Therefore, from a nutritional standpoint, it was not deemed necessary to require compositional data that compare beer made with Sourvisiae® with beer made with traditional brewer’s yeast.

As previously mentioned, Sourvisiae® is produced through fermentation of a genetically modified S. cerevisiae strain that expresses LDH. LDH is ubiquitous in nature and is found in plants, animals, and microorganisms, and therefore, is commonly consumed in foods as well as endogenously produced in the body. When ingested, LDH is expected to be digested similarly to other dietary proteins. Specifically, it is expected to be hydrolyzed in the small intestine by proteolytic enzymes into smaller peptides and individual amino acids that can be absorbed and utilized by the body.

The reaction product of LDH, lactate, is also commonly consumed in the diet (e.g., dairy products, sourdough bread, pickled vegetables) and is endogenously produced in the body. Ingested lactate is expected to be readily absorbed from the gut and transported to the liver where it will be converted to glucose or further catabolized via the lactic acid cycle.

Sourvisiae® is intended to replace conventional brewer’s yeast in the manufacture of beer and the levels of Sourvisiae® in beer will be similar to the levels of yeast in beer brewed using conventional brewer’s yeast. Further, it is expected that very little, if any, Sourvisiae® will be present in the final beer product after pasteurization and/or filtration, both of which are standard practices in the brewing industry.

Based on this information, the Bureau of Nutritional Sciences (BNS) has not identified any nutritional food safety concerns with the proposed uses of Sourvisiae®.

Chemistry

The petitioner provided specifications for Sourvisiae® demonstrating that the ingredients meet the lead specification for dried yeast (brewer’s yeast) in the Food Chemical Codex (FCC). They have also established limits for arsenic, cadmium and mercury in the absence of FCC specifications for these trace elements.

The petitioner analyzed for trace elements (arsenic, cadmium, lead, and mercury) in multiple batches of Sourvisiae® brewer’s yeast and reported either ‘not detected’ or ‘very low concentrations’. The analytical limits of detection (LOD) employed for all contaminants evaluated were deemed to be suitably low. Using either the quantified analytical results or assuming the specification values, the Food Contaminants Section (FCS) determined that the proposed use of Sourvisiae® is not expected to result in increased dietary exposure to these contaminants. On this basis, the FCS is of the opinion that Sourvisiae® is not expected to pose a food safety concern from a chemical contaminants perspective.

A list of substances used during the manufacturing of Sourvisiae® was included in the submission. Constituents of the fermentation media are not usually classified as food additives and as such there is no requirement under the Food and Drug Regulations for their pre-market review. Based on the information provided as part of the submission, substances from the growth and fermentation media are not expected to remain in the final product.

The petitioner did not submit heat lability or digestion studies for Sourvisiae® since they state very little to no viable amount of Sourvisiae® is expected to remain in the beer products after pasteurization and/or filtration, both of which are standard practices in the brewing industry. S. cerevisiae has a long history of use in food production (i.e., baking, wine making, beer production) and is known to be degraded during pasteurization of beer based on the temperatures and time periods used during pasteurization. If LDH is present in the final beer product then the gastric acid in the stomach has a pH between 1.5 and 3.5 and will decrease the LDH enzymatic activity due to the low pH. There are studies in the literature that support this argument. For example, Vallee and Williams (1975)^{Footnote 4} studied LDH from beef and determined that at a pH of 2.0 or 3.0, beef B4 LDH lost enzymatic activity within 15 seconds of exposure.

Based on this information, the Bureau of Chemical Safety (BCS) has not identified any chemical food safety concerns with the proposed uses of Sourvisiae®.

Toxicology

The petitioner did not provide the results of toxicological tests for LDH produced from S. cerevisiae strain M16141 (Sourvisiae®). Instead, the petitioner provided a narrative to support the safety of Sourvisiae®. This narrative was based on the history of safe food use of S. cerevisiae strains, demonstrated safety of the genetic modification used to create the production microorganism, and whole-genome sequencing (WGS) analyses. The BMH has confirmed that the production microorganism, Sourvisiae®, is safe for food use from a molecular and microbiological safety perspective. Therefore, the focus of the toxicological assessment was on the history of use of the parental strain (non-genetically modified S. cerevisiae), and the donor strain (Rhizopus oryzae) as the source of the coding sequence for LDH.

LDH is naturally present in foods (e.g., certain fruits and vegetables (i.e., avocado, pear, lettuce, and strawberry), meat (beef and chicken), and fermented foods) and is in most cells of the human body at low levels.

S. cerevisiae has Qualified Presumption of Safety (QPS) status in the European Union.^{Footnote 5} As previously mentioned, the parental strain used to construct the production microorganism is a commercial strain of brewer’s yeast that has been used in the brewing industry for over 20 years. Yeasts used in food production, especially baker’s or brewer’s yeast, are among the safest of microorganisms. S. cerevisiae strains are approved in Canada as production microorganisms for other food enzymes^{Footnote 6}, namely alpha-amylase, glucose oxidase and lipase, while Saccharomyces species are approved for invertase and lactase.

R. oryzae is not known to produce any mycotoxins or other harmful secondary metabolites. The petitioner provided a comparison of the amino acid sequence of LDH from R. oryzae to known protein toxins in the UniProtKB database (May 17, 2023). No significant matches were found. It was concluded by the petitioner that LDH from R. oryzae does not share significant sequence similarity to known toxins and the Pre-market Toxicology Assessment Section (PTAS) agrees that there does not appear to be similarity to known protein toxins. This information further supports the conclusion that there are no safety concerns regarding LDH from R. oryzae.

Based on this information, the PTAS has not identified any toxicological food safety concerns with the proposed uses of Sourvisiae®.

Allergenicity

To evaluate potential allergenicity of the LDH protein from R. oryzae, the petitioner conducted bioinformatic analyses. Using Version 21 (February 23, 2023)^{Footnote 7} and Version 22 (August 1, 2023)^{Footnote 8} of the www.AllergenOnline.org database and following WHO/FAO (2020) guidelines^{Footnote 9}, no relevant matches were found. Therefore, the LDH protein from S. cerevisiae strain M16141 is unlikely to trigger a cross-reaction with known allergens.

A literature search conducted by the petitioner did not identify reports of allergenicity associated with the LDH protein. Consumers have a history of low-level exposure to the LDH enzyme in food since it occurs naturally in low amounts in certain fruits and vegetables (i.e., avocado, pear, lettuce, and strawberry), meat (beef and chicken), and fermented foods. Therefore, humans are commonly exposed to low levels of LDH via consumption of food without any known reports of allergenic reactions.

Based on this information, the PTAS has not identified any allergenic food safety concerns with the proposed uses of Sourvisiae®.

Conclusion

Health Canada’s review of the information presented in support of the use of Sourvisiae® does not raise concerns related to food safety.

Health Canada's opinion refers only to the food use of Sourvisiae®.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food and Nutrition 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 and Nutrition Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca