Safety evaluation of the food enzyme asparaginase from the non‐genetically modified Saccharomyces cerevisiae strain ARY‐2

TL;DR

This study evaluates the safety of a food enzyme from yeast to reduce acrylamide in processed foods, finding it generally safe but with a possible risk of allergic reactions.

Contribution

The study provides a safety evaluation of a non-genetically modified asparaginase enzyme for food use.

Findings

The enzyme does not require separation from yeast cells during production.

Dietary exposure estimates were up to 32.246 mg TOS/kg body weight per day.

No safety concerns were identified under intended use conditions, though allergic reactions remain a potential risk.

Abstract

The asparaginase (L‐asparagine amidohydrolase, EC 3.5.1.1) is produced by the non‐genetically modified Saccharomyces cerevisiae strain ARY‐2 by Renaissance BioScience Corporation. The food enzyme is not separated from the yeast cells during the enzyme production. The food enzyme is intended to be used to reduce acrylamide formation during food processing at high temperature and low moisture conditions by hydrolysing asparagine. Dietary exposure was estimated to be up to 32.246 mg TOS/kg body weight per day in European populations. Toxicity tests were considered unnecessary by the Panel because the production strain was considered safe and no issues of concern resulting from the food enzyme manufacturing process were identified. A search for the homology of the amino acid sequence of the asparaginase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Asparaginase activity	ASNU/g a	550.6	534.8	481.3
Protein	%	33.6	34.1	36.4
Ash	%	6.1	5.9	5.9
Water	%	5.5	5.0	5.2
Total organic solids (TOS) b	%	88.4	89.1	88.9
Activity/TOS ratio	ASNU/mg TOS	0.62	0.60	0.54

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 22

Food manufacturing process a	Raw material (RM)	Recommended use level (g TOS/kg RM) b , c
Prevention of acrylamide formation in foods
Fine bakery wares	Flour	0.36–1.15
Bread and rolls	Flour
Biscuits, rusks and cookies for children	Flour
Ready‐to‐eat meal for children, cereal based	Flour
Pretzel	Flour
Snack foods	Flour	0.36–8.88
Cereal‐based products (e.g. breakfast cereals)	Cereals
Ready‐to‐eat meal for children	Fruit and vegetables
French fries	Potato flour
Potato‐based snacks (e.g. sliced crisps)	Potatoes
Chips	Cereals
Coffee beverages	Coffee beans

TABLE 3. Summary of the estimated dietary exposure to food enzyme‐TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	3.679–14.789 (12)	5.953–15.552 (15)	2.827–13.099 (19)	1.043–8.267 (21)	2.703–6.019 (22)	2.514–5.081 (23)
Min–max 95th percentile (number of surveys)	13.916–32.246 (11)	13.951–29.644 (14)	6.944–25.292 (19)	2.784–15.863 (20)	5.270–11.772 (22)	4.677–9.370 (22)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level added to the raw materials	+
Yeast cells propagate during the dough preparation, therefore, increasing the amount of TOS in the dough.	−
The highest among the two recommended maximum use levels was used in the calculation	+
In addition to the types of raw materials shown in Table 2, the calculation included also cocoa beans and plums as possible raw materials.	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Assumption that 100% of TOS remain in the final foods	+

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer.

All food enzymes currently on the EU market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

Background and terms of reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7(2) of Regulation (EC) No 1332/2008 on food enzymes.

On 23 December 2023, a new application has been introduced by the applicant ‘Renaissance BioScience Corp.’ for the authorisation of the food enzyme Asparaginase from a non‐genetically modified Saccharomyces cerevisiae (strain ARY‐2).

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: Asparaginase from a non‐genetically modified Saccharomyces cerevisiae (strain ARY‐2) in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme asparaginase from non‐GM Saccharomyces cerevisiae, Acryleast™ (ARY‐2).

Additional information, requested from the applicant during the assessment phase on 05 February 2025, was received on 19 February 2025 (see Documentation provided to EFSA).

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20023 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 07 March to 28 March 2025.4 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureAsparaginaseSystematic nameL‐asparagine amidohydrolaseSynonymsAsparaginase II; L‐asparaginase; α‐asparaginaseIUBMB NoEC 3.5.1.1CAS No9015‐68‐3EINECS No232‐765‐3

Asparaginases catalyse the hydrolysis of L‐asparagine, releasing L‐aspartic acid and ammonia. The food enzyme under assessment is intended to reduce acrylamide formation during food processing by hydrolysing asparagine as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The enzyme is produced with the non‐genetically modified yeast Saccharomyces cerevisiae strain ARY‐2, which is deposited at the ■■■■■.5 The production strain was identified as S. cerevisiae by using whole genome sequencing data, with 97.63% of reads mapping to the genome of the reference strain S. cerevisiae S288C. DNA sequences of ITS1, and 18S and 25S rRNA genes showed a 100% nucleotide sequence identity to those of S. cerevisiae S288C.6

The S. cerevisiae strain ARY‐2 was obtained from a commercially available strain of S. cerevisiae by ■■■■■.

The species S. cerevisiae is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied, provided that the absence of resistance to antimycotics used for medical treatment of yeast infections in cases where viable cells are added to the food or feed chain is verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022). As no data on the sensitivity to antimycotics has been provided, the production strain ARY‐2 cannot qualify for the QPS status.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,7 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with good manufacturing practice.

The production strain is grown as a pure culture using a typical industrial medium in a submerged, fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the yeast is separated from the fermentation broth by centrifugation and rinsed with water. ■■■■■ by filtration. ■■■■■ extruded and finally dried in fluidised beds prior to analysis.8 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.9

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The food enzyme consists of the dry biomass of the viable production strain that expresses the asparaginase. The asparaginase is a single polypeptide chain of ■■■■■ amino acids.10 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.11

No other enzyme activities were reported.

The applicant's in‐house determination of asparaginase activity is based on the hydrolysis of L‐asparagine (reaction conditions: ■■■■■). The ammonia released is measured by a colorimetric reaction. The enzyme activity is expressed in asparaginase activity units (ASNU)/g. One ASNU is defined as the amount of enzyme that produces 1 μmol of ammonia per min under the conditions of the assay.12

The L‐asparaginase has a temperature optimum around 60°C (pH 6) and a pH optimum around pH 6.5. Inactivation of the enzyme is reported above 80°C.13

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).14 The mean total organic solids (TOS) was 88.8% and the mean enzyme activity/TOS ratio was 0.59 ASNU/mg TOS.

Purity

3.3.3

The lead content in the three batches was below 0.05 mg/kg,15 ^,^ 16 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).17

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

Viable cells and DNA of the production strain

3.3.4

The applicant stated that the food enzyme contains viable yeast cells.18

Toxicological data

3.4

Although the production strain does not qualify for the QPS approach, the Panel considered that no toxicological risks are expected because the strain was unambiguously identified as S. cerevisiae, which is not expected to produce toxic compounds. In addition, no issues of concern arising from the production process of the food enzyme were identified (see Sections 3.1, 3.3). Consequently, the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

Allergenicity

3.4.1

The allergenicity assessment considered only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the asparaginase produced with the Saccharomyces cerevisiae strain ARY‐2 was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA, GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the AllergenOnline database.19

No reports on oral or respiratory sensitisation or elicitation reactions of the asparaginase under assessment have been published.20

Asparaginases administered intramuscularly or intravenously are used in the treatment of different types of cancer, where they may cause sensitisation (Bryant, 2001; Burke & Zalewska‐Szewczyk, 2022; Marini et al., 2019). However, no allergic reactions after oral exposure have been reported.

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the asparaginase under assessment.

The production strain belongs to the Saccharomyces genus, which is known to cause respiratory allergy (Baldo & Baker, 1988; Vermani et al., 2015). Allergic reactions upon dietary exposure have been observed, but are rare (Cahill et al., 2022). The food enzyme consists of the production strain biomass.

■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that allergenic proteins will be present in the food enzyme.

In conclusion, the Panel considered that, under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme, particularly for yeast allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after yeast consumption.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The yeast is used as a source of asparaginase to reduce acrylamide formation during food processing for a variety of foods at the use levels presented in Table 2. The applicant states that, due to its minor leavening capacity, this yeast will not substitute for the traditional yeast used in baking.21

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 22

The yeast can be added to a variety of raw materials to hydrolyse asparagine, reducing the formation of acrylamide during the subsequent processing at high temperature and low moisture (e.g. baking, frying, roasting). For baked/fried/toasted foods, the food enzyme is added to a variety of starch‐rich food commodities (e.g. flour, potato) at various stages of manufacturing. For bread, biscuits, breakfast cereals and crisps, it is added to flour during dough making.23 For snacks, it is added before frying.24 Fruit and vegetables in ready‐to‐eat meals for children are treated with the food enzyme during mashing.25 Green coffee beans are treated before roasting.26 Potato and vegetable chips are dipped into an enzyme solution before baking or frying.27 This enzymatic treatment reduces the formation of acrylamide from asparagine under high temperature processing conditions. The yeast remains in the final processed foods.

Based on the data provided on the temperature profile (see Section 3.3.1) and the processing steps applied in the respective food manufacturing processes, the asparaginase is expected to be inactivated in the final foods.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool28 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme‐TOS per age class, country and survey, as well as the contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 32.246 mg TOS/kg bw per day in infants at the 95th percentile.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

The conservative approach applied to estimate the dietary exposure to the food enzyme‐TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

Margin of exposure

3.6

Since no toxicological assessment was considered necessary by the Panel, a margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided and the absence of issues of concern arising from the production strain and the manufacturing process, the Panel concluded that the food enzyme asparaginase from the non‐genetically modified Saccharomyces cerevisiae strain ARY‐2 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Asparaginase from non‐GM Saccharomyces cerevisiae, Acryleast™ Pro (ARY‐2). December 2023. Submitted by Renaissance BioScience Corporation.

Additional information. February 2025. Submitted by Renaissance BioScience Corporation.ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food EnzymesGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonQPSqualified presumption of safetyTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00076

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Henk Van Loveren, Laurence Vernis and Holger Zorn.

LEGAL NOTICE

The scientific output published implements EFSA's decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA, they are consequently withheld from public disclosure by redaction.

Supporting information

APPENDIX A: Dietary exposure estimates to the food enzyme‐TOS in details