Safety evaluation of the food enzyme sucrose:sucrose fructosyltransferase from the genetically modified Yarrowia lipolytica strain E4772

TL;DR

This study evaluates the safety of a genetically modified enzyme used in food processing and concludes it is safe under intended use conditions.

Contribution

The study provides a safety evaluation of a novel food enzyme produced by a genetically modified yeast strain.

Findings

The enzyme's production strain meets the qualified presumption of safety (QPS) criteria.

Dietary exposure to the enzyme is low and unlikely to cause safety concerns.

No homology to known allergens was found, though a low risk of allergic reactions cannot be fully excluded.

Abstract

The food enzyme sucrose:sucrose fructosyltransferase (sucrose:sucrose 1′‐β‐d‐fructosyltransferase; EC 2.4.1.99) is produced with the genetically modified Yarrowia lipolytica strain E4772 by SEQENS. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is free from viable cells of the production organism, but not free from recombinant DNA. The food enzyme is intended to be used in the processing of sugars for the production of fructo‐oligosaccharides. The dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.088 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the sucrose: sucrose fructosyltransferase 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, but that the likelihood is low. 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
Sucrose:sucrose fructosyltransferase activity	U/g a	6595	6951	7587
Protein	%	0.2	0.2	0.2
Ash	%	0.6	0.6	0.6
Water	%	98.2	98.2	98.3
Total organic solids (TOS) b	%	1.2	1.2	1.1
Activity/TOS ratio	U/mg TOS	549.6	579.3	689.7

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 35

Food manufacturing proces a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of sugars
Production of fructo‐oligosaccharides (FOS)	Sucrose syrup	17.14–51.43

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)	0.012–0.038 (12)	0.001–0.017 (15)	0–0.001 (19)	0–0 (21)	0–0 (22)	0–0 (23)
Min–max 95th percentile (number of surveys)	0.039–0.088 (11)	0.008–0.039 (14)	0.001–0.006 (19)	0–0.001 (20)	0–0.001 (22)	0–0.002 (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	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

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 November 2023, a new application has been introduced by the applicant SEQENS for the authorization of the food enzyme Fructosyltransferase from a genetically modified Yarrowia lipolytica (strain P4654).

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: Fructosyltransferase from a genetically modified Yarrowia lipolytica (strain P‐4654), in accordance with Regulation (EC) No 1332/2008 stabilishing a common authorization procedure for food additives, food enzymes and food flavourings.^2^

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme fructosyltransferase from a genetically modified Yarrowia lipolytica strain E4772.

Additional information was requested from the applicant during the assessment process on 14 November 2024 and on 20 March 2025, and received on 20 February 2025 and on 10 April 2025, respectively (see Section 5).

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 of the application.

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 11 March to 1 April 2025 for which no comments were received.

ASSESSMENT

3

IUBMB nomenclatureSucrose:sucrose fructosyltransferaseSystematic nameSucrose:sucrose 1′‐β‐d‐fructosyltransferaseSynonymsSST; sucrose:sucrose 1‐fructosyltransferaseIUBMB NoE.C. 2.4.1.99CAS No73379‐56‐3EINECS NoNot assigned

Sucrose:sucrose fructosyltransferases catalyse the generation of fructo‐oligosaccharides (FOS) of different chain lengths by transferring the fructose moiety from sucrose to another sucrose molecule or to other FOS to generate FOS of different chain length.

The food enzyme under assessment is intended to be used in the processing of sugars for the production of fructo‐oligosaccharides.

Source of the food enzyme

3.1

The sucrose:sucrose fructosyltransferase is produced with the genetically modified yeast Y. lipolytica strain E4772 which is deposited at the Collection Nationale de Cultures de Microorganismes (CNCM, France) with deposition number ■■■■■.4 The production strain was identified as Y. lipolytica ■■■■■.5

The species Y. lipolytica is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied for production purposes only (implying the absence of viable cells of the production organisms in the final product; EFSA, 2007; EFSA BIOHAZ Panel, 2022).6 ^,^ 7 In addition, the genetic modifications should not give rise to safety concerns. As the genetic modifications are considered safe (see Section 3.1.4) and the product is free from the viable cells (see Section 3.3.4), the production strain is considered to qualify for the QPS approach and is considered safe.

Characteristics of the parental and recipient microorganisms

3.1.1

The parental organism is ■■■■■.8

The recipient strain ■■■■■.9 ^,^ 10

Characteristics of introduced sequences

3.1.2

The sequence encoding the sucrose:sucrose fructosyltransferase (FTase) derives from ■■■■■.

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to enable the production strain to synthesise sucrose:sucrose fructosyltransferase. For this purpose, ■■■■■.11 ^,^ 12 ■■■■■

■■■■■.13 ^,^ 14

Safety aspects of the genetic modification

3.1.4

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.

The production strain Y. lipolytica strain P4654 differs from the recipient strain in its capacity to produce sucrose:sucrose fructosyltransferase ■■■■■. No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,15 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.16

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by centrifugation. The supernatant containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.17 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.18

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 sucrose:sucrose fructosyltransferase is a single polypeptide chain of 613 amino acids.19 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.20 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.21 A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between ■■■■■ and ■■■■■ kDa markers.

No other enzyme activities were reported.

The applicant's in‐house determination of sucrose:sucrose fructosyltransferase activity is based on hydrolysis of sucrose (reaction conditions: pH ■■■■■, ■■■■■°C, ■■■■■ min). The enzyme activity is determined by quantifying the release of glucose using high‐performance liquid chromatography. The enzyme activity is quantified relative to an internal enzyme standard and expressed in U/mL.22

The food enzyme has a temperature optimum around 60°C (pH 5.6) and a pH optimum around pH 5.5 (37°C).23 Thermostability was tested after pre‐incubation of the food enzyme at different temperatures. Enzyme activity decreased at 50°C, showing no residual activity after 10 min at 80°C or 3 min at 90°C.24

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation (Table 1).25 The mean total organic solids (TOS) of the three batches for commercialisation was 1.2% and the mean enzyme activity/TOS ratio was 606.2 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.05 mg/kg,26 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, arsenic, cadmium and mercury contents were below the limits of quantification of the employed methods.27 ^,^ 28

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).29

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 absence of viable cells of the production strain in the food enzyme was demonstrated ■■■■■. No colonies were produced. A positive control was included.30 ^,^ 31

The presence of recombinant DNA in the food enzyme was assessed ■■■■■.32

As the production strain does not carry any genes of concern, the presence of traces of recombinant DNA in this food enzyme is not considered a safety concern.

Toxicological data

3.4

The production strain qualifies for the QPS approach to safety assessment (see Section 3.1). In addition, no issue of concern arising from the production process of the food enzyme was identified (see Sections 3.2 and 3.3). Thus, 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 sucrose:sucrose fructosyltransferase produced with the Y. lipolytica strain P4654 was assessed by comparing its amino acid sequence with those of known allergens as described in the 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.33

No reports on oral or respiratory sensitisation or elicitation reactions of the sucrose:sucrose fructosyltransferase under assessment have been published. No allergic reactions upon dietary exposure to any fructosyltransferase have been reported in the literature.34

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

■■■■■ products, known sources of allergens, are present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain. The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in one food manufacturing process at the recommended use level summarised in Table 2.

TABLE 2: Intended use and recommended use level of the food enzyme as provided by the applicant. 35

In this process, the food enzyme is added to sucrose syrup (67–70° brix) to produce FOS by transfructosylation.36 The resulting FOS syrups are subsequently treated by heat for 3 min at 90°C.37 FOS can be used directly or (optional) purified38 as an ingredient in a variety of foods. The food enzyme–TOS remain in the FOS syrups.

The applicant carried out two experiments to show that no residual sucrose:sucrose fructosyltransferase activity is present in FOS syrups produced in the process described above. An enzymatically treated FOS syrup was pasteurised at the processing conditions (90°C, 3 min) and then stored at −20°C. In the first experiment, the FOS syrup was re‐incubated at 50°C and its compositional change was monitored for 2 h by analysing the glucose and sucrose content. No changes were detected. In the second experiment, the FOS syrup was diluted and supplemented with sucrose syrup, then re‐incubated at 50°C and its compositional change was monitored for 5 h. Also no changes were detected.39

On the basis of these data, together with the data provided on thermostability in Section 3.3.1, the Panel considered that the food enzyme is inactivated during the production of FOS.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool40 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 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 0.088 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 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, the margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the food enzyme manufacturing process, the Panel concluded that the food enzyme sucrose: sucrose fructosyltransferase produced with the genetically modified Y. lipolytica strain P4654 does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism but not free of recombinant DNA.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the authorisation of fructosyltransferase from a genetically modified Yarrowia lipolytica as a new food enzyme. January 2024. Submitted by SEQENS.

Additional information. February 2025. Submitted by SEQENS.

Additional information. April 2025. Submitted by SEQENS.

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 NationsFOSFructo‐oligosaccharidesGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionqPCRquantitative polymerase chain reactionQPSqualified presumption of safetyTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00010

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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, Monika Sramkova, 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