Safety evaluation of an extension of use of the food enzyme pectinesterase from the genetically modified Trichoderma reesei strain RF6201

TL;DR

This study evaluates the safety of extending the use of a food enzyme produced by a genetically modified fungus in additional food manufacturing processes.

Contribution

The study provides a revised safety assessment for extended use of pectinesterase in seven food processes.

Findings

Dietary exposure to the enzyme was estimated at up to 0.488 mg TOS/kg body weight per day.

The margin of exposure was calculated to be at least 2049, indicating no safety concerns.

The enzyme is not carried into the final product in three of the seven processes.

Abstract

The food enzyme pectinesterase (pectin pectylhydrolase, EC 3.1.1.11) is produced with the genetically modified Trichoderma reesei strain RF6201 by AB Enzymes GmbH. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in five food manufacturing processes. Subsequently, the applicant has requested to extend its use to include two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from or not carried into the final foods in three food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining four processes. The dietary exposure was calculated to be up to 0.488 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 2049. Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Tables

TABLE 1. Updated intended uses and use levels of the food enzyme. 7

Food manufacturing process a		Raw material (RM)	Recommended use level (mg TOS/kg RM)
			Current evaluation b	Previous evaluation b , c
Processing of fruits and vegetables
Production of juices		Fruit and vegetables	3	3
Production of fruit and vegetable products other than juices	Puree	Fruit and vegetables	24	24
	Fruit firming		12	12
Production of wine and wine vinegar		Grapes	1	1
Production of alcoholic beverages other than grape wine		Fruit and vegetables	2–3
Production of distilled alcoholic beverages d		Fruit	2–3
Processing of plant‐ and fungal‐derived products
Production of green coffee beans by demucilation		Coffee cherries	0.5	0.5
Production of plant extracts as flavouring preparations		Fruit and vegetables	265	265

TABLE 2. Updated dietary exposure to the 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)	0.006–0.287 (12)	0.030–0.179 (15)	0.022–0.107 (19)	0.006–0.054 (21)	0.004–0.037 (22)	0.003–0.029 (23)
Min–max 95th percentile (number of surveys)	0.028–0.403 (11)	0.104–0.488 (14)	0.078–0.298 (19)	0.023–0.168 (20)	0.017–0.115 (22)	0.013–0.085 (22)

TABLE 3. 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	+
Although two different used levels were given for puree and firming, only the higher one was used in the calculation for the production of fruit and vegetable products other than juices	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of three processes from the exposure estimation: ‐ Production of distilled alcoholic beverages ‐ Production of green coffee beans by demucilation ‐ Production of plant extracts as flavouring preparations	−

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 European Union (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.

Pectin esterase produced from genetically modified Trichoderma reesei (strain RF6201) is a food enzyme included in the Register of food enzymes3 to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 18 September 2024, a new application was introduced by the applicant “AB Enzymes Gmbh” for an extension of the condition of use of the food enzyme Pectin esterase produced from genetically modified Trichoderma reesei (strain RF6201).

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: extension of the condition of use of Pectin esterase produced from genetically modified Trichoderma reesei (strain RF6201) in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings.4

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of the food enzyme pectinesterase from a genetically modified Trichoderma reesei strain RF6201.

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 existing guidance documents of 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, 2023a) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20025 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 4 to 25 March 2025.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclaturePectinesteraseSystematic namePectin pectylhydrolaseSynonymsPectin methyl esterase; pectin methoxylase; pectin demethoxylaseIUBMB NoEC 3.1.1.11CAS No9025‐98‐3EINECS No232‐807‐0

Pectinesterases catalyse the de‐esterification of pectin, resulting in the generation of pectic acid and methanol.

All aspects concerning the safety of this food enzyme, when used in five food manufacturing processes, were evaluated in June 2023 (EFSA CEP Panel, 2023b).

Following a request to update the intended uses (adding two processes), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme, when used in seven food manufacturing processes.

Dietary exposure

3.1

The current dietary exposure supersedes section 3.5 of the previous evaluation (EFSA CEP Panel, 2023b).

Revised intended use of the food enzyme

3.1.1

The food enzyme is intended to be used in seven food manufacturing processes at the use levels summarised in Table 1.

TABLE 1: Updated intended uses and use levels of the food enzyme. 7

The additional two uses of the food enzyme are described below.

In the production of alcoholic beverages other than grape wine, the food enzyme is added to fruit or vegetables during the milling/crushing and to treat the mash.8 The action of the pectinesterase de‐esterifies the pectin in the cell walls, increasing the processability and the juice yield.9 The food enzyme–total organic solids (TOS) remain in the final alcoholic beverages such as perry and cider.

In the production of distilled alcoholic beverages, the food enzyme is added to fruit during crushing and treating the mash before fermentation.10 The action of the pectinesterase de‐esterifies the pectin in the cell walls, increasing the processability and the juice yield.11 The food enzyme–TOS are not carried over with the distilled alcoholic beverages (EFSA CEP Panel, 2023a).

The food enzyme has a temperature optimum around 40°C (pH 4.5) and a pH optimum around pH 4.5 (30°C). After a pre‐incubation of the food enzyme at 85°C for different time periods (pH 4.5), no enzyme activity was detected after 2 min (EFSA CEP Panel, 2023b). Based on these data, the Panel concluded that the food enzyme is inactivated during the production of fruit and vegetable products other than juices, but may remain in its active form in wine and wine vinegar, alcoholic beverages other than grape wine, as well as in juices, depending on the processing conditions.

Dietary exposure estimation

3.1.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the four food manufacturing processes where the food enzyme–TOS remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool12 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, 2023a). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only one day per subject were excluded and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).

Table 2 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 0.488 mg TOS/kg bw per day in toddlers at the 95th percentile.

Uncertainty analysis

3.1.3

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 3.

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.

The exclusion of three food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

Margin of exposure

3.2

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1000 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure (MoE) of at least 1880 (EFSA CEP Panel, 2023b).

A comparison of the NOAEL with the newly derived exposure estimates of 0.003–0.287 mg TOS/kg bw per day at the mean and from 0.013–0.488 mg TOS/kg bw per day at the 95th percentile resulted in a MoE of at least 2049.

Despite more food manufacturing processes were considered in the current assessment, the newly derived MoE is higher than the one previously calculated. This is mainly due to the revision of food groups and technical factors used for each food manufacturing process (EFSA CEP Panel, 2023a).

CONCLUSION

4

Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that the food enzyme pectinesterase produced with the genetically modified Trichoderma reesei strain RF6201 does not give rise to safety concerns under the revised intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for modification of an already authorised pectin esterase produced with a genetically modified Trichoderma reesei strain RF6201 in accordance with Regulation (EC) No 1331/2008. January 2025. Submitted by AB Enzymes GmbH.ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionIUBMBInternational Union of Biochemistry and Molecular BiologyMoEmargin of exposureNOAELno observed adverse effect levelRMraw materialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00677

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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.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details