Safety evaluation of an extension of use of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR‐962

Abstract

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus oryzae strain AR‐962 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 requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of seven food manufacturing processes. As the food enzyme–total organic solids (TOS) is removed from the final foods in three food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining four processes. Dietary exposure was up to 0.575 mg TOS/kg body weight (bw) per day in European populations. When combined with the NOAEL reported in the previous opinion (1000 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 1739. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ 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 1331/2008 ² 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.

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background as provided by the European Commission

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 1332/2008¹ on food enzymes.

Pectin esterase from a genetically modified strain of Aspergillus oryzae (strain AR‐962) is a food enzyme to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 24 March 2023, a new application has been introduced by the applicant “AB Enzymes Gmbh” for the extension of the conditions of use for the above food enzyme in several food processes.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of an extension of the condition of use for the following food enzyme: Pectin esterase from a genetically modified strain of Aspergillus oryzae (strain AR‐962), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings. ³

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for the authorisation of the extension of the use of food enzyme pectinesterase from a genetically modified Aspergillus oryzae strain AR‐962.

Additional information was requested from the applicant during the assessment process on 30 October 2023 and received on 20 November 2023 (see ‘Documentation provided to EFSA’).

2.2. Methodologies

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 to evaluate this application.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁴ 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 15 November to 6 December 2023. ⁵ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Pectinesterase
Systematic name	Pectin pectylhydrolase
Synonyms	Pectin methylesterase, pectin methoxylase, pectin demethoxylase
IUBMB No	EC 3.1.1.11
CAS No	9025‐98‐3
EINECS No	232–807‐0

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 February 2023 (EFSA CEP Panel, 2023b). Following an application for two additional food manufacturing processes, EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in seven food manufacturing processes.

3.1. Dietary exposure

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

3.1.1. Revised intended use of the food enzyme

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.

Food manufacturing process a	Raw material (RM)	Maximum 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	2		2
Production of fruit and vegetable products other than juices	Fruit and vegetables	13	Puree	13
		26	Fruit firming	26
Production of wine and wine vinegar	Grapes	1		1
Production of distilled alcoholic beverages d	Fruit	2
Production of alcoholic beverages other than from grapes	Fruit	2
Processing of plant‐ and fungal‐derived products
Production of green coffee beans by demucilation	Coffee cherry	0.5		0.5
Production of plant extracts as flavouring preparations	Fruit and vegetables	2		2

^a The name has been harmonised by EFSA according to the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023a).

^b The numbers in bold were used for calculation.

^c The previous evaluation is made for the food enzyme application EFSA‐Q‐2021‐00072.

^d The food manufacturing process was not included in the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023a).

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

In the production of distilled alcoholic beverages and non‐distilled alcoholic beverages other than from grapes, the food enzyme is added to fruits, such as apples and pears, during the peeling and crushing. It is also added to the fruit must before fermentation. ⁷ The enzymatic treatment contributes to the degradation of pectin that, in turn, can increase the processability and the yield of the fruit must. ⁸ The food enzyme‐TOS is removed by distillation in brandies but remains in other alcoholic beverages like cider and perry. ⁹

Based on the thermostability evaluated previously (EFSA CEP Panel, 2023b) and the downstream processing steps applied in the food manufacturing processes, it was expected that the pectinesterase was inactivated during most of the food manufacturing processes, but may remain active in wine and in fruit and vegetable juices, depending on the pasteurisation conditions.

3.1.2. Dietary exposure estimation

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

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the 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 (bw). This was done for all individuals across all surveys, resulting in the 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 0.575 mg TOS/kg bw per day in infants at the 95th percentile.

TABLE 2.

Updated dietary exposure to the food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg bw 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)	0.007–0.342 (12)	0.03–0.216 (15)	0.021–0.218 (19)	0.004–0.117 (21)	0.003–0.057 (22)	0.002–0.063 (23)
Min–max 95th percentile (number of surveys)	0.032–0.575 (11)	0.085–0.540 (14)	0.074–0.455 (19)	0.018–0.264 (20)	0.013–0.144 (22)	0.010–0.144 (22)

3.1.3. Uncertainty analysis

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.

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
Exposure from the production of fruit and vegetable products other than juices, including puree, was calculated using the TOS indicated for fruit firming	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
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	−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

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 on the overall estimate derived.

3.2. Margin of exposure

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1000 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure (MOE) of at least 1546 (EFSA CEP Panel, 2023b).

A comparison of the NOAEL with the newly derived exposure estimates of 0.002–0.342 mg TOS/kg bw per day at the mean and from 0.010 to 0.575 mg TOS/kg bw per day at the 95th percentile resulted in a MOE of at least 1739.

Despite that more uses were considered in the current assessment, the newly derived MOE is higher than that previously calculated. This is due to the revision of food groups and technical factors used for each food manufacturing process (EFSA CEP Panel, 2023a). In addition, dietary surveys have been updated in the EFSA food consumption database.

4. CONCLUSION

Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that the food enzyme pectinesterase produced with the genetically modified Aspergillus oryzae strain AR‐962 does not give rise to safety concerns under the revised intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for authorisation of Pectinesterase from a genetically modified Aspergillus oryzae (strain AR‐962). March 2023. Submitted by AB Enzymes GmbH.

Additional information. November 2023. Submitted by AB Enzymes GmbH.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

European Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

EU

European Union

IUBMB

International Union of Biochemistry and Molecular Biology

MOE

margin of exposure

NOAEL

no observed adverse effect level

RM

raw material

TOS

total organic solids

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00523

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Claude Lambré, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Inger‐Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

APPENDIX A. Dietary exposure estimates to the food enzyme–TOS in details

A.1.

Appendix A can be found in the online version of this output (in the ‘Supporting information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

B.1.

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia * , Serbia * , Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia * , Serbia * , Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina * , Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro * , Netherlands, Portugal, Romania, Serbia * , Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina * , Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro * , Netherlands, Portugal, Romania, Serbia * , Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro * , Netherlands, Portugal, Romania, Serbia * , Slovenia, Spain, Sweden

^* Consumption data from these pre‐accession countries are not reported in Table 2 of this opinion; however, they are included in Appendix A for testing purpose.

^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

EFSA CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré, C. , Barat Baviera, J. M. , Bolognesi, C. , Cocconcelli, P. S. , Crebelli, R. , Gott, D. M. , Grob, K. , Lampi, E. , Mengelers, M. , Mortensen, A. , Rivière, G. , Steffensen, I.‐L. , Tlustos, C. , Van Loveren, H. , Vernis, L. , Zorn, H. , Roos, Y. , Cavanna, D. ,… Chesson, A. (2024). Safety evaluation of an extension of use of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR‐962. EFSA Journal, 22(4), e8700. 10.2903/j.efsa.2024.8700