Revised safety evaluation of the food enzyme leucyl aminopeptidase from the non‐genetically modified Aspergillus sp. strain AE‐MB

TL;DR

A revised safety evaluation concludes that a food enzyme from a non-genetically modified fungus is safe under new usage conditions.

Contribution

New safety assessment of leucyl aminopeptidase in seven food processes with updated exposure data.

Findings

Dietary exposure to the enzyme was calculated at up to 0.367 mg TOS/kg body weight per day.

Revised margin of exposure was at least 499, indicating no safety concerns under new conditions.

Safety conclusion is based on integrating new data into prior evaluations.

Abstract

The food enzyme leucyl aminopeptidase (EC 3.4.11.1) is produced with the non‐genetically modified Aspergillus sp. strain AE‐MB by Amano Enzyme Inc. In a previous evaluation, the Panel concluded that the food enzyme could not be considered safe when used in five food manufacturing processes, due to insufficient margins of exposure estimated for all age groups. Subsequently, the applicant requested to withdraw one use, to include three additional food manufacturing processes, and to revise the use levels. In the present assessment, EFSA revised the safety evaluation of this food enzyme when used in a total of seven food manufacturing processes. The dietary exposure to the food enzyme–total organic solids (TOS) was calculated to be up to 0.367 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (183 mg TOS/kg bw per day, the lowest dose tested), the revised margin of exposure was at least 499. Having integrated new data into 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. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 9

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of dairy products
Production of flavouring preparations from dairy products	Dairy ingredients (cheese, cream, butter, etc.)	65.6	32.8
Production of modified milk proteins	Milk proteins (casein, whey, etc)	65.6
Processing of meat and fish products
Production of modified meat and fish products	Meat or fish		32.8
Production of protein hydrolysates from meat and fish proteins	Animal proteins	164	3427
Processing of cereals and other grains
Production of distilled alcohol	Cereals	32.8
Processing of plant‐ and fungal‐derived products
Production of tea and other herbal and fruit infusions	Tea leaf extract	32.8
Production of protein hydrolysates from plants and fungi	Plant proteins	164	3427
Processing of yeast and yeast products	Yeast cells	131.2	32.8

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)	0.012–0.087 (12)	0.018–0.056 (15)	0.011–0.073 (19)	0.002–0.024 (21)	0.002–0.014 (22)	0.001–0.015 (23)
Min–max 95th percentile (number of surveys)	0.028–0.187 (11)	0.052–0.143 (14)	0.043–0.367 (19)	0.009–0.087 (20)	0.006–0.048 (22)	0.003–0.046 (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	+
For yeast processing, although yeast extract is the only final food ingredients shown in flowchart, 21 the food categories chosen for calculation included also those containing mannoproteins resulted from the treatment of yeast cell wall	+
The use of a conservative scenario to consider that the food enzyme–TOS fully remain in infant and follow‐on formulae	+
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	+/−
Exclusion of one processes from the exposure estimation: – production of distilled alcohol	−

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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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.

An application has been introduced by the applicant “Amano Enzyme Inc.” for the authorisation of the food enzyme Leucyl aminopeptidase from a non‐genetically modified strain of Aspergillus oryzae (strain AE‐MB).

The food enzyme has been assessed by EFSA with a negative opinion on 6 December 2023. This new application contains supplementary information to address the data gaps identified in the scientific opinion (EFSA‐Q‐2014‐00114).

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the application falls within the scope of the food enzyme Regulation and contains all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The Commission requests the European Food Safety Authority (EFSA) to verify the requirements of Article 32b of Regulation (EC) No 178/2002, the suitability of the data for risk assessment following the requirements of Article 12(2) of Regulation (EU) No 234/2011 and to carry out the safety assessment and the assessment of possible confidentiality requests on the new data only for the following food enzyme: Leucyl aminopeptidase from a non‐genetically modified strain of Aspergillus oryzae (strain AEMB) in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.4

Interpretation of the Terms of Reference

1.2

EFSA has already issued a scientific opinion for the safety of the food enzyme Leucyl aminopeptidase from a non‐GM Aspergillus sp. strain AE‐MB in December 2023 (EFSA CEP Panel, 2024). The present opinion is a follow‐up of the previous opinion and assesses only new information provided in the current food enzyme application (EFSA‐Q‐2024‐00210). Other aspects of the safety of the food enzyme were addressed in the previous Scientific Opinion (EFSA CEP Panel, 2024).

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a revised dossier in support of the application for authorisation of the food enzyme leucyl aminopeptidase from Aspergillus sp. strain AE‐MB. Three new annexes were included.

Additional information, requested from the applicant during the assessment process on 24‐September 2024, was received on 23 December 2024 (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 178/20025 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 29 January to 19 February 2025.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureLeucyl aminopeptidaseSystematic name–SynonymsCytosol aminopeptidase, leucine aminopeptidase, peptidase SIUBMB NoEC 3.4.11.1CAS No9001‐61‐0EINECS No232‐618‐3

Leucyl aminopeptidases catalyse the hydrolysis of the peptide bonds of N‐terminal amino acid residues of proteins or peptides, with a preference for leucine residues, resulting in the release of free amino acids. In December 2023, the production strain could not be be assigned unambiguously as Aspergillus flavus or Aspergillus oryzae, and the food enzyme could not be considered safe under the intended conditions of use, given the insufficient margins of exposure (EFSA CEP Panel, 2024).

As a follow‐up, the applicant requested to withdraw one use, to include three additional food manufacturing processes, and to revise the use levels. In the current evaluation, EFSA revised the exposure assessment and updated the safety evaluation of this food enzyme, when used in seven food manufacturing processes. EFSA also requested the applicant to provide new data on the taxonomic identification of the production strain.

Source of the food enzyme

3.1

The current characterisation of the source organism supersedes section 3.1 of the previous evaluation (EFSA CEP Panel, 2024).

The production strain is deposited at the ■■■■■ with the deposition number ■■■■■.7 Previous ■■■■■ analysis based on ■■■■■ did not allow its assignment to Aspergillus flavus or Aspergillus oryzae. Consequently, EFSA referred to the production strain as Aspergillus sp. (EFSA CEP Panel, 2024). In the current evaluation, EFSA requested to refine the ■■■■■ analysis by using a set of concatenated core gene sequences. ■■■■■.8 The new data were still insufficient for the assignment of the production strain to either A. oryzae or A. flavus. Therefore, the production strain remains designated as Aspergillus sp. in this opinion.

Production of the food enzyme

3.2

See the previous evaluation (EFSA CEP Panel, 2024).

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

See the previous evaluation (EFSA CEP Panel, 2024).

Chemical parameters

3.3.2

See the previous evaluation (EFSA CEP Panel, 2024).

Purity

3.3.3

See the previous evaluation (EFSA CEP Panel, 2024).

Viable cells of the production strain

3.3.4

See the previous evaluation (EFSA CEP Panel, 2024).

Toxicological data

3.4

See the previous evaluation (EFSA CEP Panel, 2024).

Allergenicity

3.4.1

See the previous evaluation (EFSA CEP Panel, 2024).

Dietary exposure

3.5

The current dietary exposure supersedes Section 3.5 of the previous evaluation (EFSA CEP Panel, 2024).

Revised intended use of the food enzyme

3.5.1

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

TABLE 1: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 9

The applicant withdrew one use of this food enzyme (production of modified meat and fish products10), and added three food manufacturing processes (production of modified milk proteins, production of distilled alcohol, and production of tea and other herbal and fruit infusions11).

The Panel noted substantial changes in the use levels recommended in the current application when compared to the previously reported levels. The applicant ascribes these changes to the availability of more recent information on actual use levels.12

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

In the production of modified milk proteins, the food enzyme is used to treat milk proteins.13 The protein hydrolysates are used in infant formulae, follow‐on formulae and food for special medical purposes as an ingredient.14 The hydrolysis of peptide bonds in proteins releases peptides and amino acids, facilitating their absorption by infants and patients.15

The applicant performed an ultrafiltration of the food enzyme solution with a ■■■■■ membrane with the objective to support the food enzyme removal in infant and follow‐on formulae. The amount of food enzyme proteins before and after the ultrafiltration was measured ■■■■■. No proteins were detected in the permeate fraction.16 These data were considered by the Panel as insufficient to confirm the absence of total organic solids (TOS) in infant and follow‐on formulae, considering that the majority of the TOS components, that have a low molecular mass, may pass through the ultrafiltration membrane. Therefore, the Panel opted for a conservative scenario by considering that all of the food enzyme–TOS remain in these formulae.

In the production of distilled alcohol, the food enzyme may be added to cereals during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps.17 The enzymatic treatment improves the yield and enhances the access of amylolytic enzymes to the starch granules, facilitating the degradation of starch and non‐starch polysaccharides into fermentable sugars. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaf extracts at the beginning of the food manufacturing process.18 The hydrolysis catalysed by this food enzyme improves the sensory properties of tea extracts.19 The food enzyme–TOS remain in the final products.

The food enzyme has a temperature optimum around 70°C (pH 7) and a pH optimum around 7.0 (37°C). After a pre‐incubation of the food enzyme for 1 h at different temperatures (pH 7), the leucyl aminopeptidase activity decreased above 60°C, showing no residual activity above 75°C (EFSA CEP Panel, 2024). Based on these data, the Panel considered that the food enzyme is inactivated in the food manufacturing processes listed in Table 1 in which the food enzyme–TOS remain in the final foods.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the six 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 webtool20 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). 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 0.367 mg TOS/kg bw per day in children 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 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 one food manufacturing process from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact on the overall estimate derived.

Margin of exposure

3.6

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 183 mg TOS/kg bw per day, the lowest dose tested, resulting in a margin of exposure (MoE) for infants, toddlers, children, adolescents, adults and the elderly of at least 135, 81, 83, 109, 160 and 184, respectively (EFSA CEP Panel, 2024).

A comparison of the NOAEL with the newly derived exposure estimates of 0.001–0.087 mg TOS/kg bw per day at the mean and from 0.003 to 0.367 at the 95th percentile resulted in a margin of exposure of at least 499.

Despite more uses were considered in the current assessment, the newly derived MoE is higher than the one previously calculated. This is mainly due to the significant reduction of use levels in the highly contributing food manufacturing processes.

CONCLUSIONS

4

Having integrated new data into the previous evaluation, the Panel concludes that the food enzyme leucyl aminopeptidase produced with the non‐genetically modified Aspergillus sp. strain AE‐MB does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of Leucyl aminopeptidase from Aspergillus oryzae AE‐MB in accordance with the Regulation (EC) No 1331/2008. April 2024. Submitted by Amano Enzyme Inc.

Additional information. December 2024. Submitted by Amano Enzyme Inc.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFEZEFSA Panel on Food EnzymesIUBMBInternational Union of Biochemistry and Molecular BiologyMOEmargin of exposureNOAELno observed adverse effect levelRMRaw MaterialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00210

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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