Revised safety evaluation of the food enzyme aqualysin 1 from the genetically modified Bacillus subtilis strain LMG S‐25520 produced by a modified process

Abstract

The food enzyme aqualysin 1 (EC 3.4.21.111) is produced with the genetically modified Bacillus subtilis strain LMG S‐25520 by Puratos N.V. In a previous evaluation, the Panel could not exclude safety concerns due to an insufficient margin of exposure and noted the presence of recombinant DNA in all food enzyme batches tested. As a follow‐up, the applicant changed the manufacturing process and provided new data. These data established that the production strain meets the requirements for qualified presumption of safety (QPS) status, and no concerns arose from the modified food enzyme manufacturing process; hence, toxicological tests were not considered necessary. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in baking processes. Dietary exposure is estimated to be up to 0.385 mg TOS/kg body weight per day in European populations. A new search for the homology of the amino acid sequence of the aqualysin 1 to known allergens was made, and matches with one food allergen, 20 respiratory allergens and three contact allergens were found. Based on the new data provided, the Panel concluded that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. The Panel concluded that the food enzyme does not give rise to safety concerns under the 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 I foods, in accordance with the specification and condition of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes. ³

On 12 January 2023, a new application has been introduced by the applicant “Puratos NV” for the authorization of the food enzyme Aqualysin from a genetically modified strain of Bacillus subtilis (LMG S‐25520).

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 the following food enzyme: Aqualysin from a genetically modified strain of Bacillus subtilis (LMG S‐25520), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings. ⁴

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Aqualysin from a genetically modified strain of Bacillus subtilis strain LMG S‐25520.

Additional information requested from the applicant during the assessment process on 1 March 2024 and on 25 September 2024, were received on 29 May 2024 and 23 October, respectively (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, 2009b) 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.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁵ and to the Decision of EFSA's Executive Director laying down the practical arrangements on the pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 7 May to 28 May 2024. ⁶ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Aqualysin 1
Synonyms	Caldolysin
IUBMB No	EC 3.4.21.111
CAS No	88747‐68‐6
EINECS No	Not available

Aqualysins are serine endopeptidases that catalyse the hydrolysis of peptide bonds at the carboxy‐terminal side of the amino acids alanine and phenylalanine, releasing peptides. The food enzyme under assessment is intended to be used in the processing of cereals and other grains for the production of baked products.

In the previous application (EFSA‐Q‐2014‐00920), the EFSA CEF Panel could not exclude safety concerns of the food enzyme aqualysin 1 produced with the genetically modified Bacillus subtilis strain LMG S‐25520 due to an insufficient margin of exposure, calculated from a no observed adverse effect level (NOAEL) identified from a repeated dose 90‐day oral toxicity study and the estimated dietary exposure. Furthermore, the Panel noted that recombinant DNA was present in all batches of the food enzyme tested (EFSA CEF Panel, 2018).

In the present follow‐up application (EFSA‐Q‐2023‐00303), the food enzyme manufacturing process was modified by the introduction of ■■■■■ and ■■■■■ steps. The applicant provided additional information to characterise the production strain; analysed three new food enzyme batches for chemical composition, purity, viable cells and DNA of the production strain, and updated the homology search against known allergens and the literature search. The present opinion assessed these new data. Other aspects of the safety of the food enzyme were addressed in the previous scientific opinion under EFSA‐Q‐2014‐00920 and cross‐referenced in the present opinion.

3.1. Source of the food enzyme

The current evaluation supersedes section 3.1.4 of the previous evaluation (EFSA CEF Panel, 2018).

The aqualysin 1 is produced with the genetically modified bacterium Bacillus subtilis strain LMG S‐25520, which is deposited at the Culture collection of the Belgian Co‐ordinated Collection of Microorganisms (BCCM/LMG) under the deposition number LMG S‐25520. ⁷ The production strain was identified as B. subtilis by whole genome sequence (WGS) analysis and de novo assembly analysis of seven closely related genomes, with an average nucleotide identity (ANI) of 99.95% compared to the B. subtilis ■■■■■ from which it derives. ^{8 , 9}

The species B. subtilis is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied, provided that the absence of acquired antimicrobial resistance genes and toxigenic activity are verified for the specific strain used, and the genetic modifications do not raise concerns (EFSA BIOHAZ Panel, 2020, 2022). A cytotoxicity test made with supernatants indicated that the production strain B. subtilis LMG S‐25520 did not induce cell damage to VERO cells using the lactate dehydrogenase assay. ^{10 , 11} WGS analysis of the production strain was conducted against two regularly updated databases using thresholds of 80% sequence identity and 70% coverage. ¹² No antimicrobial resistance genes of concern were identified.

3.1.1. Characteristics of the parental microorganisms

The parental microorganism is the bacterium B. subtilis ■■■■■. The recipient strain B. subtilis ■■■■■ was developed from the parental strain by subsequent steps of ■■■■■ and ■■■■■, resulting in a strain with mutations in the ■■■■■ encoding ■■■■■ ■■■■■ and ■■■■■, (partial) deletion of the ■■■■■ ■■■■■, ■■■■■ and ■■■■■, and mutations in the ■■■■■ and ■■■■■. All mutations were obtained by ■■■■■, and ■■■■■ were derivatives of B. subtilis ■■■■■.

During the development of the recipient strain, plasmids were used containing genes conferring resistance to chloramphenicol and kanamycin, which did not remain in the genome, as confirmed by WGS analysis of the production strain. ¹³

3.1.2. Characteristics of introduced sequences

The sequence encoding the aqualysin 1 peptidase, including ■■■■■ was obtained from the ■■■■■. It was placed under the control of the ■■■■■ and preceded by the ■■■■■, both from ■■■■■ ■■■■■. ^{14 , 15}

3.1.3. Description of the genetic modification

The purpose of the genetic modification was to enable the production strain to synthesise aqualysin 1. For this purpose, the recipient strain ■■■■■ was ■■■■■, resulting in the successive integration of ■■■■■ of the aqualysin 1 ■■■■■. The presence and the location of the aqualysin 1 encoding genes were confirmed by WGS analysis. As a consequence of ■■■■■, the ■■■■■ were disrupted.

3.1.4. Safety aspects of the genetic modification

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

The production strain B. subtilis strain LMG S‐25520 differs from the recipient strain in its capacity to produce high amounts of aqualysin 1. The absence of the antimicrobial resistance genes used during the genetic modifications, including those used for the development of the recipient strain, was confirmed by WGS analysis. ^{16 , 17}

The Panel confirms its previous conclusions that no issues of safety concern arose from the genetic modifications (EFSA CEF Panel, 2018). As the other qualifications have been met, the production strain is considered to qualify for the QPS approach.

3.2. Production of the food enzyme

The current evaluation supersedes the corresponding part in Section 3.1.5 of the previous evaluation (EFSA CEF Panel, 2018).

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

The production strain is grown as a pure culture using a typical industrial medium in a contained, submerged, fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation and heat treatment, the solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is 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. Afterwards, a ■■■■■ step is applied ■■■■■, followed by ■■■■■ and heat treatment. ¹⁸

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. ¹⁹

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.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The current evaluation supersedes the corresponding parts in Sections 3.1.2 and 3.1.3 of the previous evaluation (EFSA CEF Panel, 2018).

The aqualysin 1 is a ■■■■■ chain of ■■■■■ amino acids. The molecular mass of the mature protein, calculated from the amino acid sequence, is around ■■■■■. ²⁰ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gel showed a main protein band corresponding to an apparent molecular mass of about ■■■■■, consistent with the expected molecular mass of the enzyme. ²¹

The food enzyme was tested for xylanase and α‐amylase activities, and neither were detected. ²² No other enzyme activities were reported.

The applicant's in‐house determination of aqualysin 1 activity is based on the hydrolysis of N‐succinyl‐Ala‐Ala‐Pro‐Phe p‐nitroanilide (reaction conditions: pH 7.5, 70°C, 20 min) and is determined by measuring the release of 4‐nitroaniline spectrophotometrically at 415 nm. The enzyme activity is expressed in Protease Units (U)/mL. One U is defined as the amount of enzyme that releases 1.9 μmol of 4‐nitroaniline per minute under the assay conditions. The assay includes a pre‐incubation of the food enzyme for 90 min at 70°C in the reaction buffer (EFSA CEF Panel, 2018).

The food enzyme has a temperature optimum around 70°C (pH 9.5) and a pH optimum around pH 9.5 (70°C). Thermostability was tested after a pre‐incubation of the food enzyme at different temperatures and for different times. The aqualysin 1 shows approximately 97% residual activity after incubation for 250 min at 70°C and no residual activity after 25 min at 90°C (EFSA CEF Panel, 2018).

3.3.2. Chemical parameters

The current evaluation supersedes the corresponding part in Section 3.1.2 of the previous evaluation (EFSA CEP Panel,2018).

Data on the chemical parameters of the food enzyme were provided for three new batches intended for commercialisation (Table 1). ²³ The mean total organic solids (TOS) of the three food enzyme batches was 3.3% and the mean enzyme activity/TOS ratio was 0.4 U/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Aqualysin 1 activity	U/mL a	13.6	12.8	8.5
Protein	%	1.2	1.2	0.8
Ash	%	5.6	4.5	2.3
Water	%	92.5	91.1	94.0
Total organic solids (TOS) b	%	1.9	4.4	3.7
Activity/TOS ratio	U/mg TOS	0.7	0.3	0.2

^a U: Protease Unit.

^b TOS calculated as 100% – % water – % ash.

3.3.3. Purity

The current evaluation supersedes the corresponding part in Section 3.1.2 of the previous evaluation (EFSA CEF Panel, 2018).

The lead content in the three commercial batches was below 5 mg/kg, ^{24 , 25} 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). ²⁶ No antimicrobial activity was detected in any of the tested batches. ²⁷

The presence of aflatoxin B1, B2, G1, G2, ochratoxin A, zearalenone, deoxynivalenol, T2‐toxin and HT2‐toxin was examined in the three food enzyme batches, and all were below the limits of quantification (LoQ) of the applied analytical methods. ^{28 , 29}

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

3.3.4. Viable cells and DNA of the production strain

The current evaluation supersedes the corresponding part in section 3.1.5 of the previous evaluation (EFSA CEF Panel, 2018).

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. One mL of product was plated on two non‐selective agar medium petri dishes, which were incubated at 37°C for 72 h. No colonies were produced. A positive control was included. ^{30 , 31}

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify a ■■■■■ ■■■■■ that is specific for a region containing ■■■■■ in the ■■■■■, with a limit of detection of 10 ng spiked DNA/mL food enzyme. ³²

3.4. Toxicological data

The current evaluation supersedes Section 3.3 of the previous evaluation (EFSA CEF Panel, 2018).

As the production strain qualifies for the QPS approach of safety assessment and no issue of concern arising from the production process of the food enzyme was identified (see Sections 3.1, 3.2 and 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

3.4.1. Allergenicity

The current evaluation supersedes Section 3.4 of the previous evaluation (EFSA CEF Panel, 2018).

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

The allergenicity of aqualysin 1 produced with the genetically modified Bacillus subtilis strain LMG S‐25520 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, matches with the food allergen Bac s 1, a nattokinase from B. subtilis (48.8% sequence identity), 20 allergenic (serine) proteases from fungi and bacteria (50.0%–66.2% sequence identity) and 3 contact allergens (57.5%–65.0% sequence identity) were found in the AllergenOnline database. ³³

No reports on oral or respiratory sensitisation or elicitation reactions of the aqualysin 1 under assessment have been published.

Two studies identified Bac s 1, a nattokinase from B. subtilis as a food allergen in patients with natto allergy who tested negative for the major natto allergen poly‐γ‐glutamic acid (Awatani‐Yoshidome et al., 2022; Suzuki et al., 2023). ³⁴ The aforementioned serine proteases from fungi and bacteria are respiratory allergens. Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Cullinan et al., 1997; Poulsen,2004 ; Armentia et al., 2009). The contact allergens to which sequence homologies were found were from Trichophyton rubrum, the athlete's foot fungus, from which no allergic reactions, after oral ingestion, are expected.

The Panel considered that the results of the sequence homology and the available literature indicate a risk of allergic reactions for natto allergic individuals upon dietary exposure to the aqualysin 1 under assessment.

The production strain B. subtilis produces a poly‐γ‐glutamic acid and the nattokinase Bac s 1, a subtilisin‐like serine protease, which have both been identified as food allergens. Few case reports of allergic reactions after consumption of natto produced by B. subtilis have been reported in Japan (Inomata et al., 2012; Suzuki et al., 2023). The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the culture medium from which the food enzyme is obtained.

■■■■■, a known source of allergens, is present in the medium fed to the microorganisms. 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 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, particularly for natto allergic individuals, cannot be excluded. However, the likelihood of such reactions will not exceed the risk of reactions after natto consumption.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The current evaluation supersedes Sections 3.1.7 and 3.1.8 of the previous evaluation (EFSA CEF Panel, 2018).

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

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of cereals and other grains
Production of baked products	Flour	Up to 37.71 d	Up to 190 e

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

^b The numbers in bold were used for calculation.

^c The previous evaluation is made for the food enzyme application EFSA‐Q‐2014‐00920.

^d Based on 0.318 UNIT/ mg TOS, as reported in the EFSA‐Q‐2023‐00303.

^e Based on 0.063 UNIT/ mg TOS, as reported in the EFSA‐Q‐2014‐00920.

The use level, expressed as UNIT/kg raw material (RM), remained unchanged when compared to the previous evaluation (i.e. up to 12 UNIT/kg RM ³⁶ ). However, when expressed as mg TOS/kg RM, the use level decreased in the current evaluation. This change is due to the higher mean activity/TOS ratio found for the new food enzyme batches, which were produced by the modified food enzyme manufacturing process. According to the applicant, this is a consequence of the improved effectiveness of the manufacturing process of the food enzyme that consequently increased its purity. ³⁷

The food enzyme aqualysin 1 is added to flour during the preparation of dough. ³⁸ It is used to hydrolyse gluten proteins (glutenin, gliadin), which reduces viscosity, increases extensibility and improves the structure of the dough. This results in better processability of the dough and in more uniform products. The food enzyme–TOS remains in the final baked products.

The information provided by a kinetic model developed to predict enzyme inactivation during bread making (Lu et al. 2017) suggests that food enzymes may not be fully inactivated during baking. The Panel concluded that the food enzyme may remain in its active form during baking, depending on the processing conditions.

3.5.2. Dietary exposure estimation

The current section supersedes Sections 3.2.1, 3.2.2 and 3.2.3 of the previous evaluation (EFSA CEF Panel, 2018).

The approach and methodology to estimate the dietary exposure to the food enzyme–TOS remain the same as those reported in the previous evaluation (EFSA CEF Panel, 2018). A new calculation was made with the current use level. 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 43 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure was estimated to be 0.385 mg TOS/kg bw per day in children at the 95th percentile.

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)	0–0.087 (12)	0.005–0.208 (15)	0.003–0.191 (19)	0.001–0.107 (21)	0.030–0.071 (22)	0.034–0.063 (23)
Min–max 95th percentile (number of surveys)	0–0.250 (11)	0.020–0.356 (14)	0.011–0.385 (19)	0.004–0.218 (20)	0.069–0.163 (22)	0.071–0.125 (22)

3.5.3. Uncertainty analysis

See Section 3.2.4 of the previous evaluation (EFSA CEF Panel, 2018).

3.6. Margin of exposure

The current section supersedes the last paragraph of section 3.3.2.2 of the previous evaluation (EFSA CEF Panel, 2018).

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

4. CONCLUSIONS

New data established that the production strain meets the requirements for QPS status. Based on the QPS status of the production strain, the absence of issues of concern arising from the modified manufacturing process of the food enzyme, and the previous evaluation, the Panel concluded that the food enzyme aqualysin 1 produced with the genetically modified B. subtilis strain LMG S‐25520 does not give rise to safety concerns under the intended conditions of use.

The FEZ Panel considered the food enzyme free from recombinant DNA and viable cells of the production strain.

5. DOCUMENTATION AS PROVIDED TO EFSA

Protease (Aqualysin) from a genetically modified strain of Bacillus subtilis (LMG S‐25520). September 2023. Submitted by Puratos N.V.

Additional information. May 2024 and November 2024. Submitted by Puratos N.V.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GMM

genetically modified microorganism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

LOD

limit of detection

MOE

margin of exposure

PCR

polymerase chain reaction

QPS

qualified presumption of safety

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00303

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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 LM 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

Dietary exposure estimates to the food enzyme–TOS in details

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

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

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, the Netherlands, Portugal, 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, the Netherlands, Portugal, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, the Netherlands, Portugal, Romania, Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, the Netherlands, Portugal, Romania, 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, the Netherlands, Portugal, Romania, Slovenia, Spain, Sweden

^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 FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Peluso, S. , Andryszkiewicz, M. , Cavanna, D. , Fernàndez‐Fraguas, C. , de Nijs, R. A. , & Liu, Y. (2025). Revised safety evaluation of the food enzyme aqualysin 1 from the genetically modified Bacillus subtilis strain LMG S‐25520 produced by a modified process. EFSA Journal, 23(3), e9288. 10.2903/j.efsa.2025.9288