Safety evaluation of the food enzyme bacillolysin from the genetically modified Bacillus amyloliquefaciens strain NPR

Abstract

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the genetically modified Bacillus amyloliquefaciens strain NPR by DSM Food Specialties B.V. The production strain meets the requirements for the qualified presumption of safety (QPS) status. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in 10 food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated only for the remaining nine food manufacturing processes. It was estimated to be up to 6.450 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 bacillolysin to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but 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.

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 1332/2008 on food enzymes.

On 20 June 2023 a new application has been introduced by the applicant “DSM Food Specialties B.V.” for the authorisation of the food enzyme Bacillolysin from a genetically modified strain of Bacillus amyloliquefaciens (strain NPR).

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: Bacillolysin from a genetically modified strain of Bacillus amyloliquefaciens (strain NPR), in accordance with the Regulation (EC) No 1331/2008 establishing a common authorisation 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 authorisation of the food enzyme Bacillolysin from Bacillus amyloliquefaciens strain NPR.

Additional information, requested from the applicant during the assessment process on 18 November 2024, were received on 22 November 2024 (see Section 5).

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 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.

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 27 November to 18 December 2024. ⁵ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Bacillolysin
Systematic name	–
Synonyms	Bacillus metalloendopeptidase; Bacillus subtilis neutral proteinase
IUBMB No	EC 3.4.24.28
CAS No	9080‐56‐2
EINECS No	232‐991‐2

Bacillolysins catalyse the hydrolysis of peptide bonds of proteins with broad specificity, releasing peptides and amino acids.

The food enzyme under assessment is intended to be used in 10 food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): processing of dairy products for the production of 1) flavouring preparations from dairy products and 2) modified milk proteins; 3) processing of eggs and egg products; 4) processing of meat and fish products for the production of protein hydrolysates from meat and fish proteins; processing of cereals and grains for the production of 5) baked products, 6) cereal‐based products other than baked, 7) brewed products and 8) distilled alcohol; processing of plant‐ and fungal‐derived products for the production of 9) plant‐based analogues of milk and milk products and 10) protein hydrolysates from plants and fungi.

3.1. Source of the food enzyme

The enzyme is produced with the genetically modified bacterium Bacillus amyloliquefaciens strain NPR, which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (CBS, The Netherlands) with deposit number CBS 150612. ⁶ The production strain was identified as B. amyloliquefaciens by whole genome sequence (WGS) analysis, showing an average nucleotide identity of 99.9% to the type strain B. amyloliquefaciens DSM7. ⁷

The species B. amyloliquefaciens 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 (AMR) genes and toxigenic activity are verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022) ⁸ and the genetic modifications do not raise concerns. The production strain B. amyloliquefaciens NPR was shown not to be toxic to CHO‐K1 cells. ⁹ The WGS of the production strain was interrogated for the presence of antimicrobial resistance genes using two regularly updated databases with thresholds of > 80% identity and > 70% coverage. No genes of concern were identified. ¹⁰

3.1.1. Characteristics of the parental and recipient microorganisms

The parental microorganism is B. amyloliquefaciens strain DS 14719. The recipient strain, DS 81338, was obtained from the parental strain by classical mutagenesis, to obtain a ■■■■■, and by ■■■■■, to ■■■■■ the ■■■■■ encoding ■■■■■ and ■■■■■.

3.1.2. Characteristics of introduced sequences

The sequence encoding bacillolysin ■■■■■ including its own regulatory sequences, and the ■■■■■, which enables the ■■■■■ and was used as a ■■■■■, derive from ■■■■■.

The ■■■■■, that carried the ■■■■■ and the ■■■■■ genes, derived from ■■■■■ and did not contain antimicrobial resistance genes. ¹¹

3.1.3. Description of the genetic modification

The aim of the genetic modification was to enable the production strain to overproduce bacillolysin. For this purpose, the ■■■■■ was introduced ■■■■■. Transformants were selected by ■■■■■. One of the transformants was selected as B. amyloliquefaciens NPR.

WGS analysis showed ■■■■■ of ■■■■■ in the production strain, as well as ■■■■■ of the chromosomal copies of ■■■■■ and ■■■■■. ¹²

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. amyloliquefaciens NPR differs from the recipient strain in its capacity to overproduce bacillolysin and in its ■■■■■.

No issues of concern arising from the genetic modifications were identified by the Panel. 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 food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ¹³ with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice. ¹⁴

The production strain is grown as a pure culture using a typical industrial medium in a submerged, fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the cells are killed and 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. ¹⁵ 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. ¹⁶

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 bacillolysin is a single polypeptide chain of 520 amino acids. ¹⁷ The molecular mass of the unprocessed pre‐pro‐protein, calculated from the deduced amino acid sequence, is 56.8 kDa. ¹⁸ The food enzyme was analysed by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis. ¹⁹ A consistent protein pattern was observed across all batches. The gels showed a major protein band of about 36 kDa, consistent with the expected molecular mass of the mature protein (Wang et al., 2016). The gel also showed other bands of lesser stain intensity.

No other enzyme activities were reported. ²⁰

The applicant's in‐house determination of the protease activity is based on the hydrolysis of casein (reaction conditions: pH 7.0, 37°C) by measuring the amount of casein hydrolysate spectrophotometrically at 275 nm. The enzyme activity is expressed in protease casein total (PCT) units. One PCT is defined as the amount of enzyme that produces an amount of casein hydrolysate per minute with an absorbance (measured at 275 nm) that is equal to that of a 1.5‐μg/mL L‐tyrosine solution. ²¹

The food enzyme has a temperature optimum around 45°C (pH 7.0) and a pH optimum around pH 7.0 (37°C). ²² Thermostability was tested after a pre‐incubation of the food enzyme for 10, 15 and 30 min at different temperatures (pH 7.0). The enzyme activity decreased above 50°C showing no residual activity at 65°C after 10 min pre‐incubation. ²³

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1). ²⁴ The mean total organic solids (TOS) of the three batches intended for commercialisation was 11.8% and the mean enzyme activity/TOS ratio was 7517 PCT/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Bacillolysin activity	PCT/g a	883,000	860,000	925,000
Protein	%	8.9	8.1	10.0
Ash	%	1.0	0.8	1.0
Water	%	87.5	87.6	86.6
Total organic solids (TOS) b	%	11.5	11.6	12.4
Activity/TOS ratio	PCT/mg TOS	7678	7414	7460

^a PCT: Protease Casein Total (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three commercial batches was below 5 mg/kg ^{25 , 26} 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). ²⁷ No antimicrobial activity was detected in any of the tested batches. ²⁸

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 absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. One millilitre of food enzyme was mixed with 100 mL of non‐selective agar medium using the pour‐plate procedure. Plates were incubated at 30°C for 2 days. No colonies were produced. A positive control was included. ²⁹

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 ■■■■■ of the ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme. ³⁰

3.4. Toxicological data

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, 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

3.4.1. Allergenicity

The 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 bacillolysin produced with the Bacillus amyloliquefaciens strain NPR 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, no match was found in the AllergenOnline database. ³¹

No reports on oral or respiratory sensitisation or elicitation reactions of the bacillolysin under assessment have been published. In addition, no allergic reactions upon dietary exposure to any bacillolysin have been reported in the literature. ³²

The Panel considered that the information on the identified sequence matches and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the bacillolysin under assessment.

Soybean flour and soybean meal, products from soy that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³³ ), are used as raw materials. In addition, yeast extract, a known source of allergens, is also present in the medium fed to the microorganism. 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. ³⁴ 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, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in 10 food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2.

Intended uses and recommended use levels of the food enzyme as provided by the applicant. ³⁵

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of dairy products
Production of flavouring preparations from dairy products	Dairy products	7–20
Production of modified milk proteins	Milk proteins	24–1196
Processing of eggs and egg products	Egg white	48–120
Processing of meat and fish products
Production of protein hydrolysates from meat and fish proteins	Meat or fish protein	2.4–1196
Processing of cereals and other grains
Production of baked products	Flour	0.2–4
Production of cereal‐based products other than baked	Flour	0.2–4
Production of brewed products	Cereals	0.6–24
Production of distilled alcohol	Cereals	0.6–24
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts etc.	7–133
Production of protein hydrolysates from plants and fungi	Protein‐rich plant material	2.4–1196

^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 represent the maximum recommended use levels, which were used for calculation.

In the production of flavouring preparations from dairy products, the food enzyme is added—together with other enzymes (e.g. lipases)—either directly to curd or cheese, or after homogenisation and pasteurisation to enhance sensory properties. ³⁶ The food enzyme‐TOS remain in these enzyme‐modified dairy ingredients, which are subsequently used as an ingredient in a variety of foods such as processed cheese, cheese sauce, cheese powder, salad dressings and snacks.

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. meat, fish, bones, skin or feathers), milk (whey proteins, caseins), plant (e.g. pea flour, soy meal or rapeseed meal) or fungal sources for protein hydrolysis. ³⁷ Bacillolysin is used to increase the yield of the hydrolysis and to enhance flavour of the resulting protein hydrolysates, which are subsequently used as ingredients in a variety of foods. ³⁸ The food enzyme–TOS remain in these protein hydrolysates.

In the processing of egg and egg products, whole eggs are separated into yolk and egg white. The food enzyme is added to filtered egg white to improve their sensory and technological properties by protein hydrolysis. ³⁹ The food enzyme–TOS remain in the modified egg white.

In the production of baked products and in the production of cereal‐based products other than baked, the food enzyme is added to flour during the preparation of the dough. ⁴⁰ The bacillolysin cleaves peptide bonds in the gluten network, which reduces the rigidity of the dough. ⁴¹ The food enzyme–TOS remain in the baked and cereal‐based foods, such as bread, biscuits, pasta and noodles.

In the production of brewed products, the food enzyme is added to milled cereals during mashing. ⁴² The bacillolysin hydrolyses proteins in the cereals, enhancing the clarity of beer and releasing peptides and free amino acids as nitrogen source for the yeast during fermentation. ⁴³ The food enzyme–TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme can be added during slurry mixing, liquefaction, pre‐saccharification or fermentation steps. ⁴⁴ The bacillolysin hydrolyses proteins in the cereals to release free amino acids as nitrogen source for the yeast during fermentation. Additionally, bacillolysin hydrolyses proteins surrounding the starch granules, making them accessible to amylolytic enzymes for the conversion into fermentable sugars. ⁴⁵ The food enzyme–TOS are not carried over to distilled alcohols (EFSA CEP Panel, 2023).

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to plant‐based materials, which may be grained or pureed and mixed with water prior to enzymatic hydrolysis. ⁴⁶ The hydrolysis increases the yield and enhances the flavour of the plant‐based dairy alternatives, which include beverages, yoghurt‐like products, desserts and ice creams. The food enzyme–TOS remain in these products.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied, the Panel considered that the food enzyme is inactivated in all the food manufacturing processes listed in Table 2 in which it is not removed.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the nine 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 webtool ⁴⁷ 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 1 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 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 6.450 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)	0.101–1.473 (12)	0.213–0.970 (15)	0.151–1.117 (19)	0.039–0.223 (21)	0.032–0.171 (22)	0.017–0.135 (23)
Min–max 95th percentile (number of surveys)	0.232–3.433 (11)	0.656–2.546 (14)	0.488–6.450 (19)	0.106–0.710 (20)	0.102–0.408 (22)	0.059–0.409 (22)

3.5.3. 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 4.

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	+
Egg white is the only raw material indicated by the applicant. 48 However, for egg processing, the food categories chosen for calculation are egg yolk, egg white and raw eggs (without shells).	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of one process from the exposure estimation: –production of distilled alcohol	−

Note: +: 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 one food manufacturing process from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

3.6. Margin of exposure

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

4. CONCLUSIONS

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme bacillolysin produced with the genetically modified Bacillus amyloliquefaciens strain NPR does not give rise to safety concerns under the intended conditions of use.

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

5. DOCUMENTATION AS PROVIDED TO EFSA

Bacillolysin from a genetically modified strain of Bacillus amyloliquefaciens (strain NPR). June 2023. Submitted by DSM Food Specialties B.V.

Additional information. November 2024. Submitted by DSM Food Specialties B.V.

ABBREVIATIONS

AMR

Antimicrobial resistance

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LOD

limit of detection

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‐2023‐00905

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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.

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, 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 3 of this opinion; however, they are included in Appendix B 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 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. , Andryszkiewicz, M. , Cavanna, D. , Peluso, S. , Gallo, A. , De Nijs, R. , & Liu, Y. (2025). Safety evaluation of the food enzyme bacillolysin from the genetically modified Bacillus amyloliquefaciens strain NPR . EFSA Journal, 23(2), e9220. 10.2903/j.efsa.2025.9220