Abstract
The food enzyme subtilisin (serine endopeptidase; EC 3.4.21.62) is produced with the genetically modified Bacillus paralicheniformis strain AP‐GKY by Kerry Ingredients & Flavours Ltd. The production strain met the requirements for the qualified presumption of safety (QPS). The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in six food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in one process, dietary exposure was calculated for the remaining five food manufacturing processes. It was estimated to be up to 7.99 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 subtilisin to known allergens was made and matches with three food, twenty respiratory and three contact allergens were found. The Panel considered that the risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded. 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.
Keywords: alcalase, Bacillus paralicheniformis, EC 3.4.21.62, EFSA‐Q‐2024‐00552, genetically modified microorganism, serine endopeptidase, Subtilisin
1. INTRODUCTION
Article 3 of the Regulation (EC) No 1332/2008 1 provides definitions 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 26 August 2024, a new application was introduced by the applicant “Kerry Ingredients & Flavours Ltd” for the authorisation of the food enzyme Subtilisin produced from a genetically modified Bacillus paralicheniformis (strain AP‐GKY).
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: Subtilisin from a genetically modified Bacillus paralicheniformis (strain AP‐GKY) in accordance with 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 subtilisin from Bacillus paralicheniformis strain AP‐GKY.
Additional information was requested during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00552.
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, 2023b) 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 5 to 26 of August 2025. 5 No comments were received.
3. ASSESSMENT
| IUBMB nomenclature | Subtilisin |
| Systematic name | Serine endopeptidase |
| Synonyms | Alcalase, bacillopeptidase, alkaline proteinase, thermoase, subtilopeptidase |
| IUBMB No | EC 3.4.21.62 |
| CAS No | 9014‐01‐1 |
| EINECS No | 232‐752‐2 |
Subtilisins 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 six food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023b): processing of dairy products for the (1) production of modified milk proteins; processing of meat and fish products for (2) production of protein hydrolysates from meat and fish proteins; processing of cereals and other grains for (3) production of cereal‐based products other than baked; processing of plant‐ and fungal‐derived products for the production of (4) edible oils from plant and algae, (5) protein hydrolysates from plants and fungi and (6) processing of yeast and yeast products.
3.1. Source of the food enzyme
The subtilisin is produced with the genetically modified bacterium Bacillus paralicheniformis strain AP‐GKY, which is deposited at ■■■■■ with the deposition number ■■■■■. 6 The production strain was identified as B. paralicheniformis by whole genome sequencing (WGS) analysis, which showed an Average Nucleotide Identity (ANI) of ■■■■■ with B. paralicheniformis type strain KJ‐16. 7 , 8
The species B. paralicheniformis is included in the list of organisms for which the qualified presumption of safety (QPS) may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes, toxigenic activity, and the inability to synthesise bacitracin are verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022). 9
The production strain was shown to be not cytotoxic against Vero cells using the lactate dehydrogenase assay. 10 The WGS data from the production strain was searched against two maintained databases for antimicrobial resistance genes. No genes of concern were identified with thresholds above 80% of identity and 70% of coverage.
As the production strain B. paralicheniformis AP‐GKY does not contain AMR genes of concern, is not cytotoxic, does not contain genes involved in the synthesis of bacitracin (see Section 3.1.2), and the genetic modifications do not raise safety concerns (see Section 3.1.3), it meets the requirements for the QPS approach and is considered safe.
3.1.1. Characteristics of the parental microorganism
The parental microorganism is Bacillus paralicheniformis strain LMG S‐30155 (EFSA CEP Panel, 2023a). 11
3.1.2. Description of the genetic modification
The purpose of the genetic modification was to prevent the production strain from producing bacitracin. 12
■■■■■, ■■■■■. 13
■■■■■.
The absence of vector backbone sequence and the deletion of the bacitracin gene cluster were confirmed by WGS analysis. 14
3.1.3. 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. paralicheniformis strain AP‐GKY differs from the parental strain in its inability to produce bacitracin.
3.2. Production of the food enzyme
The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, 15 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice. 16
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 solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. 17 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. 18
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 subtilisin is a single polypeptide chain of ■■■■■ amino acids. 19 The molecular mass of the protein, calculated from the amino acid sequence, is 38.8 kDa. 20 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. 21 A consistent protein pattern was observed across all batches. The gels showed a major protein band migrating between the marker proteins of 25 and 37 kDa, consistent with the expected mass of the mature enzyme (35.6 kDa).
No other enzyme activities were reported.
The applicant's in‐house determination of subtilisin activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of ■■■■■ is measured spectrophotometrically at ■■■■■ nm. The enzyme activity is expressed in Detergent Alkaline Protease Units (DAPU)/g. One DAPU is defined as the activity that releases the equivalent of 4 μmol of tyrosine per minute under the conditions of the assay. 22
The food enzyme has a temperature optimum around 60°C (pH 8.5) and a pH optimum around 10 (40°C). Thermostability was tested by pre‐incubation of the food enzyme at different temperatures (pH 8.5) for different times. The enzyme activity decreased above 50°C, and no residual activity was detected after 5 min at 80°C. 23
3.3.2. Chemical parameters
Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation. 24 The mean total organic solids (TOS) was 7.6% and the mean enzyme activity/TOS ratio was 4674 DAPU/mg TOS.
3.3.3. Purity
The lead content in the three batches was below 0.004 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
Bacillus paralicheniformis may have the capacity to synthesise bacitracin, which is considered a hazard by EFSA as the exposure to low concentrations of antimicrobials, including sub‐inhibitory concentrations, may result in the selection of antimicrobial‐resistant bacteria (EFSA BIOHAZ Panel, 2021). Genes involved in bacitracin synthesis are deleted in the genome of the production strain (see Section 3.1.2). Accordingly, bacitracin was not detected in three food enzyme batches ■■■■■. 29 , 30
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. Ten mL of product was incubated in 90 mL of non‐selective medium. The samples were then filtered with a 0.45 μm pore size membrane, and the filters placed onto agar plates at 35°C for 5 days. No colonies were produced. A positive control was included. 31
The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction analysis of three batches in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme. 32
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, 3.2 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 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 subtilisin produced with the Bacillus paralicheniformis strain AP‐GKY 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 three food, twenty respiratory, and three contact allergens were found in the AllergenOnline database. 33
The matching food allergens were Bac s 1 (75.0% sequence identity), a nattokinase from Bacillus subtilis, Cuc m 1 (37.5% sequence identity), an alkaline serine protease from melon (Cucumis melo), and Pun g 14 (36.2% sequence identity), a chitinase from pomegranate (Punica granatum).
The matching respiratory allergens were subtilisin from B. licheniformis (100% sequence identity), B. lentus (75.0% sequence identity) and Bacillus sp. (68.8% sequence identity). Further matches were alkaline serine proteases from Penicillium chrysogenum and P. citrinum (47.1%–50.0% sequence identity), Aspergillus flavus, A. oryzae, A. fumigatus and A. versicolor (40.0%–45.0% sequence identity), and subtilisin‐like serine proteases from Curvularia lunata (45.7% sequence identity) and Alternaria alternata (45.7% sequence identity). In addition, matches to vacuolar serine proteases from Aspergillus spp., Fusarium proliferatum, Cladosporium spp. Penicillium spp. and Rhodotorula mucilaginosa (43.3%–47.5% sequence identity) were observed.
The three matching contact allergens were subtilisin‐like serine proteases (48.1%–51.2% sequence identity) from Trichophyton rubrum, T. schoenleinii, and T. benhamiae.
Bac s 1, the subtilisin‐like serine protease nattokinase from Bacillus subtilis, has been identified as a food allergen. Few cases of allergic reactions after consumption of natto have been reported (Inomata et al., 2012; Suzuki et al., 2023).
Allergic reactions to Cucumis melo have been reported and Cuc m 1 has been identified as a relevant food allergen (Cuesta‐Herranz et al., 2003). Allergic reactions to Punica granatum have been reported (Hassan & Venkatesh, 2015; Neeharika & Sunkar, 2021).
Subtilisin is a known occupational respiratory allergen (Van Kampen & Merget, 2002). Considering the matches with respiratory allergens, the Panel notes that 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 (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004).
Considering the matches with contact allergens from Trichophyton species (e.g. Tri r 2 from the Athlete's foot fungus T. rubrum), no reports of allergic reactions after oral exposure have been published.
The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for natto, melon and pomegranate allergic individuals upon dietary exposure to the subtilisin under assessment.
■■■■■, products from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 34 ), are used as raw material. In addition, ■■■■■, a known source of allergens, is present in the culture medium. 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 due to the raw material could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), 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 intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, particularly in natto, melon and pomegranate allergic individuals. However, the likelihood of such reactions will not exceed the risk of reactions after natto, melon and pomegranate consumption.
3.5. Dietary exposure
3.5.1. Intended use of the food enzyme
The food enzyme is intended to be used in six 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 | Raw material (RM) | Recommended use level (mg TOS/kg RM) b |
|---|---|---|
| Processing of dairy products | ||
|
Milk proteins | 422–1400 (other foods & food supplements) |
| 400–800 (infant formulae, follow‐on formulae and foods for special medical purposes | ||
| Processing of meat and fish products | ||
|
Meat and fish | 110–200 |
| Processing of cereals and other grains | ||
|
Flour | 3–400 |
| Processing of plant‐and fungal‐derived products | ||
|
Microalgae | 80–200 |
|
Plant, algae and fungal proteins | 60–280 |
| Processing of yeast and yeast products | Yeast, yeast extract, yeast cell wall | 180–350 |
The numbers in bold represent the maximum recommended use levels, which were used for calculation.
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, 2023b).
In the production of modified milk proteins, the food enzyme is added to milk proteins during the hydrolysis 36 to enhance the flavour of the resulting milk protein products (e.g. whey protein hydrolysates). The food enzyme‐TOS remain in the final foods.
In the production of hydrolysed proteins, the food enzyme is added to plant proteins (e.g. pea proteins) or animal products (e.g. ground beef and fish) 37 to increase the yield and to improve flavour. The food enzyme‐TOS remain in the protein hydrolysates.
In the production of cereal‐based products other than baked, the food enzyme is added to cereal flour during the dough preparation 38 to cleave the peptide bonds in the gluten network, thus improving the rheology of the dough. The food enzyme‐TOS remains in the final products.
In the treatment of algae for edible oil production, the food enzyme is added to the microalgal biomass 39 to hydrolyse proteins of the cell wall. 40 The food enzyme‐TOS are removed during the refinement processes 41 (EFSA CEP Panel, 2023b).
In the production of yeast and yeast products, the food enzyme is added during the autolysis 42 to improve the extraction process and the sensory properties of the yeast products. The food enzyme‐TOS remains in the yeast and yeast products.
Based on data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that the food enzyme is inactivated in all the food manufacturing processes listed in Table 2 in which the food enzyme‐TOS remain.
3.5.2. Dietary exposure estimation
In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the five food manufacturing processes where the food enzyme‐TOS remains in the final foods.
Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool 43 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, 2023b).
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 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix B). The highest dietary exposure was estimated to be 7.99 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) | 0.615–2.070 (14) | 0.442–1.882 (17) | 0.311–1.761 (21) | 0.166–0.684 (23) | 0.058–0.272 (23) | 0.047–0.331 (25) |
| Min–max 95th percentile (number of surveys) | 2.219–5.915 (13) | 1.699–3.832 (16) | 0.928–7.990 (21) | 0.419–1.694 (22) | 0.277–0.852 (23) | 0.202–0.679 (24) |
TABLE 1.
Composition of the food enzyme.
| Parameters | Unit | Batches | ||
|---|---|---|---|---|
| 1 | 2 | 3 | ||
| Subtilisin activity | DAPU/mL a | 367,000 | 335,000 | 337,000 |
| Protein | % | 6.8 | 6.7 | 7.4 |
| Ash | % | 0.8 | 1.3 | 0.6 |
| Water | % | 89.6 | 92.7 | 92.1 |
| Total organic solids (TOS) b | % | 9.6 | 6.0 | 7.3 |
| Activity/TOS ratio | DAPU/mg TOS | 3823 | 5583 | 4616 |
DAPU: Detergent Alkaline Protease Unit (see Section 3.3.1).
TOS calculated as 100% – % water – % ash.
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 | + |
| 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 edible oils from algae |
− |
Note: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.
The conservative approach applied to estimate the dietary 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 food manufacturing process, the Panel concluded that the food enzyme subtilisin produced with the genetically modified Bacillus paralicheniformis strain AP‐GKY does not give rise to safety concerns under the intended conditions of use.
The Panel considered the food enzyme free from viable cells of the production organism and recombinant DNA.
5. DOCUMENTATION AS PROVIDED TO EFSA
Additional information was requested during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00552.
ABBREVIATIONS
- AMR
Antimicrobial resistance
- bw
body weight
- CAS
Chemical Abstracts Service
- CEF
EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids
- CEP
EFSA Panel on Food Contact Materials, Enzymes and Processing Aids
- CFU
colony forming units
- DRF
dose‐range finding
- EC
European Commission
- EINECS
European Inventory of Existing Commercial Chemical Substances
- FAO
Food and Agricultural Organization of the United Nations
- FEZ
EFSA Panel on Food Enzymes
- GLP
Good Laboratory Practice
- GMM
genetically modified microorganism
- 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
- MNBN
bi‐nucleated cells with micronuclei
- OECD
Organisation for Economic Cooperation and Development
- PCR
polymerase chain reaction
- QPS
qualified presumption of safety
- SDS‐PAGE
sodium dodecyl sulfate‐polyacrylamide gel electrophoresis
- TOS
total organic solids
- WGS
whole genome sequencing
- WHO
World Health Organization
REQUESTOR
European Commission
QUESTION NUMBER
EFSA‐Q‐2024‐00552
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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, Henk Van Loveren, Laurence Vernis, 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
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 16 weeks on up to and including 11 months of age | Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Poland, Portugal, Slovenia, Spain |
| Toddlers | From 12 months up to and including 35 months of age | Belgium, Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, Portugal, Republic of North Macedonia*, Serbia*, Slovenia, Spain |
| Children | From 36 months up to and including 9 years of age | Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, 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*, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Poland, 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, Poland, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden |
| The elderly a | From 65 years of age and older | Austria, Belgium, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Poland, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden |
Consumption data from these pre‐accession countries are not reported in Table 4 of this opinion; however, they are included in Appendix B for testing purpose.
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. , Van Loveren, H. , Vernis, L. , Peluso, S. , Fraguas, C. F. , Precup, G. , & Liu, Y. (2026). Safety evaluation of the food enzyme subtilisin from the genetically modified Bacillus paralicheniformis strain AP‐GKY . EFSA Journal, 24(2), e9956. 10.2903/j.efsa.2026.9956
Adopted: 4 February 2026
Correspondence: Ask a Question
The declarations of interest of all scientific experts active in EFSA's work are available at https://open.efsa.europa.eu/experts.
Notes
Regulation (EC) No 1332/2008 of the European Parliament and of the Council of 16 December 2008 on Food Enzymes and Amending Council Directive 83/417/EEC, Council Regulation (EC) No 1493/1999, Directive 2000/13/EC, Council Directive 2001/112/EC and Regulation (EC) No 258/97. OJ L 354, 31.12.2008, pp. 7–15.
Regulation (EC) No 1331/2008 of the European Parliament and of the Council of 16 December 2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. OJ L 354, 31.12.2008, pp. 1–6.
OJ L 354, 31.12.2008, p. 1.
Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. OJ L 31, 1.2.2002, p. 1–24.
Accessible at https://open.efsa.europa.eu/consultations/a0cTk00000I3VD9IAN?search=EFSA‐Q‐2024‐00552.
Technical Dossier/Source of the food enzyme/Annex 5.1.
Technical Dossier/Source of the food enzyme/Annex_5_159188_WGS_B_paralicheniformis_CONF
pg. 9 and 18.
Technical Dossier/Source of the food enzyme/Additional Data/Annex_5_159188_WGS_B_paralicheniformis.
Technical Dossier/Source of the food enzyme/Annex 5.3.
Technical Dossier/Source of the food enzyme/Source FE pg. 7.
Technical Dossier/Source of the food enzyme/Annex 5 pg. 12–13.
Technical Dossier/Source of the food enzyme/Additional Data/Annex_5_159188_WGS_B_paralicheniformis pg. 12.
Technical Dossier/Source of the food enzyme/Annex 5 pg. 12–13 and Source FE pg. 7.
Regulation (EC) No 852/2004 of the European Parliament and of the Council of 29 April 2004 on the hygiene of foodstuffs. OJ L 226, 25.6.2004, pp. 3–21.
Technical Dossier/Manufacturing process, pp.1 and Annex_6_FSSC22000_EnMex.
Technical dossier/6.Manufacturing process, pp. 5.
Technical dossier/6.Manufacturing process and Annex_6_2_Materials.
Technical dossier/Chemical composition, properties and purity of the food enzyme/Annex 7.
Technical dossier/Chemical composition, properties and purity of the food enzyme/Annex 7.
Technical dossier/Chemical composition, properties and purity of the food enzyme/Annex 7_4.
Technical dossier/Chemical composition, properties and purity of the food enzyme/Annex 7–3.
Technical dossier/Chemical composition, properties and purity of the food enzyme/Annex 7–5.
Technical dossier/Chemical composition, properties and purity of the food enzyme/7. Chemical composition, properties, purity and Annex_7_1_CoAs.
Technical dossier/Chemical composition, properties and purity of the food enzyme/7.Chemical composition, properties, purity and Annex_7_1_CoAs.
LoD Pb = 0.00138 mg/L.
Technical dossier/Chemical composition, properties and purity of the food enzyme/7. Chemical composition, properties, purity and Annex_7_1_CoAs.
Technical dossier/Chemical composition, properties and purity of the food enzyme/7. Chemical composition, properties, purity and Annex_7_1_CoAs.
Technical Dossier/Source of the food enzyme/Annex 5.5, 5.6 and 5.7.
LoD = ■■■■■.
Technical Dossier/Source of the food enzyme/Annex 5.8.
Technical Dossier/Source of the food enzyme/Annex 5.9.
Technical Dossier/Risk Assessment/Allergenicity/Annex 10.
Regulation (EU) No 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/p. 1.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figures 5 and 10.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figures 1, 2, 3, 4.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figures 12 and 13.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figure 6.
Technical dossier/Risk assessment/Intended use(s) in food and use level(s)/pp. 3.
information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figure 7.
Additional information August 2025/Intended use(s) in food and use level(s)/13_Intended_uses_update7Aug2025/Figure 9.
Version 1.1.0.
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Associated Data
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Supplementary Materials
APPENDIX A Dietary exposure estimates to the food enzyme‐TOS in details