Safety evaluation of the food enzyme acid prolyl endopeptidase from the genetically modified Aspergillus niger strain GEP

TL;DR

This study evaluates the safety of a food enzyme produced from a genetically modified fungus and concludes it is safe for use in food manufacturing.

Contribution

A safety assessment of a genetically modified acid prolyl endopeptidase food enzyme, including toxicity and allergenicity evaluations.

Findings

Genetic modifications in the enzyme production did not raise safety concerns.

Dietary exposure was estimated at up to 0.913 mg TOS/kg body weight per day in European populations.

No observed adverse effects were found in toxicity studies, with a high margin of exposure.

Abstract

The food enzyme acid prolyl endopeptidase is produced with the genetically modified Aspergillus niger strain GEP by DSM Food Specialties B.V. The genetic modifications did 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 seven food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in two processes, dietary exposure was calculated for the remaining five food manufacturing processes. It was estimated to be up to 0.913 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 5040 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 5520. A search for the homology of the amino acid sequence of the acid prolyl endopeptidase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that 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.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a
Acid prolyl endopeptidase activity	PPU/g b	16.0	18.5	18.7	11.0
Protein	%	14.3	18.9	18.6	13.9
Ash	%	0.3	0.5	0.5	0.7
Water	%	78.9	72.3	72.5	74.1
Total organic solids (TOS) c	%	20.8	27.2	27.0	25.2
Activity/mg TOS ratio	PPU/mg TOS	0.076	0.068	0.069	0.044

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant.

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of meat and fish products
Production of protein hydrolysates from meat and fish proteins	Meat and fish proteins	939–1408
Processing of cereals and other grains
Production of brewed products	Wort	0.7–2.1
Production of glucose syrups and other starch hydrolysates	Starch	0.21–3.5
Production of distilled alcohol	Cereals	70–704
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, pulses, seeds	176–282
Production of protein hydrolysates from plants and fungi	Plant proteins	939–1408
Processing of yeast and yeast products	Yeast culture, yeast extract, cell walls or autolyzed yeast	704

TABLE 3. Summary of the estimated dietary exposure to the food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.003–0.110 (14)	0.052–0.276 (17)	0.068–0.271 (21)	0.014–0.170 (23)	0.012–0.104 (23)	0.006–0.103 (25)
Min–max 95th percentile (number of surveys)	0.005–0.290 (13)	0.187–0.913 (16)	0.271–0.876 (21)	0.064–0.656 (22)	0.049–0.384 (23)	0.027–0.385 (24)

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 two processes from the exposure estimation: ‐ production of distilled alcohol ‐ production of glucose syrups and other starch hydrolysates	−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or 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 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer.

All food enzymes currently on the European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission in 2014

1.1.1

Only food enzymes included in the European Union (EU) Community 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.

Three applications have been submitted by the companies “Takabio” and “DSM Food Specialties B.V” of the food enzymes Microbial Rennet from Rhizomucor miehei, Acid prolyl endopeptidase from a genetically modified strain of Aspergillus niger (strain GEP) and Beta‐galactosidase from a genetically modified strain of Aspergillus niger (strain TOL).

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

Terms of Reference in 2014

1.1.2

The European Commission requests the European Food Safety Authority to carry out safety assessments on the food enzymes Microbial Rennet from Rhizomucor miehei, Acid prolyl endopeptidase from a genetically modified strain of Aspergillus niger (strain GEP) and Beta‐galactosidase from a genetically modified strain of Aspergillus niger (strain TOL) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Background as provided by the European Commission in 2021

1.1.3

Only food enzymes included in the European Union (EU) Community 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.

Acid prolyl endopeptidase from a genetically modified strain of Aspergillus niger (strain GEP) is a food enzyme included in the Register of food enzymes4 to be considered for inclusion in the Union list and thus subject to risk asessment by the European Food Safety Authority (EFSA). In the initial dossier with reference EFSA‐Q‐2014‐00852, the applicant requests for the authorisation of the above food enzyme is brewing processing, potable alcohol processing, protein processing and starch processing in accordance with Regulation (EC) No 1331/2008.

On 8 June 2021, a new application has been introduced by the applicant “DSM Food Specialties B.V.” for an extension of the conditons of use for the above food enzyme in yeast processing, flavouring production and plant‐based products (dairy alternatives).

Taking into account that the above food enzyme is subject to a risk assessment by the European Food Safety Authority (EFSA), in accordance with Regulation (EC) No 1331/2008, it is appropriate to address the safety of the proposed extension of the condition of use within the scientific opinion evaluating the safety of that food enzyme.

Terms of Reference in 2021

1.1.4

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of an extension of the conditions of use for the following food enzyme: acid prolyl endopeptidase from a genetically modified strain of Aspergillus niger (strain GEP) in accordance with Regulation (EC) No 1331/2008, establishing a common authorisation procedure for food additives, food enzymes and food flavourings.5

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's requests from 2014 and 2021 to carry out the safety assessment of the food enzyme acid prolyl endopeptidase from a genetically modified strain of Aspergillus niger (strain GEP).

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme acid prolyl endopeptidase from a genetically modified strain of A. niger (strain GEP). The dossier was updated on 8 June 2021 with an application for extension of use of the food enzyme.

Additional information, requested from the applicant during the assessment phase on 15 December 2022 and 31 January 2022, was received on 22 April 2022 and 6 September 2023, respectively. Spontaneous additional information was received from the applicant on 1 December 2025 (see Section "Documentation provided to EFSA").

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009a) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009b) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

ASSESSMENT

3

IUBMB nomenclatureAcid prolyl endopeptidaseSystematic name–SynonymsProline‐specific endopeptidase; post‐proline endopeptidase; proline endopeptidase; endoprolylpeptidase; prolyl endopeptidaseIUBMB No–CAS No–EINECS No–

The acid prolyl endopeptidase catalyses the hydrolysis of peptide bonds at the C‐terminal side of proline residues, and to a lesser extent of alanine residues, of peptides and proteins, releasing peptides and amino acids. The food enzyme under assessment is intended to be used in seven food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of meat and fish products for the production of protein hydrolysates from meat and fish proteins; processing of cereals and other grains for the production of (2) brewed products, (3) glucose syrups and other starch hydrolysates and (4) distilled alcohol; processing of plant‐ and fungal‐derived products for the production of (5) plant‐based analogues of milk and milk products and (6) protein hydrolysates from plants and fungi; (7) processing of yeast and yeast products.

Source of the food enzyme

3.1

The acid prolyl endopeptidase is produced with the genetically modified filamentous fungus A. niger strain GEP (■■■■■), which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (CBS, The Netherlands), with deposition number ■■■■■.6 The production strain was identified as A. niger by whole genome sequence (WGS) analysis, showing an average nucleotide identity of > 99% with the reference strain ■■■■■.7

Characteristics of the parental and recipient microorganisms

3.1.1

■■■■■

■■■■■■■■■■

■■■■■.

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■■■■■■ ■■■■■■■■■■ ■■■■■.

Description of the genetic modification process

3.1.3

The purpose of the genetic modification was to enable the production strain to overproduce acid prolyl endopeptidase. ■■■■■.11

Safety aspects of the genetic modification

3.1.4

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

The production strain A. niger GEP differs from the recipient strain in its ability to overproduce acid prolyl endopeptidase.

No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,12 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.13

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 enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.14 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.15 ^,^ 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.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The acid prolyl endopeptidase is a single polypeptide chain of 485 amino acids.17 The molecular mass of the mature protein, calculated from the amino acid sequence, is around 56 kDa.18 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.19 A consistent protein pattern was observed across all batches. The gels showed a major protein band corresponding to an apparent molecular mass of about 66 kDa, attributed to the glycosylated form of the enzyme (Sebela et al., 2009).

No other enzymatic activities were reported.20

The applicant's in‐house determination of acid prolyl endopeptidase activity is based on the hydrolysis of the substrate N‐benzyloxycarbonyl‐glycyl‐prolyl‐p‐nitroanilide (Z‐Gly‐Pro‐pNA) (reaction conditions: pH 4.6, 37°C). The release of 4‐nitroaniline (pNA) is measured spectrophotometrically at 405 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in PPU/g. One PPU is defined as the amount of enzyme required to release one μmol of pNA from Z‐Gly‐Pro‐pNA per minute under the conditions of the assay.21

The food enzyme has a temperature optimum around 60°C (pH 4.6) and a pH optimum around pH 4.5 (37°C).22 No enzyme activity occurred at 80°C.23

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and one batch produced for the toxicological tests (Table 1).24 The mean total organic solids (TOS) of the three food enzyme batches intended for commercialisation is 25.0% and the mean enzyme activity/TOS ratio is 0.071 PPU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 5 mg/kg25 ^,^ 26 ^,^ 27 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 28 as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). No antimicrobial activity was detected in any of the tested batches.29

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of zearalenone, fumonisins, trichothecenes, aflatoxins and ochratoxin A was examined in three food enzyme batches and was below the limit of detection (LoD) of the applied method.30 ^,^ 31 Adverse effects caused by the potential presence of other secondary metabolites is addressed by the toxicological examination of the food enzyme.

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

Viable cells and DNA of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches, analysed in triplicate. ■■■■■. No colonies were detected. A positive control was included.32

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 ■■■■■.33

Toxicological data

3.4

A battery of toxicological tests including a bacterial gene mutation test (Ames test), an in vitro mammalian chromosomal aberration test and a repeated dose 90‐day oral toxicity study in rats has been provided. The batch 4 (Table 1) used in these studies has a similar enzyme activity/TOS ratio to the batches intended for commercialisation, and thus, it is considered suitable as a test item.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997a) and following Good Laboratory Practice (GLP).34 Four strains of S. Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the standard plate incorporation method (the first experiment) and the ‘treat and plate’ assay (the second experiment). The experiments were carried out in triplicate.

A first experiment was carried out using five concentrations of the food enzyme of 60, 180, 540, 1622, 4865 μg TOS/plate. Growth stimulation, as indicated by the thickening of the background bacterial lawn, and probably caused by free amino acids present in the test item, was observed at concentrations of 540 μg TOS/plate and above in TA98, TA1535 and E. coli WP2uvrA strains with or without S9‐mix and at 4865 μg TOS/plate in TA100 strain with or without S9‐mix. Upon treatment with the food enzyme, an increase in the number of revertant colonies was observed at the highest concentration tested in TA1535 strain with S9‐mix and at 1622 μg TOS/plate in TA1537 strain without S9‐mix.

The second experiment was carried out on TA1535, TA1537 and TA98 strains, using five concentrations of the food enzyme of 304, 608, 1216, 2432 and 4865 μg TOS/plate. No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme there was no biologically relevant increase in the number of revertant colonies above the control values, in any strain tested, with or without S9‐mix.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme acid prolyl endopeptidase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.2

The in vitro mammalian chromosomal aberration test was carried out according to OECD Test Guideline 473 (OECD, 1997b) and following GLP.35 Two separate experiments were performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1216, 2432 and 4865 μg TOS/mL, in a short‐term treatment (4‐h exposure and 20‐h recovery period) either with or without S9‐mix and in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix. No cytotoxicity was seen in the short‐term treatment with or without S9‐mix. In the long‐term treatment, a relative mitotic index of 45% was reported at concentration of 4865 μg TOS/mL.

In the second experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 1946, 3892 and 4865 μg TOS/mL, in two long‐term treatments (24‐h exposure without recovery period and 48‐h exposure without recovery period) without S9‐mix and 2919, 3892 and 4865 μg TOS/mL in two short‐term treatments (4‐h exposure and 20‐ or 40‐h recovery period) with S9‐mix. No cytotoxicity was seen in the short‐term treatment with or without S9‐mix. In the long‐term treatment (24‐h exposure without recovery period), a relative mitotic index of 43% and 49% was reported at the concentrations of 3892 and 4865 μg TOS/mL, respectively. In the long‐term treatment (48‐h exposure without recovery period), a relative mitotic index of 47% was reported at the concentrations of 3892 μg TOS/mL.

The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme acid prolyl endopeptidase did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 1998).36

Groups of 10 male and 10 female Wistar (HsdCpb:WU) rats received the food enzyme by gavage in doses of 504, 1764 or 5040 mg TOS/kg bw per day. Controls received the vehicle (distilled water).

No mortality was observed.

The body weight was statistically significantly increased in weeks 6, 8, 9 and 10 of administration in high‐dose females (+8% to +9%). The body weight gain was statistically significantly increased in weeks 4–13 of administration in high‐dose females (+18% to +27%). The Panel considered the changes as not toxicologically relevant, as they were recorded at single time intervals, they were only observed in one sex (both parameters) and were transient (body weight), the change was small (body weight) and the changes were without a statistically significant effect on the final body weight (both parameters).

The feed consumption was statistically significantly decreased in weeks 1, 8, 9, 10, 11 and 13 of administration in high‐dose males (−6% to −11%) and in week 1 of administration in mid‐ and high‐dose females (−22% both). The Panel considered the change as not toxicologically relevant, because of no consistency between the time of the occurrence of the changes in males and females and there was no statistically significant effect on the final body weight.

In the functional observations, a statistically significant decrease in the hind limb foot splay was observed in mid‐ and high‐dose males (−12.5% both) and females (−10.3% both). The Panel considered the change not toxicologically relevant, as there were no dose–response relationship and no statistically significant changes in other related endpoints (e.g. a grip strength or motor activity).

Haematological investigations revealed a statistically significant decrease in red blood cell count (RBC, −7%) in high‐dose females; in white blood cell count (WBC, −26% and −19%) in low‐ and mid‐dose males; in monocytes (Mon, −75%; −83% and −33%) in low‐, mid‐ and high‐dose females; a statistically significant increase in mean corpuscular haemoglobin (MCH, +5%; +7%) in mid‐ and high‐dose females and a statistically significant decrease in platelet count (Plt, −22% and −23%) in mid‐ and high‐dose females and in prothrombin time (PT, −5%) in low‐dose females. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there was no dose–response relationship (WBC, Mon, PT) and the changes were small (RBC, MCH, PT).

Clinical chemistry investigations revealed a statistically significant increase in total bilirubin (+17%) in high‐dose males, of glucose (Glc; +17% and +15%) in mid‐ and high‐dose females; and of cholesterol (+33%) in high‐dose females; a statistically significant decrease in creatinine (Crea, −5% each) in low‐ and mid‐dose males; in blood urea nitrogen (BUN) and urea (−23% and −20%) in low‐dose females. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there was no dose–response relationship (Glc, Crea, BUN, urea) and the change was small (Crea).

Statistically significant changes detected in organ weights were an increase in the relative liver weight (+6% and +8%) in high‐dose males and females, and in absolute liver weight (+17%) in high‐dose females. The Panel considered the changes as not toxicologically relevant, as it was only observed in one sex (absolute liver weight), the change was small (relative liver weights) and there were no gross or histopathological changes in the organ.

No other statistically significant or toxicologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 5040 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

3.4.3

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

The potential allergenicity of the acid propyl endopeptidase produced with the A. niger strain GEP 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 using the AllergenOnline database.37

No reports on oral or respiratory sensitisation or elicitation reactions of the acid propyl endopeptidase under assessment have been published. No allergic reactions upon dietary exposure to any acid prolyl endopeptidase have been reported in the literature.38

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the acid propyl endopeptidase under assessment.

The production strain belongs to the Aspergillus genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen & Han, 1998; Vermani et al., 2015). Allergic reactions upon dietary exposure have been observed, but are rare (Xing et al., 2022). 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 culture medium. 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), 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 alcohol, 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.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in seven food manufacturing processes at the recommended use levels summarised in Table 2.39

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. meat, fish, gelatine, collagen) or plant sources (e.g. wheat, corn).40 The food enzyme–TOS remain in the protein hydrolysates.

In the production of brewed products, the food enzyme is added to wort during fermentation41 to prevent haze formation. The food enzyme–TOS remain in the brewed products.

In the production of glucose syrups and other starch hydrolysates, the food enzyme is added to starch during saccharification42 to reduce viscosity and increase yield. The Panel considered that the food enzyme–TOS are removed from glucose syrups and other starch hydrolysates (EFSA CEP Panel, 2023).

In the production of distilled alcohol, the food enzyme is added to milled cereals during pre‐saccharification or fermentation.43 The enzymatic treatment enhances the access of amylolytic enzymes to the starch granules and improves the yield. The food enzyme–TOS are not carried over into the distilled alcohol (EFSA CEP Panel, 2023).

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to a slurry of plant materials44 to increase the yield and to enhance the flavour of the plant‐based dairy alternatives. The food enzyme–TOS remain in the final foods.

In the processing of yeast and yeast products, the food enzyme is added to the yeast biomass during autolysis, or directly to autolysed yeast, yeast extracts or yeast cell walls45 to enhance the savoury taste of the yeast products, in which the food enzyme–TOS remain.

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 most of the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain. However, it may remain in its active form in brewed products, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

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 remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool46 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).

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 0.913 mg TOS/kg bw per day in toddlers at the 95th percentile.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 4.

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 two food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

Margin of exposure

3.6

A comparison of the NOAEL (5040 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.003–0.276 mg TOS/kg bw per day at the mean and from 0.005 to 0.913 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 5520.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme acid prolyl endopeptidase from the genetically modified A. niger strain GEP does not give rise to safety concerns under the intended conditions of use.

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

DOCUMENTATION AS PROVIDED TO EFSA

5

Request for EFSA to perform a scientific risk assessment on the food enzyme: Acid prolyl endopeptidase from a genetically modified strain of A. niger (GEP). April 2015. Submitted by DSM Food Specialties B.V.

Application for the extension of use of the food enzyme acid prolyl endopeptidase from a genetically modified Aspergillus niger strain GEP. June 2021. Submitted by DSM Food Specialties B.V.

Additional data. April 2022. Submitted by DSM Food Specialties B.V.

Additional data. September 2023. Submitted by DSM Food Specialties B.V.

Spontaneous submission. December 2025. Submitted by DSM Food Specialties B.V.

Summary report on genetically modified microorganism. December 2014. Delivered by Technical University of Denmark (Lungby, Denmark).

Summary report on technical data and dietary exposure. January 2016. Delivered by Hylobates Consulting and BiCT (Rome and Villanova del Sillaro, Italy).

Summary report on genotoxicity and subchronic toxicity. September 2016. Delivered by FoBIG (Freiburg, Germany).

ABBREVIATIONSBUNblood urea nitrogenbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGLPGood Laboratory PracticeGMMgenetically modified microorganismGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakilodaltonLoDlimit of detectionMCHmean corpuscular haemoglobinNOAELno observed adverse effect levelOECDOrganisation for Economic Co‐operation and DevelopmentPCRpolymerase chain reactionPPUproline protease unitsPTprothrombin timeRBCred blood cellRMraw materialTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2014‐00852, EFSA‐Q‐2021‐00423

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

PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Henk Van Loveren, Laurence Vernis and Holger Zorn.

NOTE

The full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality is received from the European Commission.

Supporting information

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