Safety evaluation of the food enzyme aspergillopepsin I from the non‐genetically modified Aspergillus sp. strain AE‐PRHF

Abstract

The food enzyme aspergillopepsin I (EC 3.4.23.18) is produced with the non‐genetically modified Aspergillus sp. strain AE‐PRHF by AMANO ENZYME INC. The food enzyme was free from viable cells of the production organism. It is intended to be used in 15 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 13 food manufacturing processes. It was estimated to be up to 2.172 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 2003 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 922. A search for the homology of the amino acid sequence of the aspergillopepsin I to known allergens was made and matches with three respiratory allergens and one injected allergen were 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.

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 1332/2008¹ 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 1332/2008¹ on food enzymes.

On 17 October 2023, a new application has been introduced by the applicant “AMANO ENZYME INC.” for the authorization of the food enzyme Aspergillopepsin I from a non‐genetically modified Aspergillus oryzae (strain AE‐PRHF).

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: Aspergillopepsin I from a non‐genetically modified Aspergillus oryzae (strain AE‐PRHF) in accordance with Regulation (EC) No 1331/2008² establishing a common authorization procedure for food additives, food enzymes and food flavourings. ³

1.2. Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme aspergillopepsin I from a non‐genetically modified Aspergillus oryzae strain AE‐PRHF.

Recent data identified the production microorganism as Aspergillus sp. (Section 3.1). Therefore, this name will be used in this opinion instead of A. oryzae.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme aspergillopepsin I from a non‐genetically modified A. oryzae strain AE‐PRHF.

Additional information, requested from the applicant during the assessment phase on 21 January 2025, were received on 18 April 2025 (see ‘Documentation provided to EFSA’).

2.2. Methodologies

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 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 PC‐1379 on the non‐confidential version of the technical dossier from 20 March 2025 to 10 April 2025. ⁵ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	Aspergillopepsin I
Systematic name	Not assigned
Synonyms	Aspergillus aspartic proteinase, carboxyl proteinase, pepsin‐type aspartic proteinase
IUBMB No	3.4.23.18
CAS No	9025‐49‐4
EINECS No	232‐796‐2

Aspergillopepsins I are aspartic endopeptidases that catalyse the hydrolysis of peptide bonds in proteins with broad specificity, favouring hydrophobic residues in P1 and P1’, but also accepting Lys in P1.

The food enzyme under assessment is intended to be used in 15 food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of dairy products for the production of (1) flavouring preparations and (2) modified milk proteins; (3) processing of eggs and egg products; processing of meat and fish products for the production of (4) modified meat and fish products and (5) protein hydrolysates; processing of cereals and other grains for the production of (6) baked products, (7) cereal‐based products other than baked, (8) brewed products and (9) distilled alcohol; processing of plant‐ and fungal‐derived products for the production of (10) edible oils from plant and algae, (11) tea and other herbal and fruit infusions, (12) plant extracts, (13) plant‐based analogues of milk and milk products and (14) protein hydrolysates; and (15) processing of yeast and yeast products.

3.1. Source of the food enzyme ⁶

The aspergillopepsin I is produced with the non‐genetically modified filamentous fungus Aspergillus sp. strain AE‐PRHF, which is deposited as A. oryzae at the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan) with the deposition number ■■■■■. ⁷ ■■■■■ was provided, but proved to be insufficient to identify the production strain at the species level. The production strain was identified at the genus level and, therefore, it will be considered as Aspergillus sp. in this opinion.

3.2. Production of the food enzyme ⁸

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ⁹ with food safety procedures based on Hazard Analysis and Critical Control Points and in accordance with Good Manufacturing Practice. ¹⁰

The production strain is grown as a pure culture using a typical industrial medium in ■■■■■ fermentation system with conventional process controls in place. ¹¹ After completion of the fermentation, the enzyme is ■■■■■ and the solid biomass is removed from the suspension broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including ■■■■■ in which the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. ¹² Finally, the food enzyme is ■■■■■. 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. ¹³

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 aspergillopepsin I is a single polypeptide chain of ■■■■■ amino acids. ¹⁴ The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁵ The food enzyme was analysed by size exclusion chromatography. ¹⁶ The chromatograms showed similar profiles.

No other enzyme activities were reported. ¹⁷

The applicant's in‐house determination of aspergillopepsin I activity is based on ■■■■■ (reaction conditions: pH ■■■■■, ■■■■■°C, ■■■■■ min). ■■■■■ was measured by a spectrophotometric method. The enzyme activity is expressed in protease activity units (UNIT)/g. One Unit is defined as the amount of enzyme that produces 1 μg/min of ■■■■■ under the conditions of the assay. ¹⁸

The food enzyme has a temperature optimum around ■■■■■°C (pH ■■■■■) and a pH optimum around pH ■■■■■ (■■■■■°C). ¹⁹ Thermostability was tested after pre‐incubation of the food enzyme for 60 min at different temperatures (pH 3.0). The enzyme activity decreased above 30°C showing no residual activity at 70°C. ²⁰

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation, one of which was used for the toxicological tests (Table 1). ²¹ The mean total organic solids (TOS) of the three batches was 94.5% and the mean enzyme activity/TOS ratio was 262.7 UNIT/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batch
		1	2	3 a
Aspergillopepsin I activity	UNIT/g b	260,000	247,000	238,000
Protein	%	78.8	76.3	75.6
Ash	%	0.5	0.5	0.5
Water	%	5.2	4.7	5.1
Total organic solids (TOS) c	%	94.3	94.8	94.4
Activity/TOS ratio	UNIT/mg TOS	275.7	260.5	252.1

^a Batch used for the toxicological studies.

^b UNIT: Protease activity Unit (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three commercial batches was below 0.05 mg/kg ^{22 , 23} 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). ²⁴ No antimicrobial activity was detected in any of the tested batches. ²⁵

Strains of the 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 aflatoxin B1, B2, G1, G2, deoxynivalenol, T‐2 toxin, HT‐2 toxin, zearalenone, ochratoxin A, sterigmatocystin was examined in all food enzyme batches and all were below the limit of quantification (LoQ) of the applied methods. ^{26 , 27} Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

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

3.3.4. Viable cells 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. ■■■■■. No colonies of the production strain were detected. A positive control was included. ²⁸

3.4. Toxicological data

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian cell micronucleus test and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batch 3 (Table 1) used in these studies is one of the batches used for commercialisation and is considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020), Japanese Guidance Notification 0920 (Japanese Guidance Notification 0920, 2012) and following Good Laboratory Practice (GLP). ²⁹

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method.

Based on the results of a range‐finding test, two main experiments were carried out in triplicate, using five concentrations of the food enzyme of 313, 625, 1250, 2500 and 5000 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 restriction and the results of high relevance.

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

3.4.1.2. In vitro mammalian cell micronucleus test

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2016), Japanese Guidance Notification 0920 (Japanese Guidance Notification 0920, 2012) and following GLP. ³⁰ An experiment was performed with duplicate cultures of human lymphoblastoid (TK6) cells. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In a range‐finding test, no cytotoxicity above 50% was seen at any concentration tested up to 5000 μg TOS/mL with and without S9‐mix.

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for the frequency of micronucleated cells at concentrations of 1250, 2500 and 5000 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.

A cytotoxicity of 30% (based on relative population doubling) was seen in the short‐term treatment and in the long‐term treatment without S9‐mix at the highest concentration tested. The frequency of micronucleated cells was not statistically significantly different to the negative controls at all concentrations tested.

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

The Panel concluded that the food enzyme aspergillopepsin I did not induce an increase in the frequency of micronucleated cells under the test conditions applied in this study.

3.4.2. Repeated dose 90‐day oral toxicity study in rodents

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018) ³¹ with the following deviations: the oestrus cycle of females was not determined by taking vaginal smears; several parameters were not measured in all females, i.e. prothrombin time, activated partial thromboplastin time, fibrinogen; histopathological examination of the parathyroid gland was not performed in one control and one high‐dose females. The Panel considered that these deviations are minor and do not impact on the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley Crl:CD(SD) rats received the food enzyme by gavage in doses of 501, 1002 or 2003 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

One male in the vehicle control group was euthanised in moribund condition on day 70 of administration, due to an incidental lymphoma.

The feed consumption was statistically significantly decreased on day 21 of administration in high‐dose females (−5%). The Panel considered the change as not toxicologically relevant, as it was only recorded at a single time interval, it was only observed in one sex, there was no statistically significant change in the final feed consumption, body weight or the body weight gain and the change was within the historical control values.

In the functional observations, a statistically significant decrease in the rearing count was observed in high‐dose males (−50%) in week 8, an increase in motor activity in the intervals 10–20 min (+315%, +246%) and 30–40 min (+538%, +413%) and in total motor activity (+87%, +61%) in low‐ and high‐dose males; an increase in motor activity in the 30–40 min interval was observed in low‐ and mid‐dose females (+1000%, +650%). The Panel considered the changes as not toxicologically relevant, as they were only recorded at a single time interval (rearing count), they were only observed in one sex (except of motor activity in 30–40 min interval), there was no dose–response relationship (motor activity), the change was within the historical control values (motor activity in males) and there were no other functional parameters affected.

Haematological investigations revealed a statistically significant decrease in haematocrit (−4%) and reticulocyte count (−44%) in high‐dose males. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), there were no changes in other relevant parameters (red blood cell count), the change was small (haematocrit), there were no histopathological changes in bone marrow and the changes were within the historical control values (both parameters).

Clinical chemistry investigations revealed a statistically significant decrease in lactate dehydrogenase (LDH) in high‐dose males (−47%), an increase in glucose in low‐, mid‐ and high‐dose females (+13%, +15%, +14%) and a decrease in inorganic phosphorus (P) in mid‐dose males (−10%). 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 (P, glucose), there were no changes in other relevant parameters (other liver enzymes, electrolytes), there were no histopathological changes in liver and kidneys and the changes were within the historical control values (LDH, glucose).

Statistically significant changes in hormone levels included a decrease in thyroid stimulating hormone (TSH) in mid‐dose females (−32%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship and there were no histopathological changes in thyroid and pituitary glands.

Statistically significant changes detected in organ weights were a decrease in the relative spleen weight in high‐dose males (−36%), a decrease in the relative lung weight in low‐dose males (−7%) and a decrease in the relative heart weight in mid‐dose females (−6%). 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 (the relative lung and heart weights), there were no haematological changes correlated with the decreased spleen weight, there were no histopathological changes in relevant organs and the change was within the historical control values (relative spleen weight).

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

The Panel identified a no observed adverse effect level (NOAEL) of 2003 mg TOS/kg bw per day, the highest dose tested.

3.4.3. 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 aspergillopepsin I produced with the Aspergillus sp. strain AE‐PRHF 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 four allergens were found in the AllergenOnline database and the Allergen Database for Food Safety. ³²

The matching respiratory allergens were Asp f 10 (88.7% sequence identity), an aspergillopepsin from A. fumigatus, pepsin A (39.0% sequence identity) from pig (Sus scrofa) and Rhi o 1 (35.6% sequence identity), an aspartyl endopeptidase from Rhizopus oryzae. The matching injected allergen was Aed a 11 (40.7% sequence identity), a lysosomal aspartic protease from yellow fever mosquito (Aedes aegypti). ³³

No reports on oral or respiratory sensitisation or elicitation reactions of the aspergillopepsin I under assessment have been published. ³⁴

The aspartic protease Asp f 10 is an inhalant allergen from A. fumigatus associated with allergic aspergillosis (Crameri, 1998). Pepsin A from S. scrofa is associated with occupational asthma and rhinitis (Añíbarro Bausela & Fontela, 1996; Cartier et al., 1984). Rhi o 1 is an inhalant aspartic protease identified in R. oryzae (Sircar et al., 2015). Several studies have shown that individuals respiratorily sensitised to an enzyme are usually able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Cullinan et al., 1997; Poulsen, 2004; Armentia et al., 2009;). Aspartic proteases are associated with allergies to insects, but no allergic reactions upon dietary exposure have been reported (Cantillo et al., 2017). ³⁵

Aspergillopepsins are respiratory fungal allergens (Porter et al., 2009; Rementeria et al., 2005). ³⁶ No allergic reactions upon dietary exposure to any aspergillopepsin have been reported in the literature.

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 aspergillopepsin I 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.

■■■■■ and ■■■■■, products from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³⁷ ) are used as raw materials. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, 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 15 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. ³⁸

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of dairy products
• Production of flavouring preparation from dairy products	Dairy products	30.6–61.3
• Production of modified milk proteins	Milk proteins	122.5–306.4
Processing of eggs and egg products	Eggs	30.6–61.3
Processing of meat and fish products
• Production of modified meat and fish products	Meat and fish	30.6–61.3
• Production of protein hydrolysates from meat and fish proteins	Meat and fish materials	122.5–306.4
Processing of cereals and other grains
• Production of baked products	Flour	24.5–61.3
• Production of cereal‐based products other than baked	Cereals	30.6–61.3
• Production of brewed products	Cereals	30.6–61.3
• Production of distilled alcohol	Cereals	61.3–122.5
Processing of plant‐ and fungal‐derived products
• Production of edible oils from plant and algae	Algal cells	147.1–1467.5
• Production of tea and other herbal and fruit infusions	Tea leaves, herbs, fruits	18.4–36.8
• Production of plant extracts	Plant material	18.4–36.8
• Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseed, nuts	2.5–12.3
• Production of protein hydrolysates from plants and fungi	Plant proteins	122.5–306.4
Processing of yeast and yeast products	Yeast biomass 39	30.6–61.3

Abbreviation: TOS, total organic solids.

^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 to dairy products (e.g. curd, cheese) ⁴⁰ to enhance sensory properties. ⁴¹ The food enzyme–TOS remain in these enzyme‐modified dairy ingredients, which are subsequently used as an ingredient to formulate a variety of foods such as processed cheese, cheese sauce, cheese powder, salad dressings and snacks.

In the production of modified milk proteins, the food enzyme is added to milk proteins after the mixing with water. ⁴² The hydrolysis improves solubility and taste. The food enzyme–TOS remain in the modified milk proteins, which are ingredients of a variety of final foods, including infant formulae, follow‐on formulae and foods for special medical purposes.

In the processing of eggs and egg products, the food enzyme is added to the whole liquid egg, to the egg white or to the egg yolk during the incubation step. ⁴³ The hydrolysis by aspergillopepsin I improves the sensory and technological properties of these products. ⁴⁴ The food enzyme–TOS remain in the final egg products.

In the production of modified meat and fish products, the food enzyme is added to meat and fish during the mincing phase to obtain products like steaks or hamburgers. ⁴⁵ The enzymatic treatment softens meat. ⁴⁶ The food enzyme–TOS remain in the final foods.

In the production of hydrolysed proteins, the food enzyme is added to plant (e.g. legumes and cereals) and animal source materials (e.g. meat, fish) during hydrolysis. ⁴⁷ Aspergillopepsin I is used to achieve the desired degree of hydrolysis and to enhance the flavour of the resulting protein hydrolysates, which are subsequently used as ingredients in a variety of foods. The food enzyme–TOS remain in these hydrolysates.

In the production of baked products and cereal‐based products other than baked, the food enzyme is added to flour during the preparation of the dough or batter. ⁴⁸ The aspergillopepsin I cleaves peptide bonds in the gluten network, thus reducing the rigidity of the dough. The food enzyme–TOS remain in the final products.

In the production of low‐protein cereals, the aspergillopepsin I is added to the rinsed cereals ⁴⁹ to hydrolyse proteins on their surface. ⁵⁰ The treated cereals are then washed with running water for at least ■■■■■ (equivalent to ■■■■■ of washing, according to the applicant), ⁵¹ by which the food enzyme–TOS are expected to be removed. To substantiate this removal, the amount of total proteins present after each washing step was measured by ■■■■■ ⁵² and by ■■■■■ (■■■■■. ⁵³ The data from both methods showed that after the 5th wash, the amount of total protein in the washing solution is less than 1%. ⁵⁴ These data were considered by the Panel as sufficient to confirm the removal of the food enzyme–TOS in these low‐protein cereals.

In the production of brewed products, the food enzyme is added to the grains during the mashing step for beer production. ⁵⁵ In the production of fermented beverages such as sake, it may be added during slurry mixing, liquefaction, saccharification or fermentation steps. ⁵⁶ The aspergillopepsin I hydrolyses proteins of the cereals to release free amino nitrogen for the growth of yeast during fermentation. The food enzyme–TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme is added during the liquefaction and fermentation steps, and may also be added during slurry mixing and saccharification. ⁵⁷ The enzymatic treatment improves the yield and enhances the access of amylolytic enzymes to the starch granules, facilitating the degradation of starch and non‐starch polysaccharides into fermentable sugars. The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of edible oils from algae, the food enzyme is added to the algal oil during the degumming and deacidification step ⁵⁸ to hydrolyse proteins of the algal cell wall. The food enzyme–TOS are removed from the final processed oils by refining processes (EFSA CEP Panel, 2023).

In the processing of tea, herbal and fruit infusions, the food enzyme is added to tea leaves, herbs and fruits ⁵⁹ to enhance the sensory properties of the infusions. ⁶⁰ The food enzyme–TOS remain in the final products.

In the processing of plant extracts, the food enzyme is added ⁶¹ to improve the sensory properties of the final food ingredients, ⁶² in which the food enzyme–TOS remain.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to the plant‐based drinks ⁶³ to increase the yield and to enhance the flavour. ⁶⁴ The food enzyme–TOS remain in these plant‐based analogues of milk and milk products.

In the processing of yeast and yeast products, the food enzyme is added to the yeast biomass during the lysis or during the incubation steps ⁶⁵ to intensify the savoury taste of the yeast extracts ⁶⁶ that are used as ingredients in a wide range of foods. The food enzyme–TOS remain in the final food products.

Based on the data provided on thermostability (see Section 3.3.1) and the processing steps applied in the respective food manufacturing processes, the Panel considered that the food enzyme is inactivated in the majority 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 baked products, brewed products and hamburgers, depending on the heat treatment conditions.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the 13 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 ⁶⁷ 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 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 2.172 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)	0.310–0.604 (12)	0.532–0.744 (15)	0.272–0.769 (19)	0.121–0.385 (21)	0.157–0.283 (22)	0.117–0.222 (23)
Min–max 95th percentile (number of surveys)	0.838–1.227 (11)	0.933–1.291 (14)	0.516–2.172 (19)	0.248–0.679 (20)	0.313–0.629 (22)	0.240–0.408 (22)

Abbreviation: TOS, total organic solids.

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	+
Although the food enzyme–TOS are removed in the low‐protein cereals, the calculation did not differentiate the low‐protein cereals from regular cereal products. The consumption of low‐protein cereals is not separately recorded in dietary surveys designed for whole population.	+
For yeast processing, although only yeast extract is produced 68 , the food categories chosen for calculation cover also those containing mannoproteins resulting from the treatment of yeast cell walls.	+
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 edible oils from plant and algae	−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure; TOS, total organic solids.

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.

3.6. Margin of exposure

A comparison of the NOAEL (2003 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.117–0.769 mg TOS/kg bw per day at the mean and from 0.240 to 2.172 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MoE) of at least 922.

4. CONCLUSIONS

Based on the data provided, the absence of issues of concern arising from the production process, the removal of TOS during two processes and the derived margin of exposure for the remaining 13 food manufacturing processes, the Panel concluded that the food enzyme aspergillopepsin I produced with the non‐genetically modified Aspergillus sp. strain AE‐PRHF does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for an authorisation of a new food enzyme aspergillopepsin I from Aspergillus oryzae strain AE‐PRHF. 17 October 2023. Submitted by AMANO ENZYME INC.

Additional information. 18 April 2025. Submitted by AMANO ENZYME INC.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

EFSA CEP Panel

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EFSA FEZ Panel

EFSA Panel on Food Enzymes

EFSA GMO Panel

EFSA Panel on Genetically Modified Organisms

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FEIM

food enzyme intake model

FoodEx

standardised food classification and description system

GLP

Good Laboratory Practice

GM

genetically modified

GMO

genetically modified organism

■■■■■

■■■■■

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LDH

lactate dehydrogenase

LoQ

limit of quantification

MHLW

Ministry of Health, Labour and Welfare, Japan

MoE

margin of exposure

NITE

National Institute of Technology and Evaluation Biological Resource Center

NOAEL

no observed adverse effect level

non‐GM

non‐genetically modified

OECD

Organisation for Economic Co‐operation and Development

P

inorganic phosphorus

RM

raw material

■■■■■

■■■■■

TK6

human lymphoblastoid cells

TOS

total organic solids

TSH

thyroid stimulating hormone

Unit

protease activity units

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00205

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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, and Holger Zorn.

LEGAL NOTICE

The scientific output published implements EFSA's decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA they are consequently withheld from public disclosure by redaction.

Supporting information

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

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

Appendix A can be found in the online version of this output (in the ‘Supporting information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, the Netherlands, Portugal, 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, the 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*, the 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*, the 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*, the 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. , Van Loveren, H. , Vernis, L. , Kovalkovičová, N. , Cavanna, D. , Fernàndez‐Fraguas, C. , Lunardi, S. , Peluso, S. , & Liu, Y. (2025). Safety evaluation of the food enzyme aspergillopepsin I from the non‐genetically modified Aspergillus sp. strain AE‐PRHF . EFSA Journal, 23(7), e9561. 10.2903/j.efsa.2025.9561