Safety evaluation of the food enzyme aspergillopepsin I from the non‐genetically modified Aspergillus sp. strain LPL 3U‐3‐10

Abstract

The food enzyme aspergillopepsin I (EC 3.4.23.18) is produced with the non‐genetically modified Aspergillus sp. strain LPL 3U‐3‐10 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in 16 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 14 food manufacturing processes. It was estimated to be up to 4.673 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 1800 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 385. 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 this 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 1332/2008 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 1332/2008¹ on food enzymes

Five applications have been introduced by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzyme consisting of Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I from Aspergillus oryzae and the companies “BENEO‐Palatinit Gmbh” for the authorisation of the food enzyme Isomaltulose synthase from Protaminobacter rubrum (strain Z12A), “Naganase (Europa) GmbH” for the authorisation of the food enzyme Chitinase from a genetically modified strain of Streptomyces violaceoruber (strain pChi), “Clasado Ingredients Ltd.” for the authorisation of the food enzyme Beta‐galactosidase from a genetically modified strain of Escherichia coli (strain BglA MCB3) and “Meiji Seika Pharma Co., Ltd” for the authorisation of the food enzyme consisting of Aspergillopepsin I and II from Aspergillus niger var. macrosporus (strain DBD‐0406).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008,² the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contains all the elements required under Chapter II of that Regulation

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I from Aspergillus oryzae, Isomaltulose synthase from Protaminobacter rubrum (strain Z12A), Chitinase from a genetically modified strain of Streptomyces violaceoruber (strain pChi), Beta‐galactosidase from a genetically modified strain of Escherichia coli (strain BglA MCB3) and Aspergillopepsin I and II from Aspergillus niger var. macrosporus (strain DBD‐0406) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

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 enzymes Protease, Leucyl amino‐peptidase, Oryzin and Aspergillopepsin I from A. oryzae submitted by the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).

The application was submitted initially as a joint dossier ⁴ and identified as the EFSA‐Q‐2018‐01030. During a meeting between EFSA, the European Commission and AMFEP, ⁵ it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00268 and concerns the food enzyme Aspergillopepsin I produced with the A. oryzae strain LPL 3U‐3‐10 and submitted by Shin Nihon Chemical Co., Ltd.

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 LPL 3U‐3‐10.

Additional information, requested from the applicant during the assessment process on 8 February 2024 and 1 June 2026 was received on 8 August 2024 and 17 June 2026, respectively (see ‘Documentation provided to EFSA’).

2.2. Methodologies

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 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).

3. ASSESSMENT

IUBMB nomenclature	Aspergillopepsin I
Systematic name	–
Synonyms	Aspergillus aspartic proteinase, carboxyl proteinase, pepsin‐type aspartic proteinase
IUBMB No	EC 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, resulting in the generation of peptides and amino acids.

The food enzyme is intended to be used in 16 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 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 (4) baked products, (5) cereal‐based products other than baked, (6) brewed products and (7) distilled alcohol; processing of fruits and vegetables for the production of (8) wine and wine vinegars and (9) non‐wine vinegar; 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, (14) soy sauce and (15) protein hydrolysates from plants and fungi; (16) processing of yeast and yeast products.

3.1. Source of the food enzyme

The aspergillopepsin I is produced with the non‐genetically modified filamentous fungal strain LPL 3U‐3‐10 which is deposited as A. oryzae at the National Institute of Technology and Evaluation's Biological Resource Center (NBRC, Japan) with deposition number ■■■■■. ⁶ The phylogenetic analyses provided for the identification of the production strain did not allow an unambiguous distinction among Aspergillus species. Therefore, the strain is reported here as Aspergillus sp. ⁷

Aspergillus sp. strain LPL 3U‐3‐10 was shown to ■■■■■ ⁸

3.2. Production of the food enzyme

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 a ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the enzyme is extracted with ■■■■■ and the solid biomass is removed from the suspension by centrifugation. The supernatant 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 membrane and is discarded. ¹¹ 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. ¹²

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 composed of ■■■■■ amino acids ¹³ and the molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁴ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. ¹⁵ A consistent protein pattern was observed across all batches.

No other enzyme activities were reported. ¹⁶

The determination of the aspergillopepsin I activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The release of ■■■■■ is measured spectrophotometrically at ■■■■■ nm. The enzyme activity is expressed in Protease activity units (HUT)/g. One HUT is defined as the amount of enzyme that produces ■■■■■ equivalent to 1.10 μg/mL of ■■■■■ per min under the assay conditions. ¹⁷

To determine the pH profile of the enzyme, a different activity assay was used, substituting ■■■■■ by ■■■■■ as substrate. The aspergillopepsin I has a temperature optimum around 50°C (■■■■■) and a pH optimum around 6.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures. Enzyme activity decreased above 30°C, showing no residual activity at 70°C. ¹⁸

3.3.2. Chemical parameters

Data were provided for four food enzyme batches intended for commercialisation, among which batch 4 was also used for the toxicological tests (Table 1). ¹⁹ The mean Total Organic Solids (TOS) of the four food enzyme batches for commercialisation was 18.1% and the mean enzyme activity/TOS ratio was 1720 U/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3	4 a
Aspergillopepsin I activity	U/g b	306,000	319,000	316,000	305,000
Protein	%	11.6	11.6	11.4	11.7
Ash	%	0.3	0.2	0.3	0.7
Water	%	81.4	81.3	81.2	82.1
Total Organic Solids (TOS) c	%	18.3	18.5	18.5	17.2
Aspergillopepsin I activity/TOS ratio	U/mg TOS	1672	1724	1708	1773

^a Batch used for the toxicological studies.

^b HUT/g: Protease Activity, Fungal (the same as U/g) (see Section 3.3.1).

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

3.3.3. Purity

The lead content in all batches was below 0.05 mg/kg ²⁰ which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, the arsenic content was below the limit of quantification (LoQ) of the employed method. ^{21 , 22}

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). ²³ No antimicrobial activity was detected in any of the tested batches. ²⁴

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 aflatoxins (B1, B2, G1, G2), ochratoxin A, sterigmatocystin, T‐2 toxin, zearalenone, kojic acid, cyclopiazonic acid and 3‐nitropropionic acid was examined in batches 1, 2 and 3 and all were below the limit of detection (LoD) of the applied methods. ^{25 , 26} 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 is 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. Ten g of product was diluted in 40 mL of sterile water. From this, 5 mL was spread onto non‐selective agar plates and incubated at 30°C for up to 6 days. No colonies were detected. A positive control was included. ²⁷

3.4. Toxicological data

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

The batch 4 (Table 1) used in these studies is one of the batches intended for commercialisation, and has a similar enzyme activity/TOS ratio to the other batches and it 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, 1997) and following Good Laboratory Practice (GLP). ²⁸ 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 pre‐incubation method or ‘treat and wash’ method. All experiments were carried out in triplicate.

The dose‐finding study applying the pre‐incubation method was carried out using eight concentrations of the food enzyme of 8, 25, 74, 222, 667, 2000, 6000 and 18,000 μg TOS/plate. Toxic effects, evident as a reduction of background bacterial lawn, occurred in strains TA100, TA1537 and E. coli WP2uvrA without S9‐mix at ≥ 6000 μg TOS/plate and with S9‐mix in strain TA100 at ≥ 6000 μg TOS/plate and at 18,000 μg TOS/plate in strain E. coli WP2uvrA. Upon treatment with the food enzyme, an increase in the number of revertant colonies was observed at 6000 μg TOS/plate in strains TA100 and TA1535 without S9‐mix and at 18,000 μg TOS/plate in strain TA100 with S9‐mix. Growth stimulation, as indicated by the thickening of the background bacterial lawn, was observed in the absence of S9‐mix: at ≥ 6000 μg TOS/plate in strains TA98, TA100, TA1535 and TA1537 and at ≥ 2000 μg TOS/plate in strain E. coli WP2uvrA and in the presence of S9‐mix: at ≥ 2000 μg TOS/plate in strains E. coli WP2uvrA and TA1537 and at ≥ 6000 μg TOS/plate in strains TA98, TA100 and TA1535.

The main study applying the pre‐incubation method was carried out using eight concentrations of the food enzyme of 141, 281, 563, 1130, 2250, 4500, 9000 and 18,000 μg TOS/plate in strains E. coli WP2uvrA and TA98, TA1537 without S9‐mix and seven concentrations of the food enzyme of 281, 563, 1130, 2250, 4500, 9000 and 18,000 μg TOS/plate in strains E. coli WP2uvrA and TA98, TA1535, TA1537 with S9‐mix. Toxic effects, evident as a reduction of background bacterial lawn, occurred at 9000 μg TOS/plate in strain TA1537, at ≥ 4500 μg TOS/plate in strain E. coli WP2uvrA without S9‐mix and at 18,000 μg TOS/plate in strain E. coli WP2uvrA with S9‐mix. Upon treatment with the food enzyme, an increase in the number of revertant colonies was observed at 18,000 μg TOS/plate in strain TA1535 with S9‐mix. Growth stimulation, as indicated by the thickening of the background bacterial lawn, was observed in the absence of S9‐mix: at ≥ 2250 μg TOS/plate in strain E. coli WP2uvrA, at ≥ 4500 μg TOS/plate in strains TA98 and TA1537 and in the presence of S9‐mix: at ≥ 2250 μg TOS/plate in strains E. coli WP2uvrA and TA1537, at ≥ 4500 μg TOS/plate in strains TA1535 and TA98.

The growth stimulation of the background bacterial lawn and the increase of revertant colony numbers, observed in dose‐finding and main study applying the pre‐incubation method, were attributed by the study authors to the presence of free amino acids such as histidine, in the medium.

The dose‐finding study applying the treat and wash method was carried out using seven concentrations of the food enzyme of 25, 74, 222, 667, 2000, 6000 and 18,000 μg TOS/plate in strains TA100 and TA1535 without and with S9‐mix. 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 main study applying the treat and wash method was carried out using six concentrations of the food enzyme of 563, 1130, 2250, 4500, 9000 and 18,000 μg TOS/plate in strains TA100 and TA1535 without and with S9‐mix. 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 Panel concluded that the food enzyme did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosomal aberration test was carried out according to the OECD Test Guideline 473 (OECD, 2016a) and following GLP. ²⁹ An experiment was 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).

Based on the results of a range finding test, in the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 4500, 9000 and 18,000 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery period) either with or without S9‐mix and at concentrations of 500, 1000 and 1750 μg TOS/mL in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

No cytotoxicity was seen in the short‐term treatment either with or without S9‐mix. In the long‐term treatment, cytotoxicity of 50% (mitotic inhibition) was observed at 1750 μg TOS/mL. The frequency of structural and numerical aberrations was not statistically significantly different to the negative controls at any concentration tested.

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

3.4.1.3. In vitro mammalian cell micronucleus test

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

Cells were exposed to the food enzyme and scored for the frequency of mononucleated cells with micronuclei at concentrations of 988, 2222 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure) either with or without S9‐mix and at concentrations of 658, 1481 and 2222 μg TOS/mL in a long‐term treatment (24‐h continuous exposure) without S9‐mix. Cultures were then incubated until 1.5 to 2.0 population doublings were achieved in the concurrent solvent (vehicle) control (24–29 h).

No cytotoxicity was seen in the short‐term treatment with or without S9‐mix. In the long‐term treatment, cytotoxicity of 52% was observed at a concentration of 2222 μg TOS/mL. The frequency of micronucleated cells was not statistically significantly different to the negative controls at any concentration tested.

The Panel concluded that the food enzyme 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

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 200, 600 or 1800 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water for injection). In addition, heat‐inactivated food enzyme (inactivation conditions: at 70°C for 30 min) was given to the rats as the comparative high‐dose group of 1800 mg TOS/kg bw per day.

No mortality was observed.

In the functional observations, on day 13 a statistically significant decrease in the number of defecation in high‐dose males (−92%) compared to the control group and on day 89 a decrease in the forelimb grip strength in high‐dose females (−23%) in comparison to the comparative high‐dose group were observed, but not compared to the control group. The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically and they were only observed in one sex.

Haematological investigations revealed a statistically significant decrease in basophil count (−50%) and large unstained cell count (LUC) (−43%) and an increase in the prothrombin time (PT; +13%) in high‐dose males as compared to those of the comparative high‐dose group, but not compared to the control group. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there were no changes in other relevant parameters (other white blood cell parameters, activated partial thromboplastin time) and there were no histopathological changes in bone marrow, spleen, liver and lymph nodes.

Clinical chemistry investigations revealed a statistically significant decrease in the total protein concentration in high‐dose males (−5%) as compared to that of the comparative high‐dose group, but not compared to the control group. The Panel considered the change as not toxicologically relevant, as it was only observed in one sex and there were no histopathological changes in liver and kidneys.

The urinalysis using fresh urine revealed an increased number of protein‐ and urobilinogen‐positive high‐dose males and females and ketone body‐positive males in all treated groups. The Panel considered the changes as not toxicologically relevant, as there were no histopathological changes in kidneys.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1800 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 from A. oryzae strain LPL 3U‐3‐10 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 respiratory allergens and one injected allergen were found in the AllergenOnline database. ³²

The matching respiratory allergens were Asp f 10 (88.7% sequence identity), an aspergillopepsin from Aspergillus fumigatus, Rhi o 1 (35.6% sequence identity), an aspartic protease from Rhizopus oryzae and pepsin A (39.0% sequence identity) from pig (Sus scrofa).

The matching injected allergen was Aed a 11 (40.7% sequence identity), an aspartic protease from Yellow fever mosquito (Aedes aegypti).

No reports on oral or respiratory sensitisation or elicitation reactions of the food enzyme under assessment have been published. ³³

Asp f 10 is an aspergillopepsin from A. fumigatus, associated with allergic aspergillosis (Crameri, 1998), Rhi o 1 is an inhalant cross‐reactive aspartic protease identified in R. oryzae (Sircar et al., 2015) and pepsin A from pig (Sus scrofa) is associated with occupational asthma and rhinitis (Añíbarro Bausela & Fontela, 1996; Cartier et al., 1984). 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 (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004).

The matching injected allergen was Aed a 11, an aspartic protease from Yellow fever mosquito (A. aegypti). Aspartic proteases are associated with allergies to insects, but no allergic reactions upon dietary exposure have been reported (Cantillo et al., 2017).

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 food enzyme 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 product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³⁴ ) is used as raw material. In addition, ■■■■■ and ■■■■■, known sources of allergens, are present in the culture medium. 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), 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.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in 16 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 , 36}

Food manufacturing process a	Raw material (RM)	Maximum use level (mg TOS/kg RM) b
Processing of dairy products
Production of flavouring preparations from dairy products	Dairy products 37	3650 38
Production of modified milk proteins 39	Milk proteins 40	30 41
Processing of meat and fish products
Production of protein hydrolysates from meat and fish proteins 42	Animal skin, fish skin, fish scale	300
Processing of cereals and other grains
Production of baked products	Flour	30
Production of cereal‐based products other than baked	Flour, Cereals	330
Production of brewed products	Cereals	120
Production of distilled alcohol	Cereals	120
Processing of fruits and vegetables
Production of wine and wine vinegar	Grapes	120
Production of non‐wine vinegar	Cereals, apple	300
Processing of plant‐ and fungal‐derived products
Production of edible oils from plant and algae	Olive fruit 43	48.4
Production of tea and other herbal and fruit infusions	Tea leaves	300
Production of plant extracts 44	Vegetables	300
Production of plant‐based analogues of milk and milk products	Cereals	146
Production of soy sauce	Wheat, soya bean	300
Production of protein hydrolysates from plants and fungi 45	Soy, pea	300
Processing of yeast and yeast products	Yeast	1460

Abbreviation: TOS, Total Organic Solids.

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

^b The numbers in bold were used for calculation.

In the production of flavouring preparations from dairy products, the food enzyme is added to dairy products (e.g. butter, cheese) after the first pasteurisation ⁴⁶ to enhance sensory properties. The food enzyme–TOS remain in the final products.

In the production of modified milk proteins, the food enzyme is added to casein or whey proteins prior to pasteurisation ⁴⁷ to enhance solubility and taste. The food enzyme–TOS remain in the modified milk proteins.

In the production of protein hydrolysates, the food enzyme is added to a variety of protein‐rich materials from animal (e.g. animal skin, fish scales) ⁴⁸ or plant (e.g. soy, pea) ⁴⁹ sources. Protein hydrolysates derived from plant or animal sources are used interchangeably as an ingredient to formulate foods. The food enzyme–TOS remain in these protein hydrolysates.

In the production of baked products and in the production of cereal‐based products other than baked, the food enzyme is added to flour during dough formation ⁵⁰ to catalyse the hydrolysis of gluten proteins, facilitating its handling. The food enzyme–TOS remain in the final products.

In the production of brewed products, the food enzyme is added to cereals during the mashing. ⁵¹ In the production of fermented beverages such as sake, it may be added before or after the koji fermentation ⁵² to catalyse the release of free amino nitrogen for the growth of yeast during alcoholic fermentation. The food enzyme–TOS remain in the brewed products.

In the production of distilled alcohol, the food enzyme may be added to cereals during slurry mixing, liquefaction, saccharification or fermentation ⁵³ to improve the yield and to enhance the access of amylolytic enzymes to the starch granules, facilitating the hydrolysis of starch and non‐starch polysaccharides into fermentable sugars. The food enzyme–TOS are not carried over into the distilled alcohols (EFSA CEP Panel, 2023).

In the production of wine and wine vinegar, the food enzyme may be added to grapes during pressing, fermentation and clarification ⁵⁴ to increase the yield and to catalyse the hydrolysis of the haze‐forming proteins, such as chitinases and thaumatin‐like proteins. According to the applicant, filtration in the subsequent purification steps can remove the food enzyme–TOS. However, in the absence of analytical data to demonstrate the extent of such removal, ⁵⁵ the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS are transferred in wines and wine vinegars.

In the production of non‐wine vinegar, the food enzyme is added to cereals or to fruits during the saccharification and fermentation ⁵⁶ to enhance alcoholic fermentation and prevent haze formation during storage. ⁵⁷ The food enzyme–TOS remain in vinegars.

In the production of edible oils from plants and algae, the food enzyme is used to treat olives ⁵⁸ during malaxation. ⁵⁹ The food enzyme–TOS are removed in the refining process (EFSA CEP Panel, 2023).

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaves during the extraction ⁶⁰ to enhance the sensory properties of the infusion. The food enzyme–TOS remain in the final products.

In the production of plant extracts, ⁶¹ the food enzyme is added to the aqueous slurry before the filtration ⁶² to enhance the sensory properties of the final food ingredients, in which the food enzyme–TOS remain.

In the production of plant‐based analogues of milk and milk products, the food enzyme may be added to plant materials after the milling or during the saccharification ⁶³ to increase the yield and to enhance the flavour. The food enzyme–TOS remain in the final products.

In the production of soy sauce, the food enzyme is added after the fermentation ⁶⁴ to enhance the sensory properties of the final products. ⁶⁵ The food enzyme–TOS remain in the soy sauce.

In the processing of yeast and yeast products, the food enzyme is added to the yeast biomass during the lysis ⁶⁶ to intensify the savoury taste of the resulting yeast extracts, ⁶⁷ 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 baked products, wine and wine vinegars 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 14 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 ⁶⁸ 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 4.673 mg TOS/kg bw per day in infants at the 95th percentile.

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)	0.354–1.489 (14)	0.591–1.340 (17)	0.398–0.903 (21)	0.154–0.653 (23)	0.163–0.464 (23)	0.118–0.529 (25)
Min–max 95th percentile (number of surveys)	0.955–4.673 (13)	1.458–3.155 (16)	0.964–2.322 (21)	0.372–1.464 (22)	0.468–1.104 (23)	0.313–1.304 (24)

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
For yeast processing, although only yeast extract is produced, 69 the food categories chosen for calculation cover also those containing mannoproteins resulting from the treatment of yeast cell walls.	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Assumption that the food enzyme–TOS are fully transferred into wines and wine vinegars	+
Selection of broad FoodEx categories for the exposure assessment	+
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 plants and algae	−

TOS, Total Organic Solids; +, 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 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 (1800 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.118–1.489 mg TOS/kg bw per day at the mean and from 0.313 to 4.673 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 385.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme aspergillopepsin I produced with the non‐genetically modified Aspergillus sp. strain LPL 3U‐3‐10 does not give rise to safety concerns under the intended conditions of use.

5. REMARK

The term ‘olive oil’ is defined in the Regulation (EU) No 1308/2013 ⁷⁰ as “composed of refined olive oils and virgin olive oils”. The term ‘virgin olive oils’ means ‘oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alterations in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oils obtained using solvents or using adjuvants having a chemical or biochemical action, or by re‐esterification process and any mixture with oils of other kinds’. In accordance with the law, the use of enzymes is not permitted in the production of virgin olive oils in the European Union. Therefore, the use of this food enzyme in the production of virgin olive oils is excluded from this assessment.

6. DOCUMENTATION AS PROVIDED TO EFSA

Application for the authorisation of protease from aspergillus oryzae strain LPL 3U‐3‐10 as a food enzyme in the European union. March 2023. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information. August 2024. Submitted by Shin Nihon Chemical Co.

Additional information. June 2026. Submitted by Shin Nihon Chemical Co.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

Euroepan Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

EU

European Union

FAO

Food and Agricultural Organization of the United Nations

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

LoQ

limit of quantification

LUC

large unstained cell

MoE

margin of exposure

OECD

Organisation for Economic Co‐operation and Development

PCR

polymerase chain reaction

TOS

Total Organic Solids

WGS

whole genome sequencing

WHO

World Health Organization

AUTHOR CONTRIBUTIONS

Under Regulation 178/2002, the Scientific Panel is the author of the scientific output. Contributions include Working Groups who provide the draft assessment which is reviewed and adopted by the Panel; EFSA staff members who provide scientific support including inter alia data collection and analysis and literature searching; and other contributors such as staff of national food safety agencies, EFSA trainees and interim staff members who have contributed meaningfully to the work.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00268

COPYRIGHT FOR NON‐EFSA CONTENT

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.

LEGAL NOTICE

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

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,* the 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,* the 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,* the 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,* the 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,* the Netherlands, Poland, 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. , Andreoli, C. , Terron, A. , Yurkov, A. , Cavanna, D. , Aguilera, J. , Peluso, S. , … Liu, Y. (2026). Safety evaluation of the food enzyme aspergillopepsin I from the non‐genetically modified Aspergillus sp. strain LPL 3U‐3‐10. EFSA Journal, 24(9), e10338. 10.2903/j.efsa.2026.10338