Safety evaluation of the food enzyme aspergillopepsin I from the genetically modified Trichoderma reesei strain DP‐Nzq40

TL;DR

A genetically modified enzyme from Trichoderma reesei is evaluated for safety in alcohol production, with no safety concerns identified.

Contribution

The safety evaluation of a genetically modified aspergillopepsin I enzyme for use in distilled alcohol production is presented.

Findings

The genetic modifications in Trichoderma reesei do not pose safety concerns.

The enzyme does not contain viable cells or DNA from the production organism.

Allergic reaction risks are considered negligible for the intended use.

Abstract

The food enzyme aspergillopepsin I (EC 3.4.23.18) is produced with the genetically modified microorganismTrichoderma reesei strain DP‐Nzq40 by Danisco US Inc. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in the processing of grains and other cereals for the production of distilled alcohol. Since residual amounts of total organic solids (TOS) are removed by distillation, toxicological data were not considered necessary and dietary exposure was not calculated. A search for the homology of the amino acid sequence of the aspergillopepsin I to known allergens was made and matches with two respiratory allergens were found. The Panel considered that a risk of allergic reactions to the food enzyme can be excluded for the production of distilled alcohol. 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
Aspergillopepsin I activity	SAPU/g a	7811	8337	8279
Protein	%	29.9	29.7	30.6
Ash	%	0.1	0.04	0.1
Water	%	64.4	64.5	64.1
Total organic solids (TOS) b	%	35.5	35.5	35.8
Activity/ TOS ratio	SAPU/mg TOS	22.0	23.5	23.1

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 34 , 35

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM)
Processing of cereals and other grains
Production of distilled alcohol	Cereals	5–50

TABLE 3. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model assumptions and factors
Exclusion of one process from the exposure estimation:	–
– production of distilled alcohol

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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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 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.

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7(2) of Regulation (EC) No 1332/2008 on food enzymes.

Five applications have been introduced by the companies “Meiji Seika Pharma Co., Ltd.” for the authorisation of the food enzyme Cellulase from Talaromyces cellulolyticus/Talaromyces pinophilus (strain Acremonium cellulolyticus); “Danisco US Inc.” for the authorisation of the food enzymes Aspergillopepsin I from a genetically modified strain of Trichoderma reesei (strain DP‐Nzq40) and Triacylglycerol lipase from a genetically modified strain of Hansenula polymorpha (strain DP‐Jzk33); “Neova Technologies Inc.” for the authorisation of the food enzyme Trypsin and Chymotrypsin from porcine pancreatic glands, and “Novozymes A/S.” for the authorisation of the food enzyme Peptidase from a strain of Aspergillus oryzae (strain NZYM‐EX).

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 five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter 11 of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Cellulase from Talaromyces cellulolyticus/Talaromyces pinophilus (strain Acremonium cellulolyticus); Aspergillopepsin I from a genetically modified strain of Trichoderma reesei (strain DP‐ Nzq40), Triacylglycerol lipase from a genetically modified strain of Hansenula polymorpha (strain DP‐Jzk33); Trypsin and Chymotrypsin from porcine pancreatic glands and Peptidase from a strain of Aspergillus oryzae (strain NZYM‐EX) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme aspergillopepsin I from the genetically modified strain of Trichoderma reesei strain DP‐ Nzq40.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme aspergillopepsin I from a genetically modified strain of Trichoderma reesei (strain DP‐ Nzq40).

Additional information was requested from the applicant during the assessment process on 17 January 2023, and 23 January 2024 and received on 20 October 2023 and 22 October 2024 (see ‘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 nomenclatureAspergillopepsin ISystematic name–Synonyms Aspergillus aspartic proteinase; carboxyl proteinase; pepsin‐type aspartic proteinaseIUBMB NoEC 3.4.23.18CAS No9025‐49‐4EINECS No232‐796‐2

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

The food enzyme under assessment is intended to be used in the processing of cereals and other grains for the production of distilled alcohol, as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The aspergillopepsin I is produced with the genetically modified filamentous fungus Trichoderma reesei strain DP‐Nzq40 (■■■■■), which is deposited at the Westerdijk Fungal Biodiversity Institute (the Netherlands) with the deposition number ■■■■■.4 The production strain was identified as T. reesei by phylogenomic analysis using whole genome sequence (WGS) data.5

Characteristics of the parental and recipient microorganisms

3.1.1

The parental strain ■■■■■ is derived from ■■■■■ (■■■■■) by ■■■■■

The recipient strain is ■■■■■6 has been developed from the parental strain ■■■■■ ■■■■■

Characteristics of introduced sequences

3.1.2

■■■■■■■■■■ ■■■■■

■■■■■■■■■■ ■■■■■■■■■■ ■■■■■

Description of the genetic modification process

3.1.3

The purpose of genetic modification was to enable the production strain to overproduce aspergillopepsin I.

For this purpose, ■■■■■.

■■■■■.10

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 T. reesei DP‐Nzq40 differs from the recipient strain in its ability to overproduce aspergillopepsin I, ■■■■■.

The absence of vector backbone sequences including the ■■■■■ gene was confirmed by whole genome sequence analysis.11

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, batch or 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

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 aspergillopepsin I is a single polypeptide chain of ■■■■■ amino acids.16 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.17 The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis.18 A consistent protein pattern was observed across all batches. The gel showed a single protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.19

The applicant's in‐house determination of aspergillopepsin I activity is based on the hydrolysis of casein (reaction conditions: pH 3.0, 37°C, 30 min). The release of peptides and amino acids is measured spectrophotometrically at 275 nm. The enzyme activity is expressed in Spectrophotometric Acid Protease Units (SAPU)/g. One SAPU is defined as the amount of enzyme that produces 1 μmol of tyrosine equivalents per minute under the conditions of the assay.20

The food enzyme has a temperature optimum around 60°C (pH 3.0) and a pH optimum around 3.5 (37°C). Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures (pH 4.5).21 Enzyme activity decreased above 45°C showing no residual activity at 71°C.22

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).23 The mean total organic solids (TOS) of the three batches was 35.6% and the mean enzyme activity/TOS ratio was 22.9 SAPU/mg TOS.

Purity

3.3.3

The lead content in all batches was below 0.01 mg/kg24 ^,^ 25 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).26 No antimicrobial activity was detected in any of the tested batches.27

Strains of the Trichoderma species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of T‐2 toxin was examined in all food enzyme batches and was below the limit of quantification (LoQ) of the applied method.28 ^,^ 29

The Panel considered that the information provided on the purity of the food enzyme was 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 of the food enzyme analysed in triplicate. ■■■■■ No colonies were produced. A positive control was included.30

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction analysis of three batches in triplicate. No DNA was detected with primers that would amplify a ■■■■■ with a limit of detection of 10 ng spiked DNA/mL food enzyme.31

Toxicological data

3.4

No Toxicological studies were provided by the applicant. In line with the currently applicable guidance (EFSA CEP Panel, 2021), as the food enzyme–TOS are removed (> 99%) in the final foods (see Section 3.5), the Panel considered that a toxicological testing is not necessary for this food enzyme under the intended conditions of use, except for the assessment of allergenicity.

Allergenicity

3.4.1

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 T. reesei strain DP‐Nzq40 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 two respiratory allergens were found in AllergenOnline database.32 The matching allergens were Asp f 10 (63.8% sequence identity), an aspergillopepsin from Aspergillus fumigatus and pepsin A (42.5% sequence identity) from pig (Sus scrofa).

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

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). Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). 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.

■■■■■, 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.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the intended use (see Section 3.5), this would not result in their presence in the final foods.

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.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in one food manufacturing process at the recommended use level presented in Table 2.

**TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 34

,

35**

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.36 The use of the food enzyme 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).

Dietary exposure estimation

3.5.2

The Panel accepted the evidence provided as sufficient to conclude that the residual amounts of food enzyme–TOS in the distilled alcohol is negligible (EFSA CEP Panel, 2023). Consequently, a dietary exposure was not calculated.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2007), the following sources of uncertainties have been considered and are summarised in Table 3.

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

Margin of exposure

3.6

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

CONCLUSIONS

4

Based on the data provided and the removal of TOS during the production of distilled alcohol, the Panel concluded that the food enzyme Aspergillopepsin I produced with the genetically modified Trichoderma reesei strain DP‐ Nzq40 does not give rise to safety concerns under the intended conditions of use.

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

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of Aspergillopepsin I from a genetically modified strain of Trichoderma reesei (DP‐Nzq40). February 2015. Submitted by Danisco US Inc.

Additional information. October 2023. Submitted by IFF Danisco (UK) Ltd.

Additional information. October 2024. Submitted by IFF Danisco (UK) Ltd.

Summary report on technical data and dietary exposure related to aspergillopepsin I from a strain of Trichoderma reesei (strain DP‐Nzq40). June 2016. Delivered by Hylobates Consulting and BiCT (Rome and Villanova del Sillaro, Italy).

Summary report on genotoxicity and subchronic toxicity study related to Aspergillopepsin I produced with a strain of Trichoderma reesei (strain DP‐Nzq40). July 2016. Delivered by FoBiG GmbH (Freiburg, Germany).

ABBREVIATIONSCASChemical 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 NationsGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLoDlimit of detectionOECDOrganisation for Economic Cooperation and DevelopmentPCRpolymerase chain reactionTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2015‐00371

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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.

NOTE

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.