Safety evaluation of the food enzyme glucan 1,4‐α‐maltohydrolase from the genetically modified Trichoderma reesei strain DP‐Nyn90

TL;DR

This study evaluates the safety of a food enzyme produced from a genetically modified fungus used in alcohol production and finds it safe for its intended use.

Contribution

The study provides a safety evaluation of a novel food enzyme from a genetically modified Trichoderma reesei strain.

Findings

The genetic modifications in the production strain do not pose safety concerns.

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

Allergenic potential was identified, but no dietary risk is expected due to processing.

Abstract

The food enzyme glucan 1,4‐α‐maltohydrolase (4‐α‐D‐glucan α‐maltohydrolase; EC 3.2.1.133) is produced with the genetically modified Trichoderma reesei strain DP‐Nyn90 by Genencor International B.V. The genetic modifications do not give rise to safety concerns. The food enzyme was considered free from viable cells of the production organism and its DNA. The food enzyme is intended to be used in the processing of cereals and other grains for the production of distilled alcohol. Since residual amounts of food enzyme–total organic solids are removed during this food manufacturing process, dietary exposure was not calculated. A search for the homology of the amino acid sequence of the glucan 1,4‐α‐maltohydrolase to known allergens was made and matches with three respiratory allergens were found. However, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Glucan 1,4‐α‐maltohydrolase activity	SSU/g a	48,831	47,989	48,399
Protein	%	24.3	23.9	26.5
Ash	%	0.2	0.2	0.3
Water	%	73.6	74.0	70.3
Total organic solids (TOS) b	%	26.2	25.8	29.4
Activity/TOS ratio	SSU/mg TOS	186.4	186.0	164.6

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 37

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM)
Processing of cereals and other grains
Production of distilled alcohol	Fermentable carbohydrates	54.3–108.6

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 a definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof including a product obtained by a fermentation process using microorganisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction; and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008 on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

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

All food enzymes currently on the 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 specification and condition of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes.3

On 4 December 2023, a new application has been introduced by the applicant “Genencor International B. V” for the authorization of the food enzyme Glucan 1,4‐α‐maltohydrolase from a genetically modified strain of Trichoderma reesei (strain DP‐Nyn90).

Terms of Reference

1.1.2

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: Glucan 1,4‐α‐maltohydrolase from a genetically modified strain of Trichoderma reesei (strain DP‐Nyn90), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings.4

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Glucan 1,4‐α‐maltohydrolase from a genetically modified strain of Trichoderma reesei (strain DP‐Nyn90).

Additional information, requested from the applicant during the assessment process on 4 November 2024, was received on 13 February 2025 (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 ‘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.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20025 and to the Decision of EFSA's Executive Director laying down the practical arrangements on the pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 28 March to 18 April 2025.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureGlucan 1,4‐α‐maltohydrolaseSystematic name4‐α‐D‐glucan α‐maltohydrolaseSynonymsMaltogenic α‐amylase; 1,4‐α‐D‐glucan α‐maltohydrolaseIUBMB No3.2.1.133CAS No160611‐47‐2EINECS NoNA

Glucan‐1,4‐α‐maltohydrolases catalyse the hydrolysis of (1,4)‐α‐D‐glucosidic linkages in starch polysaccharides to successively remove maltose residues from the non‐reducing chain ends. 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 glucan‐1,4‐α‐maltohydrolase is produced with the genetically modified filamentous fungus Trichoderma reesei strain DP‐Nyn90, which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (CBS, the Netherlands) with the deposition number ■■■■■.7 The production strain was identified as Trichoderma reesei by phylogenomic analysis using whole genome sequence data.8

Characteristics of the parental and recipient microorganism

3.1.1

The parental strain T. reesei RL‐P37 has been derived from strain QM6a ■■■■■ by classical mutagenesis and selection for high cellulase activity (Sheir‐Neiss & Montenecourt, 1984).9

The recipient strain T■■■■■

Subsequently, ■■■■■10

During the development of the recipient strain, plasmids were used that contained antimicrobial resistance genes.

Characteristics of introduced sequences

3.1.2

The sequence encoding the glucan 1,4‐α‐maltohydrolase ■■■■■

■■■■■

■■■■■.11

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to enable the production strain to synthesise the glucan 1,4‐α‐maltohydrolase from ■■■■■.

■■■■■.12

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 strain DP‐Nyn90 differs from the recipient strain in its capacity to produce the glucan 1,4‐α‐maltohydrolase from ■■■■■ and ■■■■■.

The absence of vector backbone sequences, including the AMR genes, was demonstrated by Southern blot and whole genome sequencing analyses.13

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,14 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with good manufacturing practice.15

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 the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.16 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.17

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 glucan 1,4‐α‐maltohydrolase is a single polypeptide chain of ■■■■■ amino acids.18 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.19 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.20 A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between ■■■■■ kDa markers, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.21

The applicant's in‐house determination of glucan 1,4‐α‐maltohydrolase activity is based on the hydrolysis of 4‐nitrophenyl maltoheptoside by a coupled enzymatic reaction (reaction conditions: pH 4.5, 30°C, 5 min). The release of 4‐nitrophenol is measured colorimetrically at 405 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in Soluble Starch Units (SSU)/g.22 ^,^ 23

To determine the properties of the enzyme, a different activity assay was used substituting 4‐nitrophenyl maltoheptoside for potato amylopectin as a substrate. The food enzyme has a temperature optimum around 60–70°C (pH 5.0, 10 min) and a pH optimum around pH 4.0 (50°C). Thermostability was tested by pre‐incubation of the food enzyme for 120 min at different temperatures (pH 5.0). The enzyme activity rapidly decreased above 50°C, showing no residual activity above 70°C.24

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).25 The mean total organic solids (TOS) was 27.1% and the mean enzyme activity/TOS ratio was 179.0 SSU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was equal to or below 0.05 mg/kg26 ^,^ 27 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).28 No antimicrobial activity was detected in any of the tested batches.29

Strains of 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 aflatoxins (B1, B2, G1 and G2), fumonisins (B1 and B2), ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in all food enzyme batches, and all were below the limit of detection/quantification (LoD/LoQ) of the applied methods.30 ^,^ 31 ^,^ 32

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 analysed in triplicate. ■■■■■. No colonies were detected. A positive control was included.33

The absence of recombinant DNA in the food enzyme was demonstrated ■■■■■ of three batches in triplicate. No DNA was detected with ■■■■■, with a limit of detection of 1 ng spiked DNA/g food enzyme.34

Toxicological data

3.4

The food enzyme is intended to be used only in a food manufacturing process in which the food enzyme–TOS are removed from the end products by the distillation step (see Section 3.5). Consequently, toxicological studies other than the assessment of allergenicity were not considered necessary for the assessment of this food enzyme (EFSA CEP Panel, 2021).

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 glucan 1,4‐α‐maltohydrolase produced with the Trichoderma reesei strain DP‐Nyn90 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 were found in the AllergenOnline database.35

The matching respiratory allergens were Asp o 21 (83.8% sequence identity), an α‐amylase from Aspergillus oryzae; Sch c 1 (51.3% sequence identity), a glucoamylase from Schizophyllum commune; and Per a 11 (37.0% sequence identity), an α‐amylase from the American cockroach (Periplaneta americana).

No reports on oral or respiratory sensitisation or elicitation reactions of the glucan 1,4‐α‐maltohydrolase under assessment have been published.

α‐Amylases and glucoamylases have been shown to cause respiratory allergy. 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) as also described for α‐amylase from A. oryzae. Taking into account the wide use of α‐amylases as food enzymes, only a low number of case reports of allergic reactions upon oral exposure to α‐amylase in individuals respiratorily sensitised to α‐amylase have been described in the literature (Baur & Czuppon, 1995; Kanny & Moneret‐Vautrin, 1995; Losada et al., 1992; Moreno‐Ancillo et al., 2004; Quirce et al., 1992). Such information has not been reported for glucoamylases. No allergic reactions upon dietary exposure to any glucan 1,4‐α‐maltohydrolase have been reported in the literature.36

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 glucan 1,4‐α‐maltohydrolase under assessment.

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 summarised in Table 2.

TABLE 2: Intended use and recommended use level of the food enzyme as provided by the applicant. 37

In the production of distilled alcohol, the food enzyme may be added during the slurry mixing, liquefaction or saccharification steps.38 The hydrolysis of starch by the glucan‐1,4‐α‐maltohydrolase releases fermentable sugars, especially maltose.39 The food enzyme–TOS are not carried over into the distilled alcohol (EFSA CEP Panel, 2023).

Dietary exposure estimation

3.5.2

The Panel accepted the evidence provided as sufficient to conclude that the residual amount 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, 2006), 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.

Margin of exposure

3.6

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

CONCLUSIONS

4

Based on the data provided, the absence of issues of concern arising from the production process and the removal of TOS during the production of distilled alcohol, the Panel concluded that the food enzyme glucan 1,4‐α‐maltohydrolase produced with the genetically modified Trichoderma reesei strain DP‐Nyn90 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 its DNA.

DOCUMENTATION AS PROVIDED TO EFSA

5

Glucan 1,4‐α‐maltohydrolase from a genetically modified Trichoderma reesei strain DP‐Nyn90. April 2024. Submitted by Genencor International B.V.

Additional information. February 2025. Submitted by Genencor International B.V.

ABBREVIATIONSAMRantimicrobial resistanceCASChemical Abstracts ServiceEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsIUBMBInternational Union of Biochemistry and Molecular BiologykDakiloDaltonLODlimit of detectionLOQlimit of quantificationMOEmargin of exposurePCRpolymerase chain reactionTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00014

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PANEL MEMBERS

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

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.