Safety evaluation of the food enzyme α‐amylase from the genetically modified Bacillus licheniformis strain DP‐Dzb105

TL;DR

This paper evaluates the safety of a genetically modified α-amylase enzyme used in food manufacturing and concludes it is safe under intended use conditions.

Contribution

The study provides a safety assessment of a genetically modified Bacillus licheniformis α-amylase enzyme for food use.

Findings

The production strain has Qualified Presumption of Safety (QPS) status.

The enzyme does not pose dietary safety concerns under intended use conditions.

Potential allergenicity was identified, but the risk is considered low.

Abstract

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase, EC 3.2.1.1) is produced with the genetically modified Bacillus licheniformis strain DP‐Dzb105 by Genencor International B.V. The production strain met the requirements for the qualified presumption of safety (QPS). The food enzyme was considered free from viable cells of the production organism, but not from its DNA. It is intended to be used in two food manufacturing processes. Since residual amounts of food enzyme‐total organic solids are removed in these two processes, dietary exposure was not calculated. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, the Panel considered toxicity tests unnecessary. A search for the homology of the amino acid sequence of the α‐amylase to known allergens was made and matches with one respiratory allergen and one contact 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.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Alpha‐amylase activity	LPAU/g a	48,250	45,303	37,732
Protein	%	1.1	0.7	0.8
Ash	%	0.3	0.3	0.3
Water	%	94.0	96.0	94.9
Total organic solids (TOS) b	%	5.7	3.7	4.8
Activity/TOS ratio	LPAU /mg TOS	846.5	1224.4	786.1

TABLE 2. Intended uses and use levels of the food enzyme as provided by the applicant. 40

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM)
Processing of cereals and other grains
• Production of glucose syrups and other starch hydrolysates	Starch	1.0–15.84
• Production of distilled alcohol	Cereals	1.0–15.84

Full text

INTRODUCTION

1

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

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

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

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

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

All food enzymes currently on the 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.

On 11 December 2023, a new application has been introduced by the applicant “Genencor International B.V.” for the authorisation of the food enzyme Alpha‐amylase from a genetically modified Bacillus licheniformis (strain DP‐Dzb105).

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: Alpha‐amylase from a genetically modified Bacillus licheniformis (strain DP‐Dzb105), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.3

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme alpha amylase from Bacillus licheniformis DP‐Dzb105.

Additional information, requested from the applicant during the assessment phase on 30 September and 18 November 2024, were received on 29 October 2024 and 17 March 2025, respectively (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 18 November 2024, the applicant requested a clarification teleconference on 17 January 2025 and provided additional data on 17 March 2025.

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, 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.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20024 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 on the non‐confidential version of the technical dossier from 26 November to 17 December 2024.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureα‐amylaseSystematic name4‐α‐d‐glucan glucanohydrolaseSynonymsGlycogenaseIUBMB No3.2.1.1CAS No9000‐90‐2EINECS No232‐565‐6

α‐Amylases catalyse the hydrolysis of 1,4‐α‐glucosidic linkages in starch (amylose and amylopectin), glycogen and related polysaccharides and oligosaccharides, resulting in the generation of soluble dextrins and other oligosaccharides.

The food enzyme under assessment is intended to be used in two food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) glucose syrups and other starch hydrolysates and (2) distilled alcohol.

Source of the food enzyme

3.1

The α‐amylase is produced with the genetically modified bacterium Bacillus licheniformis strain DP‐Dzb105 (■■■■■),6 which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (the Netherlands) with deposition number ■■■■■7 The production strain was identified as B. licheniformis by phylogenomic analysis using whole genome sequence (WGS) data, which grouped the DP‐Dzb105 strain with the B. licheniformis type strain DSM 13.8

The species B. licheniformis is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used, and the genetic modifications do not raise concerns (EFSA, 2007; EFSA BIOHAZ Panel, 2022).9 A cytotoxicity test made with culture supernatants indicated that the production strain B. licheniformis DP‐Dzb105 did not induce cell damage to Vero cells using the lactate dehydrogenase assay.10 The WGS data of the production strain was interrogated for the presence of antimicrobial genes against three regularly maintained databases.11 ^,^ 12 ^,^ 13 No genes of concern encoding acquired antimicrobial resistance genes were identified using thresholds of 80% identity and 70% length coverage. Moreover, no genes encoding virulence factors were identified. In addition, the genetic modifications are considered safe (see Section 3.1.4). Therefore, the production strain meets the requirements for the QPS approach and is considered safe.

Characteristics of the parental microorganism

3.1.1

The parental strain is the bacterium B. licheniformis strain Bra7.14 ■■■■■

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■

■■■■■15

Description of the genetic modification

3.1.3

A comparison between the WGS of production and parental strains16 ^,^ 17 revealed that ■■■■■

■■■■■

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 B. licheniformis DP‐Dzb105 differs from the parental strain in its capacity to produce the α‐amylase ■■■■■18

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 852/2004,19 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.20

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 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.21 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.22

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 α‐amylase is a single polypeptide chain of ■■■■■ amino acids.23 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.24 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.25 A consistent protein pattern was observed across all batches. The gels showed a major protein band migrating between the protein markers of ■■■■■ and ■■■■■ kDa in all batches, consistent with the expected mass of the enzyme.26

No other enzyme activities were reported.

The applicant's in‐house determination of α‐amylase activity is based on the hydrolysis of 4‐nitrophenyl maltoheptoside (reaction conditions: pH 5.6, 30°C, 5 min). The release of 4‐nitrophenol is measured by a coupled colorimetric reaction. The enzyme activity is quantified relative to an internal enzyme standard and expressed in LPAU Units (LPAU)/g27

To determine the temperature and pH optima and thermostability profile of the enzyme, a different activity assay was used, substituting 4‐nitrophenyl maltoheptoside by corn starch as substrate and quantifying the amount of released reducing sugars. The food enzyme has a temperature optimum between 60°C and 80°C (pH 5.8, 10 min) and a pH optimum around pH 6 (60°C, 5 min). Thermostability was tested after pre‐incubation of the food enzyme for 30 min at different temperatures (pH 5.8). The enzyme activity decreased above 50°C, showing no residual activity at 90°C.28

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).29 The mean total organic solids (TOS) was 4.7% and the mean enzyme activity/TOS ratio was 952.3 LPAU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.3 mg/kg30, which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).31 ^,^ 32

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).33 No antimicrobial activity was detected in any of the tested batches.34

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. Ten millilitres ■■■■■ non‐selective agar plates and incubated at 37°C for 2 days. No colonies were produced. A positive control was included.35

The presence of recombinant DNA in the food enzyme was tested by polymerase chain reaction analysis of three batches in triplicate. DNA was detected in one batch with primers ■■■■■ with a limit of detection below 10 ng spiked DNA/mL food enzyme.36

Toxicological data

3.4

As the production strain qualifies for the QPS approach of safety assessment and no issue of concern arose from the production process of the food enzyme (see Sections 3.1, 3.2 and 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (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 α‐amylase produced with the Bacillus licheniformis strain DP‐Dzb105 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 allergens were found in the AllergenOnline database.37

The matching respiratory allergen was Per a 11 (36.3% sequence identity), an α‐amylase from American cockroach (Periplaneta americana). The matching contact allergen was Hev b 9 (36.2% sequence identity), an enolase from latex tree (Hevea brasiliensis).

No reports on oral or respiratory sensitisation or elicitation reactions of the α‐amylase under assessment have been published.

α‐Amylases 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 the α‐amylase from A. oryzae. Taking into account the wide use of α‐amylases as food enzymes, only a low number 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).38 ^,^ 39

Food allergic reactions in latex allergic individuals do occur but are rare. No involvement of Hev b 9 in the latex food allergy syndrome is reported (Pfeiffer & Swoboda, 2025).

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 α‐amylase under assessment.

■■■■■, 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. 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 use, the Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in two food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and use levels of the food enzyme as provided by the applicant. 40

In the production of glucose syrups and other starch hydrolysates, the food enzyme may be added during the mixing and liquefaction steps.41 It hydrolyses starch into dextrins and reduces the viscosity of the gelatinised starch.42 The Panel considers that the food enzyme‐TOS are removed from glucose syrups and other starch hydrolysates (EFSA CEP Panel, 2023).

In the production of distilled alcohol, the food enzyme may be added to cereals during the mixing, liquefaction and pre‐saccharification steps.43 The α‐amylase facilitates the release of fermentable sugars for higher alcohol yields. The food enzyme–TOS is not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

Dietary exposure estimation

3.5.2

The Panel has established that the residual amounts of food enzyme‐TOS in the glucose syrups and other starch hydrolysates and distilled alcohols are negligible (EFSA CEP Panel, 2023). Consequently, a dietary exposure was not calculated.

Margin of exposure

3.6

Since toxicological testing was considered unnecessary 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 QPS status of the production strain, the absence of issues of concern arising from the food enzyme production process, and the removal of TOS during the two food manufacturing processes, the Panel concluded that the food enzyme α‐amylase produced with the genetically modified Bacillus licheniformis strain DP‐Dzb105 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 but noted the presence of recombinant DNA in one of the three food enzyme batches tested.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the authorisation of alpha‐amylase from a genetically modified Bacillus licheniformis strain DP‐Dzb105 as a new food enzyme. May 2024. Submitted by Genencor International B.V.

Additional information. October 2024. Submitted by IFF ‐ Genencor International B.V.

Additional information. March 2025. Submitted by IFF ‐ Genencor International B.V.

ABBREVIATIONSAMRAntimicrobial resistanceCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food EnzymesGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLODlimit of detectionQPSqualified presumption of safetyTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00087

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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.