Safety evaluation of the food enzyme pullulanase from the genetically modified Bacillus licheniformis strain DP‐Dzp107

TL;DR

This study evaluates the safety of a genetically modified pullulanase enzyme used in food manufacturing and concludes it is safe under intended use conditions.

Contribution

The novelty lies in the safety assessment of a genetically modified Bacillus licheniformis strain-produced pullulanase for food use.

Findings

The production strain meets the qualified presumption of safety (QPS) criteria.

Dietary exposure was estimated at up to 0.053 mg TOS/kg body weight per day in European populations.

No homology to known allergens was found, though a low likelihood of allergic reactions cannot be ruled out.

Abstract

The food enzyme pullulanase (pullulan 6‐α‐glucanohydrolase, EC 3.2.1.41) is produced with the genetically modified Bacillus licheniformis strain DP‐Dzp107 by Genencor International B.V. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme was considered free from viable cells of the production organism, but not its DNA. The food enzyme is intended to be used in three food manufacturing processes. Since residual amounts of food enzyme‐total organic solids (TOS) are removed in two processes, dietary exposure was calculated only for the remaining food manufacturing process. It was estimated to be up to 0.053 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, the Panel considers toxicity tests unnecessary. A search for the homology of the amino acid sequence of the pullulanase to known allergens was made and no match was 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
Pullulanase activity	ASPU/g a	36,644	32,018	30,713
Protein	%	18.7	20.2	19.0
Ash	%	2.3	2.2	2.1
Water	%	74.2	72.3	74.3
Total organic solids (TOS) b	%	23.5	25.5	23.6
Activity/TOS ratio	ASPU/mg TOS	155.9	125.6	130.1

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 42

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of cereals and other grains
Production of brewed products	Cereals	1.15–11.5
Production of glucose syrups and other starch hydrolysates	Starch	0.005–0.05
Production of distilled alcohol	Cereals	1.09–1.4

TABLE 3. Summary of the estimated dietary exposure to food enzyme‐TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0–0 (12)	0–0.1 (15)	0–0.1 (19)	0–0.002 (21)	0.001–0.012 (22)	0–0.006 (23)
Min–max 95th percentile (number of surveys)	0–0 (11)	0–0.005 (14)	0–0.005 (19)	0–0.009 (20)	0.006–0.053 (22)	0–0.025 (22)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme‐TOS always calculated based on the recommended maximum use level	+
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 glucose syrups and other starch hydrolysates – 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 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 18 December 2023, a new application has been introduced by the applicant ‘Genencor International B.V.’ for the authorisation of the food enzyme Pullulanase from a genetically modified Bacillus licheniformis (strain DP‐Dzp107).

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: Pullulanase from a genetically modified Bacillus licheniformis (strain DP‐Dzp107), 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 pullulanase from Bacillus licheniformis DP‐Dzp107.

Additional information requested from the applicant during the assessment process on 02 December 2024 were received on 03 March 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, 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 19 February to 12 March 2025.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclaturePullulanaseSystematic namePullulan 6‐α‐glucanohydrolaseSynonymsα‐Dextrin endo‐1,6‐α‐glucosidaseIUBMB No3.2.1.41CAS No9075‐68‐7EINECS No232‐983‐9

Pullulanases catalyse the hydrolysis of (1–6)‐α‐d‐glucosidic linkages in pullulan, amylopectin and glycogen and in α‐ and β‐limit dextrins of amylopectin and glycogen, releasing oligosaccharides and glucose. The food enzyme under assessment is intended to be used in three food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) brewed products, (2) distilled alcohol and (3) glucose syrups and other starch hydrolysates.

Source of the food enzyme

3.1

The enzyme is produced with the genetically modified bacterium Bacillus licheniformis strain DP‐Dzp107 (■■■■■)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.8 ^,^ 9

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 is verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022).10 A cytotoxicity test made with culture supernatants indicated that the production strain B. licheniformis DP‐Dzp107 did not induce damage to Vero cells using the lactate dehydrogenase assay.11 The WGS data of the production strain was interrogated for the presence of antimicrobial resistance genes against two regularly maintained databases.12 ^,^ 13 No genes of concern were identified with thresholds above 80% of identity and 70% of coverage. In addition, the genetic modifications are considered safe (see Section 3.1.4). Therefore, the production strain meets the requirements to qualify for the QPS approach and is considered safe.

Characteristics of the parental microorganism

3.1.1

The parental strain is Bacillus licheniformis Bra7.14 ■■■■■

Characteristics of introduced sequences

3.1.2

■■■■■15 ■■■■■

■■■■■16

Description of the genetic modification

3.1.3

A comparison between the WGS of the production and parental strains revealed that ■■■■■

■■■■■17 ^,^ 18

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 Bacillus licheniformis DP‐Dzp107 differs from the parental strain in its capacity to produce ■■■■■ pullulanase ■■■■■ ■■■■■19

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 852/2004,20 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice.21

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.22 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.23

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 pullulanase is a single polypeptide chain of ■■■■■ amino acids.24 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.25 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.26 A consistent protein pattern was observed across all batches. The gels showed a major protein band migrating between the marker proteins of ■■■■■ and ■■■■■ kDa, consistent with the expected mass of the enzyme.27

No other enzyme activities were reported.

The applicant's in‐house determination of pullulanase activity is based on the hydrolysis of Red Pullulan substrate (reaction conditions: pH 5, 40°C, 20 min). The released dye is determined spectrophotometrically at 510 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in acid stable pullulanase units (ASPU)/g.28

To determine the temperature and pH optimum and thermostability profile of the enzyme, a different activity assay was used, substituting Red Pullulan by pullulan as a substrate and using formulated samples. The food enzyme has a temperature optimum around 60°C (pH 4.5) and a pH optimum around pH 4.5–5.0 (50°C). Thermostability was tested after pre‐incubation of the food enzyme for 30 min at different temperatures (pH 4.5). The enzyme activity decreased above 57°C, showing little residual activity above 66°C.29

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).30 The mean total organic solids (TOS) of the three batches was 24.2% and the mean enzyme activity/TOS ratio was 137.2 ASPU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.01 mg/kg,31 ^,^ 32 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,33 as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). 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 of the production strain were detected.35 A positive control was included.36

The presence of recombinant DNA in the food enzyme was tested by polymerase chain reaction analysis of three batches in triplicate. DNA was detected with primers ■■■■■, with a limit of detection of 10 ng spiked DNA/mL food enzyme.37

Toxicological data

3.4

As the production strain qualifies for the QPS approach of safety assessment and no issue of concern arising from the production process of the food enzyme was identified (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 pullulanase produced with the Bacillus licheniformis strain DP‐Dzp107 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, no match was found in the AllergenOnline database.38

No reports on oral or respiratory sensitisation or elicitation reactions of the pullulanase under assessment have been published. No allergic reactions upon dietary exposure to any pullulanase have been reported in the literature.39

The Panel considered that the results of the sequence homology search and the available literature search do not indicate a risk of allergic reactions upon dietary exposure to the pullulanase under assessment.

■■■■■, a product from ■■■■■, that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201140), is used as raw material. In addition, ■■■■■, a known source of allergens, are also present in the culture medium.41 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 uses, the 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 three 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. 42

In the production of brewed products, the food enzyme is added to cereals during mashing and may also be added during fermentation.43 This leads to the degradation of starch into fermentable sugars, which reduces the mashing time and increases the alcohol yield.44 The food enzyme‐TOS remain in the brewed products.

In the production of glucose syrups and other starch hydrolysates, the food enzyme is added during saccharification.45 The use of pullulanase results in an increased saccharification rate and glucose yield.46 The food enzyme‐TOS are removed from the glucose syrups by treatment with activated charcoal and ion‐exchange resins (EFSA CEP Panel, 2023). The Panel considers that this applies also to other starch hydrolysates.

In the production of distilled alcohol, the food enzyme is added to cereals during liquefaction, pre‐saccharification and fermentation.47 The pullulanase improves the hydrolysis of starch, which increases the fermentation rate and alcohol yield, and improves the performance in the pre‐saccharification process.48 The food enzyme‐TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme may remain in its active form in the production of brewed products, depending on the heat treatment conditions during the food manufacturing process.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the production of brewed products, where the food enzyme‐TOS remain in the final foods.

Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool49 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme‐TOS per age class, country, and survey, as well as contribution from each FoodEx category to the total dietary exposure, are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix A). The highest dietary exposure was estimated to be 0.053 mg TOS/kg body weight per day in adults at the 95th percentile.

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 4.

The conservative approach applied to estimate the 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.

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, 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 two food manufacturing processes, the Panel concluded that the food enzyme pullulanase produced with the genetically modified Bacillus licheniformis strain DP‐Dzp107 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 pullulanase from a genetically modified Bacillus licheniformis strain DP‐Dzp107. May 2024. Submitted by 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 Organisation 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‐2024‐00088

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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, Monika Sramkova, Henk Van Loveren, Laurence Vernis and Holger Zorn.

LEGAL NOTICE

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

Supporting information

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