Safety evaluation of the food enzyme endo‐1,4‐β‐xylanase from the genetically modified Bacillus subtilis strain DP‐Ezd119

TL;DR

This study evaluates the safety of a food enzyme produced by a genetically modified bacterium and concludes it is safe for use in baking.

Contribution

The study confirms the safety of endo-1,4-β-xylanase from a genetically modified Bacillus subtilis strain under intended food processing conditions.

Findings

The genetically modified Bacillus subtilis strain DP-Ezd119 meets the qualified presumption of safety (QPS) criteria.

No homology was found between the enzyme and known allergens, suggesting low risk of allergic reactions.

The Panel concluded the food enzyme does not pose safety concerns under its intended use.

Abstract

The food enzyme endo‐1,4‐β‐xylanase (4‐β‐D‐xylan xylanohydrolase; EC 3.2.1.8) is produced with the genetically modified Bacillus subtilis strain DP‐Ezd119 by Genencor International B.V. The production strain meets the requirements for the qualified presumption of safety (QPS) approach. 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 baked products. Dietary exposure was estimated to be up to 0.085 mg total organic solids/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, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the endo‐1,4‐β‐xylanase 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 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
Endo‐1,4‐β‐xylanase activity	DXU/g a	1,766,957	2,877,391	2,672,174
Protein	%	1.7	2.6	2.5
Ash	%	0.7	0.7	0.6
Water	%	96.7	95.0	95.2
Total organic solids (TOS) b	%	2.6	4.3	4.2
Activity/TOS ratio	DXU/mg TOS	67,960	66,916	63,623

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 32

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM) a
Processing of cereals and other grains
Production of baked products	Flour	0.31–8.28

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)	0–0.019 (12)	0.001–0.046 (15)	0.001–0.042 (19)	0–0.024 (21)	0.007–0.016 (22)	0.008–0.014 (23)
Min–max 95th percentile (number of surveys)	0–0.055 (11)	0.005–0.079 (14)	0.002–0.085 (19)	0.001–0.048 (20)	0.015–0.036 (22)	0.016–0.028 (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	+/−

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 15 December 2023, a new application has been introduced by the applicant ‘Genencor International B.V.’ for the authorisation of the food enzyme Xylanase from a genetically modified Bacillus subtilis (strain DP‐Ezd119).

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: Xylanase from a genetically modified Bacillus subtilis (strain DP‐Ezd119), 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 endo‐1,4‐β‐xylanase from a genetically modified Bacillus subtilis strain DP‐Ezd119.

Additional information, requested from the applicant during the assessment process on 4 November 2024, was received on 4 December 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, 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 31 October to 21 November 2024.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureEndo‐1,4‐β‐xylanaseSystematic name4‐β‐D‐xylan xylanohydrolaseSynonymsEndo‐(1–4)‐β‐xylan 4‐xylanohydrolase; xylanase; β‐1,4‐xylanase; β‐xylanaseIUBMB NoEC 3.2.1.8CAS No9025‐57‐4EINECS No232‐800‐2

Endo 1,4‐β‐xylanases catalyse the random hydrolysis of 1,4‐β‐D‐xylosidic linkages in xylans (including arabinoxylans), resulting in the generation of (1–4)‐β‐D‐xylan oligosaccharides of different lengths. The food enzyme under assessment is intended to be used in the processing of cereals and other grains for the production of baked products as described in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The endo‐1,4‐β‐xylanase is produced with the genetically modified bacterium Bacillus subtilis strain DP‐Ezd119 (■■■■■) which is deposited at the Westerdijk Fungal Biodiversity Institute culture collection (CBS, The Netherlands) with deposition number ■■■■■.6 The production strain was identified as B. subtilis by phylogenomic analysis using whole genome sequence (WGS) data.7

The species B. subtilis is included in the list of organisms for which the qualified presumption of safety (QPS)8 approach may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity is verified for the specific strain used, and the genetic modifications do not raise safety concerns (EFSA, 2007; EFSA BIOHAZ Panel, 2022). Strain DP‐Ezd119 was shown to be not cytotoxic to VERO cells using LDH as a marker for cell wall disruption.9 The predicted open reading frames obtained from the WGS were searched against maintained databases for antimicrobial resistance genes and virulence factors, and no match of concern was found using thresholds of 80% identity and 70% coverage.10 ^,^ 11

Characteristics of the parental microorganism

3.1.1

The parental microorganism is the bacterium B. subtilis strain BG125, which was derived from B. subtilis strain 168.

Characteristics of introduced sequences

3.1.2

■■■■■

■■■■■.

Description of the genetic modification

12

3.1.3

A comparison between the WGS of the production and the parental strains13 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. subtilis DP‐Ezd119 differs from the parental strain in its capacity to overproduce endo‐1,4‐β‐xylanase ■■■■■

No issues of concern arising from the genetic modifications were identified by the Panel. As the other qualifications have been met, the production strain is considered to qualify for the QPS approach.

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 current good manufacturing practices.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 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.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 endo‐1,4‐β‐xylanase is a single polypeptide chain of ■■■■■ amino acids.18 The molecular mass of the 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 gels showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected molecular mass of the enzyme.

No other enzyme activities were reported.

The applicant's in‐house determination of endo‐1,4‐β‐xylanase activity is based on the hydrolysis of azurine‐cross‐linked wheat arabinoxylan (reaction conditions: pH 5, 40°C, 10 min). The enzyme activity is determined by measuring the rate of the release of water‐soluble dyed fragments spectrophotometrically at 590 nm. The enzyme activity is quantified relative to an internal enzyme standard and expressed in DXU units (DXU)/g.21

The food enzyme has a temperature optimum around 55°C (pH 5, 5 min) and a pH optimum around pH 6 (40°C, 5 min). Thermostability was tested after a pre‐incubation of the food enzyme for 5 min at different temperatures (pH 5). The enzyme activity decreased above 50°C, showing no residual activity at 65°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 3.7% and the mean enzyme activity/TOS ratio was 66,166 DXU/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.01 mg/kg,24 ^,^ 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 26 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.27

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 ■■■■■ was filtered ■■■■■ non‐selective agar plates and incubated at 37°C for 2 days. No colonies were produced. A positive control was included.28

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction (PCR) analysis of three batches in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a limit of detection of 1 ng spiked DNA/g food enzyme.

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 endo‐1,4‐β‐xylanase produced with the B. subtilis strain DP‐Ezd119 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.29

No reports on oral or respiratory sensitisation or elicitation reactions of the endo‐1,4‐β‐xylanase under assessment have been published.30

Respiratory allergy, e.g. baker's asthma, following occupational exposure to xylanase has been described in some epidemiological studies (Elms et al., 2003; Martel et al., 2010) and case reports (Baur et al., 1998; Lipińska‐Ojrzanowska et al., 2016; Merget et al., 2001). 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). Adverse reactions upon dietary exposure of xylanases in individuals sensitised through the respiratory route have not been reported.

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 endo‐1,4‐β‐xylanase under assessment.

The production strain B. subtilis produces poly‐γ‐glutamic acid and nattokinase Bac s 1, a subtilisin‐like serine protease, which have both been identified as food allergens. Few case reports of allergic reactions after consumption of natto produced by B. subtilis have been reported in Japan (Inomata et al., 2012; Suzuki et al., 2023). The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the cell culture medium from which the food enzyme is obtained.

■■■■■, a product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201131), is used as a raw material. During the fermentation process, this product 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 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that 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 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. 32

In the production of baked products, the food enzyme is added to flour during the preparation of the dough.33 The hydrolysis of (arabino)xylans decreases the viscosity of the dough, which facilitates the handling of the dough and results in more uniform products with increased volume and an improved crumb structure.34 The food enzyme‐TOS remains in the final baked products.

Based on data provided on thermostability (see Section 3.3.1) and the heat treatment applied, the Panel considered that the food enzyme will be inactivated during the production of baked products.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the application of technical conversion factors (EFSA CEP Panel, 2023). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only 1 day per subject were excluded, and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow the calculation of the 95th percentile (EFSA, 2011).

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 the 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 43 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure was estimated to be 0.085 mg TOS/kg body weight (bw) per day in children 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.

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 and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme endo‐1,4‐β‐xylanase produced with the genetically modified Bacillus subtilis strain DP‐Ezd119 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 Endo‐1,4‐β‐xylanase from a genetically modified Bacillus subtilis strain DP‐Ezd119. December 2023. Submitted by Genencor international B.V.

Additional information. December 2024. Submitted by Genencor international B.V.ABBREVIATIONSAMRantimicrobial resistanceCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organisation of the United NationsGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologykDakiloDaltonPCRpolymerase chain reactionQPSqualified presumption of safetySDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00079

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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

Dietary exposure estimates to the food enzyme–TOS in detail