Revised safety evaluation of the food enzyme protein‐glutamine γ‐glutamyltransferase from the non‐genetically modified Streptomyces mobaraensis strain M2020197

TL;DR

This paper evaluates the safety of a food enzyme produced by a non-genetically modified bacteria, concluding it is safe under intended use conditions.

Contribution

New safety evaluation of a food enzyme with updated data on strain identification and exposure levels.

Findings

The enzyme is free from viable cells and DNA of the production organism.

The revised margin of exposure is at least 379, indicating a safe level.

Allergenic potential is low, but a risk of allergic reactions cannot be entirely excluded.

Abstract

The food enzyme protein‐glutamine γ‐glutamyltransferase (EC 2.3.2.13) is produced with the non‐genetically modified Streptomyces mobaraensis strain M2020197 by Dongsheng Biotech (Taixing) Co., Ltd. In a previous opinion, the Panel could not exclude safety concerns due to insufficient margins of exposure. In addition, the taxonomic identity of the production strain was not established, the absence of genes of concern was not confirmed, and the absence of viable cells of the production strain was not demonstrated. As a follow‐up, the applicant reduced the use levels in the food manufacturing processes. In addition, the applicant provided new data on the identification and characterisation of the production strain. The food enzyme was considered free from viable cells of the production organism and its DNA. The dietary exposure was revised to be up to 0.240 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (91 mg TOS/kg bw per day), the Panel derived a margin of exposure of at least 379. A search for the homology of the amino acid sequence of the protein‐glutamine γ‐glutamyltransferase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low. A risk of contribution to coeliac disease cannot be excluded. Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Tables

TABLE 1. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 15

Food manufacturing process a	Raw material (RM)	Maximum use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of dairy products
Production of cheese	Milk	3.61	97.56
Production of fermented dairy products	Milk	1.81	4.34
Production of dairy desserts d	Milk	3.61	17.34
Processing of meat and fish products
Production of modified meat and fish products	Meat and fish	18.07	65.04
Processing of cereals and other grains
Production of baked products	Flour	0.72	24.8	Bread products
			54.2	Fine bakery products
			57.8	Raw doughs and pre‐mixes
			57.8	Pizza and pizza‐like dishes
Production of cereal‐based products other than baked	Flour	0.72	57.8	Breakfast cereals
			17.34	Pasta and similar
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Soybeans, vegetable protein	7.23	43.35	Cheese alternatives
			8.67	Yoghurt alternatives
			19.87	Tofu
Production of meat alternatives d	Soybeans, vegetable and fungal protein	7.23	21.69

TABLE 2. 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)	0.019–0.080 (12)	0.057–0.120 (15)	0.047–0.105 (19)	0.024–0.052 (21)	0.018–0.037 (22)	0.014–0.030 (23)
Min–max 95th percentile (number of surveys)	0.072–0.219 (11)	0.113–0.240 (14)	0.086–0.203 (19)	0.053–0.113 (20)	0.038–0.089 (22)	0.031–0.065 (22)

TABLE 3. 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 1332/2008^1^ on food enzymes.

An application has been introduced by the applicant “Dongsheng Biotech (Taixing) Co., Ltd.” for the authorization of the food enzyme Transglutaminase from a non‐genetically modified strain of Streptomyces mobaraensis (strain M2020197).

The food enzyme was assessed by EFSA, which issued a negative opinion on 5 December 2023. The new application contains supplementary information to address the data gaps identified in the scientific opinion (EFSA‐Q‐2021‐00651).

In accordance with Article 12(1) of Regulation (EC) No 234/2011 implementing Regulation (EC) No 1331/2008, the Commission has verified that the application in question falls within the scope of the food enzyme Regulation and contains all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The Commission requests the European Food Safety Authority (EFSA) to verify the requirements of Article 32b of Regulation (EC) No 178/2002, the suitability of the data for the risk assessment in accordance with Article 12(2) of Regulation (EU) No 234/2011, and to carry out the safety assessment and the assessment of possible confidentiality requests on the new data only for the following food enzyme: Transglutaminase from a non‐genetically modified strain of Streptomyces mobaraensis (strain M2020197) in accordance with the Regulation (EC) No 1331/2008, establishing a common authorization procedure for food additives, food enzymes and food flavourings.3

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 transglutaminase from S. mobaraensis strain M2020197.

In the technical dossier, the applicant named this food enzyme as transglutaminase and indicated that its catalytic activity is described by EC 2.3.2.13 according to the IUBMB classification system. Therefore, the EFSA scientific opinion reports this food enzyme in accordance with the IUBMB classification system (EC 2.3.2.13) and the IUBMB accepted name protein‐glutamine γ‐glutamyltransferase is used throughout the opinion.

The applicant submitted previously an application for protein‐glutamine γ‐glutamyltransferase from the non‐genetically modified S. mobaraensis strain M2020197, for which EFSA concluded that the food enzyme could not be considered safe under the intended conditions of use, due to a low margin of exposure and to data gaps (EFSA CEP Panel, 2024).

As a follow‐up, the applicant submitted new data. The present opinion assesses the new studies provided and updates the safety evaluation of the food enzyme. Whenever previous evaluation remains unchanged, reference to the previous EFSA opinion is made.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme transglutaminase from a non‐genetically modified strain of S. mobaraensis strain M2020197.

Additional information was requested during the risk assessment phase. See https://open.efsa.europa.eu/questions/EFSA‐Q‐2024‐00429.

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 May to 09 June 2025.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureProtein‐glutamine γ‐glutamyltransferaseSystematic nameProtein‐glutamine: amine γ ‐glutamyltransferaseSynonymsTransglutaminase; Factor XIIIa; fibrinoligaseIUBMB NoEC 2.3.2.13CAS No80146‐85‐6EINECS No616‐952‐0

Protein‐glutamine γ‐glutamyltransferases catalyse the acyl‐transfer reaction between the γ‐glutamyl group of a glutamine residue and the ε‐amino group of lysine in proteins, resulting in intra‐ and inter‐molecular cross‐linking of proteins. In the absence of amino substrates, transglutaminases catalyse the hydrolysis of the γ‐carboxyamide group of the glutaminyl residue, resulting in deamidation.

In the previous food enzyme application (EFSA‐Q‐2021‐00651), the Panel could not exclude safety concerns of the food enzyme due to an insufficient margin of exposure, calculated from a no observed adverse effect level (NOAEL) identified from a repeated dose 90‐day oral toxicity study and the estimated dietary exposure. Furthermore, the identity of the production strain was not established, the absence of genes of concern was not confirmed and the absence of viable cells of the production strain was not demonstrated (EFSA CEP Panel, 2024).

As a follow‐up, the applicant submitted new data on the characterisation of the production strain and on the absence of viable cells. In addition, the applicant updated the use levels and provided additional experimental data on the inactivation of the food enzyme in different final foods. Furthermore, a new allergenicity assessment has been performed. The present opinion assesses these new data provided in the application EFSA‐Q‐2024‐00429 and updates the conclusion on the safety of the food enzyme.

Source of the food enzyme

3.1

The current section on the source of the food enzyme complements Section 3.1 of the previous evaluation (EFSA CEP Panel, 2024).

The production strain was identified using whole‐genome sequence (WGS) data.6 ■■■■■, the production strain CCTCC M2020197 is considered to belong to the S. mobaraensis species.

The predicted open reading frames (ORF) of the genome sequence of the production strain were searched for the presence of genes involved in toxin production, virulence factors and secondary metabolites using ■■■■■. No gene of concern was found ■■■■■.7

The WGS of the production strain was also interrogated for the presence of antimicrobial resistance (AMR) genes using ■■■■■. Genes involved in resistance to oleandomycin, ■■■■■ were found.8 The presence in the production strain of genes conferring antimicrobial resistance is considered a hazard.

Production of the food enzyme

3.2

See previous evaluation (EFSA CEP Panel, 2024).

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

See previous evaluation (EFSA CEP Panel, 2024).

Chemical parameters

3.3.2

See previous evaluation (EFSA CEP Panel, 2024).

Purity

3.3.3

The current evaluation complements section 3.3.3 of the previous evaluation (EFSA CEP Panel, 2024).

The ■■■■■ gene, involved in oleandomycin resistance, was found in the genome of the production strain. This gene is commonly associated with the presence of the oleandomycin biosynthesis cluster in genomes of Streptomyces species. For this reason, the applicant was requested to analyse the food enzyme for the presence of oleandomycin. Analytical data showed that oleandomycin was absent in three batches of the food enzyme, with a limit of detection of 0.5 μg/kg.9

Viable cells and DNA of the production strain

3.3.4

The current evaluation supersedes Section 3.3.4 of the previous evaluation (EFSA CEP Panel, 2024).

The absence of viable cells of the production strain in the food enzyme ■■■■■ was shown in three independent batches analysed in triplicate. ■■■■■. No colonies of the production strain were observed. A positive control was included.10

The absence of 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 five fragments of ca. 400‐bp each, specific for the different AMR genes identified in the genome of the production strain, with a limit of detection of 5 ng spiked DNA/g food enzyme.11

Toxicological data

3.4

See previous evaluation (EFSA CEP Panel, 2024).

Allergenicity

3.4.1

The current evaluation supersedes Section 3.4.3 of the previous evaluation (EFSA CEP Panel, 2024).

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 protein‐glutamine γ‐glutamyltransferase produced with the S. mobaraensis strain M2020197 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 using the AllergenOnline database.12

No reports on oral or respiratory sensitisation or elicitation reactions of the protein‐glutamine γ‐glutamyltransferase under assessment have been published.13

A case report has shown that protein‐glutamine γ‐glutamyltransferase can cause occupational asthma (Sander et al., 2020). However, 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).

Protein‐glutamine γ‐glutamyltransferases have been implicated in the pathogenesis of coeliac disease as an autoantigen. In addition, due to their functional similarities with endogenous protein‐glutamine γ‐glutamyltransferases, those from microbial origin may cross link with gluten proteins, enhancing their immunogenicity (Di Sabatino et al., 2012; Lerner et al., 2025; Lerner & Matthias, 2020).

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 protein‐glutamine γ‐glutamyltransferase under assessment, but the risk of contribution to coeliac disease cannot be excluded.

■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201114) is used as raw material. In addition, ■■■■■, a known source of allergens, is 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), 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 intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low. The Panel concluded that a risk of contribution to coeliac disease cannot be excluded.

Dietary exposure

3.5

The current evaluation supersedes Section 3.5 of the previous evaluation (EFSA CEP Panel, 2024).

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in eight food manufacturing processes at the recommended use levels summarised in Table 1.

TABLE 1: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 15

The Panel noted a substantial decrease in the use levels in the current assessment, when compared to the previously reported levels. The applicant ascribes this change to the availability of more recent information on actual use levels.16

In all food manufacturing processes, the protein‐glutamine γ‐glutamyltransferase catalyses the cross‐linking between glutamine and lysine residues, modifying the physical properties (e.g. breaking strength and moisture retention) of the foods and impacts their sensory attributes such as mouthfeel.17

In the production of cheese, the food enzyme is added to milk together with the starter culture and rennet during coagulation.18 The food enzyme–TOS remain in the final foods.

In the production of fermented dairy products, the food enzyme is added to milk during the fermentation.19 The food enzyme–TOS remain in the final foods.

In the production of dairy desserts, the food enzyme is added to milk after pasteurisation and during the cooling.20 The food enzyme–TOS remain in the final foods.

In the production of modified meat and fish products, the food enzyme is added to meat or fish during the mixing, cutting and emulsifying21 in all the described processes except in the production of mechanically separated meat (MSM), in which it is added during the transfer of the MSM to the dosing machine for packaging.22 The food enzyme–TOS remain in the final foods.

In the production of baked products and cereal‐based products other than baked, the food enzyme is added to flour during the mixing to make dough.23 The food enzyme–TOS remain in the final foods.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to plant materials during the fermentation.24 The food enzyme–TOS remain in the final foods.

In the production of meat alternatives, the food enzyme is added to vegetable proteins during the mixing, cutting and emulsifying.25 The food enzyme–TOS remain in the final foods.

The food enzyme has a temperature optimum between ■■■■■ and ■■■■■°C (pH ■■■■■) and a pH optimum between pH ■■■■■ and ■■■■■ (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for up to 16 min at different temperatures. The enzyme activity decreased above 55°C, showing no residual activity above 70°C (EFSA CEP Panel, 2024). The applicant measured the protein‐glutamine γ‐glutamyltransferase activity in a selection of foods produced with and without thermal treatment and found that the residual activity was negligible.26 Taken together, the Panel considered that the food enzyme is inactive in most of the processed foods (e.g. fresh and ripened cheese, sausages, canned meat products, plant‐based alternatives of meat or dairy products). However, it may remain in its active form in baked products and ice creams, depending on the specific processing conditions.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool27 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) and input data provided in Appendix C.

Table 2 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 B). The highest dietary exposure was estimated to be 0.240 mg TOS/kg body weight (bw) per day in toddlers 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 3.

The conservative approach applied to estimate the dietary 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

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 91 mg TOS/kg bw per day, the lowest dose tested, resulting in margins of exposure (MOE) for infants, toddlers, children, adolescents, adults and the elderly of at least 36, 26, 50, 99, 115 and 133, respectively (EFSA CEP Panel, 2024).

A comparison of the NOAEL with the newly derived exposure estimates of 0.014–0.120 mg TOS/kg bw per day at the mean and from 0.031 to 0.240 mg TOS/kg bw per day at the 95th percentile resulted in a MOE of at least 379.

CONCLUSIONS

4

Based on the new data provided, the evaluation of the data previously submitted and the derived margin of exposure, the Panel concluded that the food enzyme protein‐glutamine γ‐glutamyltransferase produced with the non‐genetically modified S. mobaraensis strain M2020197 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Dossier and additional information can be accessed at https://open.efsa.europa.eu/dossier/FEN‐2024‐26531.

ABBREVIATIONSAMRantimicrobial resistancebwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditivesMSMmechanically separated meatNOAELno observed adverse effect levelOECDOrganisation for Economic Cooperation and DevelopmentORFopen reading framePCRpolymerase chain reactionTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00429

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

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, 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 details.