Safety evaluation of an extension of use of the food enzyme cyclomaltodextrin glucanotransferase from the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT

TL;DR

This study evaluates the safety of extending the use of a food enzyme produced by a non-genetically modified bacteria in six food manufacturing processes.

Contribution

The study provides an updated safety evaluation for extended use of the enzyme in additional food processes.

Findings

Dietary exposure was calculated to be up to 0.025 mg TOS/kg body weight per day.

The margin of exposure was determined to be at least 89,840, indicating no safety concerns.

EFSA concluded the enzyme is safe under the revised intended conditions of use.

Abstract

The food enzyme cyclomaltodextrin glucanotransferase ((1‐4)‐α‐d‐glucan 4‐α‐d‐[(1‐4)‐α‐d‐glucano]‐transferase; EC 2.4.1.19) is produced with the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns for adolescents, adults and the elderly in two food manufacturing processes. Subsequently, the applicant requested to extend its use to include four additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of six food manufacturing processes. Dietary exposure was calculated to be up to 0.025 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 (2246 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 89,840. 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 revised intended conditions of use.

This publication is linked to the following EFSA scientific opinion: https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2025.9221

Tables

TABLE 1. Updated intended uses and use levels of the food enzyme. 7

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of cereals and other grains
Production of baked products	Flour	0.7
Production of cereal‐based products other than baked	Cereals	1.3
Production of brewed products	Cereals, rice	0.1
Processing of plant‐ and fungal‐derived products
Production of plant extracts	Leaf (herb, tea) Leaf extracts rich in steviol glycosides Fruit peels rich in glycosides such as hesperidin 8	67.4	1400–70,000
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts, etc.	1.3
Processing of sugars
Production of specialty carbohydrates (cyclodextrins)	Starch	67.4	140–2808

TABLE 2. Updated dietary exposure to the 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)	0.001–0.008 (12)	0.005–0.011 (15)	0.005–0.010 (19)	0.003–0.005 (21)	0.001–0.003 (22)	0.001–0.003 (23)
Min–max 95th percentile (number of surveys)	0.004–0.022 (11)	0.012–0.025 (14)	0.010–0.018 (19)	0.006–0.010 (20)	0.003–0.007 (22)	0.002–0.005 (22)

TABLE 3. Summary of dietary exposure to β‐cyclodextrin (E 459) from their use as food additives in the maximum level exposure assessment scenario and subsequent exposure to the food enzyme, in six population groups (minimum–maximum across the dietary surveys in mg/kg bw per day).

Population group	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
Regulatory maximum level exposure scenario (mg β‐cyclodextrin (E 459)/kg bw per day)
Min–max mean (number of surveys)	0.7–4.5 (6)	1.9–19.1 (10)	2–24.4 (18)	1.7–16.2 (17)	1–7.7 (17)	0.9–6.8 (14)
Min–max 95th percentile (number of surveys)	2.6–16.2 (5)	8.7–56.6 (7)	8–73.1 (18)	6.3–56.8 (17)	4–26.3 (17)	3.4–15.2 (14)
Estimated exposure to food enzyme–TOS (mg TOS/kg bw per day)
Min–max mean (number of surveys)	0–0 (6)	0–0.001 (10)	0–0.002 (18)	0–0.001 (17)	0–0.001 (17)	0–0 (14)
Min–max 95th percentile (number of surveys)	0–0.001 (5)	0.001–0.004 (7)	0.001–0.005 (18)	0–0.004 (17)	0–0.002 (17)	0–0.001 (14)

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
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/–
Use of technical factors in the exposure model	+/–
Assumption that 100% of TOS remains in the final foods	+
Overall intake via all sources of dietary exposure
Only the highest intake at the P95 percentile shown in Table 2 is used to derive the lowest margin of exposure	–

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 European Union 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.

Cyclomaltodextrin glucanotransferase from a non‐genetically modified strain of Geobacillus stearothermophilus (strain AE‐KCGT) is a food enzyme included in the Register of food enzymes3 to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 4 November 2022, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the conditions of use of the food enzyme Cyclomaltodextrin glucanotransferase from a non‐genetically modified strain of Geobacillus stearothermophilus (strain AE‐KCGT).

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 extension of use for the following food enzyme: Cyclomaltodextrin glucanotransferase from a non‐genetically modified strain of Geobacillus stearothermophilus (strain AE‐KCGT), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings.4

Interpretation of the Terms of Reference

1.1.3

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of an extension of the conditions of use for the cyclomaltodextrin glucanotransferase from the non‐genetically modified Geobacillus stearothermophilus strain AE‐KCGT.

The production microorganism was reclassified as Anoxybacillus caldiproteolyticus at the species level in the scientific opinion published previously (EFSA FEZ Panel, 2025). Therefore, the same species name Anoxybacillus caldiproteolyticus is used also in the present opinion.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of food enzyme cyclomaltodextrin glucanotransferase from a non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT.

Additional information, requested from the applicant during the assessment process on 24 September 2024, was received on 11 October 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 existing guidance documents of EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c (2) of Regulation (EC) No 178/20025 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 18 November to 09 December 2024.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureCyclomaltodextrin glucanotransferaseSystematic name(1–4)‐α‐d‐glucan 4‐α‐d‐[(1–4)‐α‐d‐glucano]‐transferase (cycling)SynonymsCyclodextrin glycosyltransferase; α‐cyclodextrin glucanotransferaseIUBMB NoEC 2.4.1.19CAS No9030‐09‐5EINECS No618‐522‐8

Cyclomaltodextrin glucanotransferases catalyse the transglycosylation of glucans by formation of (1‐4)‐α‐d‐glucosidic bonds, resulting in the generation of α‐, β‐ and γ‐cyclodextrins and transglycosylated glucans.

All aspects concerning the safety of this food enzyme, when used in two food manufacturing processes, were evaluated in January 2025 (EFSA FEZ Panel, 2025).

Following a request to update the intended uses (adding four food manufacturing processes and revising the use levels), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in six food manufacturing processes.

Dietary exposure

3.1

The current dietary exposure supersedes Section 3.5 of the previous evaluation (EFSA FEZ Panel, 2025).

Revised intended use of the food enzyme

3.1.1

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

TABLE 1: Updated intended uses and use levels of the food enzyme. 7

The Panel noted a substantial decrease in the use levels recommended for the production of plant extracts and specialty carbohydrates 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.9

The additional four uses of the food enzyme are described below, in which the cyclomaltodextrin glucanotransferase catalyses a transglycosylation reaction to degrade the amylose in the starch and form a mixture of α‐, β‐, γ‐cyclodextrins.

In the production of baked products, the food enzyme is added to flour during the preparation of the dough.10 The food enzyme–TOS remain in the dough and the baked foods.

In the production of cereal‐based products other than baked, the food enzyme is added to rice before cooking11 and to cereals after milling.12 The food enzyme–TOS remain in the final cereal products.

In the production of brewed products, the food enzyme is added to cereals during the mashing step in beer production.13 For the production of fermented beverages like sake or rice wine, the food enzyme is added in multiple steps (slurry mixing, liquefaction, pre‐saccharification or fermentation).14 The food enzyme–TOS remain in beer and other fermented beverages.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to the plant‐based drinks.15 The food enzyme–TOS remain in the final plant‐based milk analogues.

Based on the thermostability data evaluated previously (EFSA FEZ Panel, 2025) and the downstream processing steps applied in the food processes, the Panel considered that this cyclomaltodextrin glucanotransferase will be inactivated in the majority of the food manufacturing processes listed in Table 1. However, it may remain in its active form in baked and brewed products, depending on the processing conditions.

Dietary exposure estimation

3.1.2

Like in the previous evaluation, two sets of calculation were made to estimate the dietary exposure of the food enzyme–TOS via the production of foods or food ingredients, and via the use of cyclodextrins as a food additive and novel foods (EFSA FEZ Panel, 2025).

Dietary exposure via the production of foods or food ingredients

3.1.2.1

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool16 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). 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 one 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 calculation of the 95th percentile (EFSA, 2011).

Table 2 provides an overview of the derived exposure estimates across all surveys. They ranged from 0.001–0.011 mg TOS/kg bw per day at the mean and from 0.002–0.025 mg TOS/kg bw per day at the 95th percentile, with the highest dietary exposure being 0.025 mg TOS/kg bw per day in toddlers at the 95th percentile. 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).

Dietary exposure via cyclodextrins as a food additive and novel foods

3.1.2.2

Like the previous evaluation, the same approach was applied also in the present evaluation. Using the revised use level for this process, EFSA recalculated the dietary exposure. Table 3 provides an overview of the derived exposure estimates across all surveys. The highest dietary exposure at the 95th percentile was estimated to be 0.005 mg TOS/kg bw per day in children.

Overall dietary exposure

3.1.2.3

The sources of the food consumption data behind these two sets of exposure estimation are different. The estimates reported in Tables 2 and 3 should not be summed to derive the highest overall intake. Therefore, the results are kept separately. Since the estimates shown in Table 2 exceed those in Table 3 by one order of magnitude, to avoid excessive overestimation, the Panel chose only the estimates shown in Table 2 to derive the margin of exposure.

Uncertainty analysis

3.1.3

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.

For figures reported in Tables 2 and 3, the conservative approach applied to the exposure estimate to food enzyme–TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to overestimation of the exposure.

The choice of using only figures reported in Table 2 to derive the margin of exposure (MoE) may lead to underestimation of the overall intake. However, estimates in Table 2 exceed greatly those in Table 3; thus, the underestimation, if it occurs, would be minimal.

Margin of exposure

3.2

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 2246 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in an MoE of at least 141 for infants, 111 for toddlers, 154 for children, 362 for adolescents, 508 for adults, 713 for the elderly (EFSA FEZ Panel, 2025).

A comparison of the NOAEL with the newly derived exposure estimates of 0.001–0.011 mg TOS/kg bw per day at the mean and from 0.002–0.025 mg TOS/kg bw per day at the 95th percentile resulted in an MoE of at least 89,840.

Despite more uses were considered in the current assessment, the newly derived MoE is higher than the one previously calculated; this is due to significantly reduced recommended use levels.

CONCLUSION

4

Based on the new data, the revised margin of exposure and previous evaluation, the Panel concluded that the food enzyme cyclomaltodextrin glucanotransferase produced with the non‐genetically modified Anoxybacillus caldiproteolyticus strain AE‐KCGT does not give rise to safety concerns under the revised intended conditions of use.

REMARK

5

The production of the food additive (E 960d glucosylated steviol glycosides) as defined in Regulation (EU) No 231/2012 laying down specifications for food additives is not covered by this assessment.

This food enzyme is intended to be used in the manufacturing of cyclodextrins. The safety evaluation of cyclodextrins as food additives and novel food ingredients is outside the remit of the EFSA FEZ Panel.

DOCUMENTATION AS PROVIDED TO EFSA

6

Application for authorisation of Cyclomaltodextrin glucanotransferase from Geobacillus stearothermophilus AE‐KCGT in accordance with Regulation (EC) No 1331/2008. November 2022. Submitted by Amano Enzyme Inc.

Additional information. October 2024. Submitted by Amano Enzyme Inc.ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFEZEFSA Panel on Food EnzymesIUBMBInternational Union of Biochemistry and Molecular BiologyMoEmargin of exposureNOAELno observed adverse effect levelRMRaw MaterialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00756

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

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details