Safety evaluation of the food enzyme α‐amylase from the non‐genetically modified Bacillus licheniformis strain TTME 6280 KY

Abstract

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the non‐genetically modified Bacillus licheniformis strain TTME 6280 KY by Kerry Ingredients & Flavours Ltd. The production strain met the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in nine food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed in three processes, dietary exposure was calculated for the remaining six food manufacturing processes. It was estimated to be up to 2.273 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, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the α‐amylase to known allergens was made and a match with one respiratory allergen 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.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ 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 1331/2008 ² 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.

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background as provided by the European Commission

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 22 January 2024, a new application has been introduced by the applicant “KERRY INGREDIENTS & FLAVOURS LTD.” for the authorisation of the food enzyme Alpha amylase from a non‐genetically modified Bacillus licheniformis (strain TTME 6280 KY).

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of the following food enzyme: Alpha amylase from a non‐genetically modified B. licheniformis (strain TTME 6280 KY), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. ³

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme alpha‐amylase from B. licheniformis strain TTME 6280 KY.

Additional information, requested from the applicant during the assessment phase on 12 February 2025 and 23 September 2025, was received on 9 May 2025 and 24 September 2025, respectively (see ‘Documentation provided to EFSA’).

2.2. Methodologies

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.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁴ 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 23 May to 13 June 2025. ⁵ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	α‐Amylase
Systematic name	4‐α‐d‐glucan glucanohydrolase
Synonyms	Glycogenase
IUBMB No	EC 3.2.1.1
CAS No	9000‐90‐2
EINECS No	232‐565‐6

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

The food enzyme under assessment is intended to be used in nine food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) starch and gluten fractions, (2) cereal‐based products other than baked, (3) brewed products, (4) glucose syrups and other starch hydrolysates and (5) distilled alcohol; processing of fruits and vegetables for the production of (6) juices, (7) fruit and vegetable products other than juices and (8) alcoholic beverages other than grape wine; (9) processing of plant‐ and fungal‐derived products for the production of plant‐based analogues of milk and milk products.

3.1. Source of the food enzyme

The α‐amylase is produced with the non‐genetically modified bacterium Bacillus licheniformis strain TTME 6280 KY, which is deposited at the Belgian Coordinated Collections of Microorganisms (BCCM, Belgium) with deposition number ■■■■■. ⁶ The production strain was identified as B. licheniformis through whole‐genome sequencing (WGS) analysis, ■■■■■. ⁷

The species B. licheniformis is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied, provided that the absence of acquired antimicrobial resistance (AMR) genes and toxigenic activity are verified for the specific strain used (EFSA, 2007; EFSA BIOHAZ Panel, 2022). ⁸ The production strain was not cytotoxic to Vero cells as indicated by the lactate dehydrogenase assay. ⁹ The genome sequence of the production strain was interrogated for the presence of antimicrobial resistance genes using two regularly maintained databases. No genes of concern were identified, with thresholds above 80% of identity and 70% of coverage. ¹⁰

Therefore, the production strain meets the requirements to qualify for the QPS approach and it is considered safe.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ¹¹ with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice. ¹²

The production strain is grown as a pure culture using a typical industrial medium in a submerged, 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. ¹³ 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. ¹⁴

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.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The α‐amylase is a single polypeptide chain of ■■■■■ amino acids. ¹⁵ The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁶ The food enzyme was analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ¹⁷ A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between the protein markers of ■■■■■ kDa in all batches, consistent with the expected mass of the enzyme.

No other enzyme activities were reported.

The applicant's in‐house determination of α‐amylase activity is based on the ability of starch to complex with iodine, acquiring a blue colour. The enzyme activity is determined by measuring the decrease in blue colour due to the hydrolysis of soluble starch (■■■■■). The activity is expressed in Modified Wohlgemuth Units (MWU)/g. One MWU is defined as the activity which hydrolyses 1.0 mg of soluble starch to a defined blue value in ■■■■■ under the conditions of the assay. ¹⁸

The food enzyme has a temperature optimum between 80 and 90°C (pH 5.7) and a pH optimum between pH 4.5 and 6.5 (40°C). Thermostability was tested by pre‐incubation of the food enzyme at different temperatures for different time periods (pH 5.4). Residual activity was about 30% after 30 min at 90°C and no activity was detected after 15 min at 100°C. ¹⁹

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1). ²⁰ The mean total organic solids (TOS) was 6.0% and the mean enzyme activity/TOS ratio was 4967 MWU/mg TOS.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
α‐Amylase activity	MWU/g a	298,000	304,000	297,000
Protein	%	3.1	3.1	3.2
Ash	%	2.1	2.1	2.2
Water	%	91.9	91.8	91.8
Total organic solids (TOS) b	%	6.0	6.1	6.0
Activity/TOS ratio	MWU/mg TOS	4967	4984	4950

^a MWU: Modified Wohlgemuth Unit (see Section 3.3.1).

^b TOS calculated as 100% – % water – % ash.

3.3.3. Purity

The lead content in all batches was below 3.44 mg/kg ^{21 , 22} 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, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). ²³ No antimicrobial activity was detected in any of the tested batches. ²⁴

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

3.3.4. Viable cells and DNA of the production strain

The demonstration of absence of viable cells and DNA of the production strain in the food enzyme is not requested for non‐genetically modified production strains that qualify for the QPS approach. However, since these data were submitted by the applicant, they were evaluated and included in this opinion for completeness.

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. Ten millilitres of product was diluted in 90 mL of non‐selective medium and incubated at 37°C for 2 days. From this, 10 mL was filtered through a 0.45‐μm membrane filter; the filters were then placed on agar plates and incubated at 37°C for 70 h. No colonies were produced. A positive control was included. ²⁵

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 designed to amplify a 103‐bp fragment from the genome of the production strain, with a limit of detection of 1 ng spiked DNA/g food enzyme. ²⁶

3.4. Toxicological data

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).

3.4.1. Allergenicity

The allergenicity assessment considered only the food enzyme and not additives, carriers or other excipients that may be used in the final formulation.

The potential allergenicity of the α‐amylase produced with the Bacillus licheniformis strain TTME 6280 KY 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, a match with a respiratory allergen was found in the AllergenOnline database. ²⁷ The matching allergen was Asp o 21 (35.4% sequence identity), an α‐amylase from Aspergillus oryzae.

No reports on oral or respiratory sensitisation or elicitation reactions of the α‐amylase under assessment have been published. ²⁸

α‐Amylases have been shown to cause respiratory allergy. Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004) as also described for the α‐amylase from A. oryzae. Taking into account the wide use of α‐amylases as food enzyme, only a low number of allergic reactions upon oral exposure to α‐amylase in individuals respiratorily sensitised to α‐amylase have been described in the literature (Baur & Czuppon, 1995; Kanny & Moneret‐Vautrin, 1995; Losada et al., 1992; Moreno‐Ancillo et al., 2004; Quirce et al., 1992).

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

■■■■■, that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ²⁹ ), are used as raw materials. In addition, ■■■■■, a known source of allergens, is also present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the Panel considered that a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in nine 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. ³⁰

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM) a
Processing of cereals and other grains
Production of starch and gluten fractions	Cereals	3–69
Production of cereal‐based products other than baked	Flour	3–69
Production of brewed products	Cereals	7–69
Production of glucose syrups and other starch hydrolysates	Starch	3–69
Production of distilled alcohol	Cereals	7–69
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	7–51
Production of fruit and vegetable products other than juices	Fruit and vegetables	7–51
Production of alcoholic beverages other than grape wine	Fruits	7–69
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts etc.	7–51

Abbreviation: TOS, total organic solids.

^a The numbers in bold represent the maximum recommended use levels, which were used for calculation.

In the production of starch and gluten fractions, the food enzyme is added to cereals during the homogenisation and agglomeration step or dough preparation ³¹ to aid the separation of starch and gluten fractions. ³² Repeated washing steps applied in the down‐stream processes remove the food enzyme–TOS (EFSA CEP Panel, 2023).

In the production of cereal‐based products other than baked, the food enzyme is added to flour during dough preparation. ³³ The hydrolysis by the α‐amylase reduces viscosity of the slurry, facilitating downstream processing, such as extrusion. The food enzyme–TOS remain in the final foods.

In the production of brewed products, the food enzyme is added to cereals during the mashing step. ³⁴ Together with other saccharifying enzymes, the α‐amylase converts the liquefied starch to fermentable sugars. ³⁵ The food enzyme–TOS remain in the final brewed products.

In the production of glucose syrups and other starch hydrolysates, the food enzyme is added to starch prior to liquefaction. ³⁶ It hydrolyses starch into dextrins and reduces viscosity. ³⁷ The Panel considers that the food enzyme–TOS are removed from glucose syrups and other starch hydrolysates (EFSA CEP Panel, 2023).

In the production of distilled alcohol, the food enzyme may be added to cereals during slurry mixing, liquefaction, pre‐saccharification or fermentation steps. ³⁸ The α‐amylase facilitates the release of fermentable sugars. ³⁹ The food enzyme–TOS are not carried over into the distilled alcohols (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme is added to fruit and vegetables prior to the mash treatment. ⁴⁰ The α‐amylase catalyses starch hydrolysis in the pressed juices, improving the filtration rate, preventing haze and enhancing sweetness. The food enzyme–TOS remain in the juices.

In the production of fruit and vegetable products other than juices, the food enzyme is added to fruit and vegetables before blanching or during the re‐suspension step. ⁴¹ The hydrolysis of starch reduces the viscosity, improves solubility and enhances sweetness in the final products (e.g. jam, puree, paste and sauce). The food enzyme–TOS remain in the final food products.

In the production of alcoholic beverages other than grape wine, the food enzyme is added to fruit before fermentation. ⁴² The enzymatic treatment increases the processability and the yield of the fruit must. ⁴³ The food enzyme–TOS remain in these beverages.

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to a slurry of milled plant materials ⁴⁴ to hydrolyse the gelatinised starch and thereby reduce viscosity. ⁴⁵ The food enzyme–TOS remain in the final foods.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the respective food manufacturing processes, the Panel considered that the food enzyme may remain in its active form in the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain, depending on the heat treatment conditions.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the six food manufacturing processes where the food enzyme–TOS remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool ⁴⁶ 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 B). The highest dietary exposure was estimated to be 2.273 mg TOS/kg body weight (bw) per day in toddlers at the 95th percentile.

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)	0.123–0.784 (12)	0.357–1.473 (15)	0.126–0.886 (19)	0.087–0.549 (21)	0.063–0.331 (22)	0.032–0.223 (23)
Min–max 95th percentile (number of surveys)	0.329–1.539 (11)	0.939–2.273 (14)	0.338–2.245 (19)	0.227–1.418 (20)	0.204–1.090 (22)	0.125–0.743 (22)

3.5.3. Uncertainty analysis

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.

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 three processes from the exposure estimation: – production of starch and gluten fractions – production of glucose syrups and other starch hydrolysates – production of distilled alcohol	−

Note: +, uncertainty with potential to cause overestimation of exposure. –, uncertainty with potential to cause underestimation of exposure.

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.

The exclusion of three food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

3.6. Margin of exposure

Since no toxicological assessment was considered necessary by the Panel, a margin of exposure was not calculated.

4. CONCLUSIONS

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 α‐amylase produced with the non‐genetically modified B. licheniformis strain TTME 6280 KY does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for the authorisation of the food enzyme α‐amylase from the non‐genetically modified B. licheniformis strain TTME 6280 KY in accordance with Regulation (EC) No 1331/2008. December 2023. Submitted by Kerry Ingredients & Flavours Ltd.

Additional information. May 2025. Submitted by Kerry Ingredients & Flavours Ltd.

Additional information. September 2025. Submitted by Kerry Ingredients & Flavours Ltd.

ABBREVIATIONS

AMR

antimicrobial resistance

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FEZ

EFSA Panel on Food Enzymes

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

QPS

qualified presumption of safety

TOS

total organic solids

WGS

whole genome sequencing

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00216

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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

Relevant information or parts of this scientific output have been blackened in accordance with the confidentiality requests formulated by the applicant pending a decision thereon by EFSA. The full output has been shared with the European Commission, EU Member States (if applicable) and the applicant. The blackening may be subject to review once the decision on the confidentiality requests is adopted by EFSA and in case it rejects some of the confidentiality requests.

Supporting information

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

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

Appendix A can be found in the online version of this output (in the ‘Supporting Information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia,* Serbia,* Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia,* Serbia,* Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina,* Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina,* Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden

^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

^*Consumption data from these pre‐accession countries are not reported in Table 3 of this opinion, however, they are included in Appendix B for testing purpose.

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Van Loveren, H. , Vernis, L. , Fernández‐Fraguas, C. , Cavanna, D. , Lunardi, S. , & Liu, Y. (2025). Safety evaluation of the food enzyme α‐amylase from the non‐genetically modified Bacillus licheniformis strain TTME 6280 KY. EFSA Journal, 23(11), e9720. 10.2903/j.efsa.2025.9720