Safety evaluation of a second extension of use of the food enzyme α‐amylase from the non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT

Abstract

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase i.e. EC 3.2.1.1) is produced with the non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that the food enzyme did not give rise to safety concerns when used in seven food manufacturing processes. Subsequently, the applicant has requested to extend its use to include three additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of ten food manufacturing processes. As the food enzyme‐total organic solids (TOS) are removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme‐TOS was estimated only for the remaining nine processes. The dietary exposure was calculated to be up to 0.049 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (230 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 4694. Based on the data provided for the previous evaluation and the revised margin of exposure in the present evaluation, 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 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.

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.

Alpha‐amylase from a non‐genetically modified strain of Cellulosimicrobium funkei (strain AE‐AMT) is a food enzyme included in the Register of food enzymes ³ to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 31 August 2023, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the condition of use of the food enzyme Alpha‐amylase from a non‐genetically modified strain of Cellulosimicrobium funkei (strain AE‐AMT).

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 an extension of the condition of use for the following food enzyme: Alpha‐amylase from a non‐genetically modified strain of Cellulosimicrobium funkei (strain AE‐AMT), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings. ⁴

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of food enzyme α‐amylase from a non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT.

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 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, 2023a) have been followed for the evaluation of the application.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁵ 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 26 January to 16 February 2024. ⁶ No comments were received.

3. ASSESSMENT

IUBMB nomenclature	α‐Amylase
Systematic name	4‐α‐d‐glucan glucanohydrolase
Synonyms	Glycogenase, endo‐amylase, Taka‐amylase
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 malto‐oligosaccharides.

All aspects concerning the safety of this food enzyme regarding its source, production, characteristics and toxicology were evaluated in July 2022 (EFSA CEP Panel, 2022), then updated when its use was extended to include six additional food manufacturing processes in June 2023 (EFSA CEP Panel, 2023b). Following a request to update the intended uses (adding three additional food manufacturing processes), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in ten food manufacturing processes.

3.1. Dietary exposure

The current dietary exposure supersedes section 3.5 in the previous evaluation (EFSA CEP Panel, 2023b).

3.1.1. Revised intended use of the food enzyme

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

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	1.0	1.0
Production of cereal‐based products other than baked	Flour, cereals	1.0	1.0
Production of brewed products	Cereals	0.2	0.2
Production of glucose syrups and other starch hydrolysates	Starch	109	109
Processing of fruits and vegetables
Production of juices	Fruits and vegetables	1.0
Production of fruit and vegetable products other than juices	Fruits and vegetables	1.0
Production of non‐wine vinegar	Cereals	2.0	2.0
Processing of plant‐ and fungal‐derived products
Production of coffee substitutes	Barley	2.0
Production of tea and other herbal and fruit infusions	Leaf extracts	2.0	2.0
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts etc.	2.0	2.0

^a The name has been harmonised by EFSA according to the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023a).

^b The numbers in bold were used for calculation.

^c The previous evaluation is made for the food enzyme application EFSA‐Q‐2022‐00532.

The additional three uses of the food enzyme are described below.

In the production of juices, the food enzyme is added during the mash treatment and the depectinisation steps. ⁸ The α‐amylase degrades starch in the pressed juices, improving the filtration rate, preventing haze, and increasing 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 during both the mash treatment and prior to straining. ⁹ The hydrolysis of starch reduces viscosity, and the hydrolysates have higher solubility and sweetness in the final products (e.g. jam, puree, paste and sauce). The food enzyme‐TOS remain in the final products.

In the production of a coffee substitute, the food enzyme is added to milled barley prior to extraction. ¹⁰ The α‐amylase hydrolyses the starch, which increases sweetness and decreases viscosity. ¹¹ The food enzyme‐TOS remain in the final coffee substitutes.

Based on the thermostability evaluated previously (EFSA CEP Panel, 2023b) and the thermal treatments applied during food processing, it is expected that the enzyme is inactivated in the food manufacturing processes listed in Table 1, with the exception of juices, in which it may remain active depending on the pasteurisation conditions.

3.1.2. Dietary exposure estimation

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

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 selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023a). 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. 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 0.049 mg TOS/kg bw per day in toddlers and children at the 95th percentile.

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)	0.002–0.015 (12)	0.008–0.035 (15)	0.006–0.018 (19)	0.003–0.012 (21)	0.002–0.007 (22)	0.002–0.005 (23)
Min–max 95th percentile (number of surveys)	0.005–0.036 (11)	0.021–0.049 (14)	0.011–0.049 (19)	0.007–0.031 (20)	0.006–0.022 (22)	0.003–0.015 (22)

3.1.3. 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 3.

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
Exposure to food enzyme‐TOS was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
The consumption data covered all types of tea and herbal infusion products, not only the stevia infusion	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of one process from the exposure estimation: – Production of glucose syrups and other starch hydrolysates	−

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

The conservative approach applied to estimate the exposure to the food enzyme‐TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to an overestimation of the exposure.

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

3.2. Margin of exposure

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 230 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure of at least 19,167 (EFSA CEP Panel, 2023b).

A comparison of the NOAEL with the newly derived exposure estimates of 0.002–0.035 mg TOS/kg bw per day at the mean and from 0.003 to 0.049 at the 95th percentile resulted in a revised margin of exposure of at least 4694.

4. CONCLUSION

Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that the food enzyme α‐amylase produced with the non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT does not give rise to safety concerns under the revised intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for authorisation of α‐amylase from Cellulosimicrobium funkei AE‐AMT in accordance with the Regulation (EC) No 1331/2008. August 2023. Submitted by Amano Enzymes Inc.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

European Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

EU

European Union

IUBMB

International Union of Biochemistry and Molecular Biology

NOAEL

no observed adverse effect level

RM

raw material

TOS

total organic solids

ACKNOWLEDGEMENTS

The Panel would like to thank Andrew Chesson for the support provided to this scientific output.

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00663

COPYRIGHT FOR NON‐EFSA CONTENT

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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.

Supporting information

Dietary exposure estimates to the food enzyme‐TOS in details

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

A.1.

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

B.1.

Population	Age range	Countries with food consumption surveys covering more than one 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

^*Consumption data from these pre‐accession countries are not reported in Table 2 of this opinion; however, they are included in Appendix A for testing purpose.

^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).

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. , Sramkova, M. , Van Loveren, H. , Vernis, L. , Cavanna, D. , Liu, Y. , de Nijs, R. A. , & di Piazza, G. , (2024). Safety evaluation of a second extension of use of the food enzyme α‐amylase from the non‐genetically modified Cellulosimicrobium funkei strain AE‐AMT . EFSA Journal, 22(7), e8948. 10.2903/j.efsa.2024.8948