Safety evaluation of an extension of use of the food enzyme α‐amylase from the non‐genetically modified Bacillus licheniformis strain AE‐TA

TL;DR

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

Contribution

The study provides an updated safety assessment for the extended use of α-amylase in nine food processes.

Findings

The food enzyme is safe for use in nine food manufacturing processes.

Dietary exposure to the enzyme was estimated at up to 0.382 mg TOS/kg body weight per day.

The enzyme-TOS is removed in two processes, so exposure was only calculated for seven.

Abstract

The food enzyme α‐amylase (4‐α‐d‐glucan glucanohydrolase; EC 3.2.1.1) is produced with the non‐genetically modified microorganism Bacillus licheniformis strain AE‐TA 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 when used in eight food manufacturing processes. Subsequently, the applicant has requested to extend its use to include one additional process and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of nine food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining seven processes. Dietary exposure was calculated to be up to 0.382 mg TOS/kg body weight per day in European populations. Based on the data provided for the previous evaluation and the revised dietary exposure in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

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	2.1		2.1
Production of brewed products	Cereals	0.4		0.4
Production of non‐wine vinegar	Cereals	850.7		850.7
Production of cereal‐based products other than baked	Cereals (e.g. wheat, rice, corn, oat)	Infant cereals	29.4	4.3
		Other products	2.1	2.1
Production of glucose syrups and other starch hydrolysates	Starch	4.3		4.3
Production of distilled alcohol	Cereals	177.4		177.4
Processing of plant‐ and fungal‐derived products
Production of refined and unrefined sugar	Sugar beet and sugar cane	0.2		0.2
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts etc.	4.3		4.3
Production of plant extracts 8	Plant extracts rich in steviol glycosides	4.3

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)	0.007–0.099 (12)	0.029–0.097 (15)	0.026–0.068 (19)	0.010–0.050 (21)	0.008–0.021 (22)	0.006–0.027 (23)
Min–max 95th percentile (number of surveys)	0.038–0.382 (11)	0.079–0.241 (14)	0.068–0.171 (19)	0.026–0.119 (20)	0.021–0.060 (22)	0.017–0.054 (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	+
Exposure from production of cereal‐based products other than baked was calculated using the use level indicated for infant cereals, although a lower use level is provided for non‐infant cereal products	+
Minor FoodEx categories found to only sporadically contain molasses were excluded from the exposure assessment	−
'Brown sugar' produced through use of cane molasses or caramelised sugar syrup was excluded, due to it being a niche product on the European market	−
Assumption that dextrans, which may be present due to microbial spoilage, are always present thus necessitating treatment with dextranase	+
The transfer of food enzyme‐TOS into cane and beet molasses/syrups was assumed to be 100%	+
No distinction was made between beet molasses and cane syrups used as ingredients in foods	+/−
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
Exclusion of two processes from the exposure estimation: – Production of glucose syrups and other starch hydrolysates – Production of distilled alcohol	−

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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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 1332/2008^1^ on food enzymes.

Alpha‐amylase from a non‐genetically modified strain of Bacillus licheniformis (strain AE‐TA) 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 28 October 2022, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the conditions of use for the above food enzyme in several food processes.

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

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 the food enzyme alpha‐amylase from a non‐genetically modified strain of Bacillus licheniformis (strain AE‐TA).

Additional information, requested from the applicant during the assessment process on 21 March 2024, was received on 16 April 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 of the application.

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 19 April to 10 May 2024.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclature α‐amylaseSystematic name4‐α‐d‐glucan glucanohydrolaseSynonyms1,4‐α‐d‐glucan glucanohydrolaseIUBMB noEC 3.2.1.1CAS no9000‐90‐2EINECS no232‐565‐6

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.

All aspects concerning the safety of this food enzyme, when used in eight food manufacturing processes, were evaluated in April 2024 (EFSA CEP Panel, 2024). Following a request to update the intended uses (adding one food manufacturing process and revising the use levels), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in nine food manufacturing processes.

Dietary exposure

3.1

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

Revised intended use of the food enzyme

3.1.1

The food enzyme is intended to be used in nine 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 increase in the use level recommended in the current assessment for the production of infant cereals when compared to the previous level. The applicant ascribes this change to the availability of more recent information on actual use levels.9

The additional use of the food enzyme is described below.

In the production of plant extracts,10 this α‐amylase is used in the production of modified steviol glycosides.11 The food enzyme–TOS remain in the final foods.

Based on the thermostability evaluated previously (EFSA CEP Panel, 2024) and the downstream processing steps applied in the food processes, it is expected that this α‐amylase may remain active in the food manufacturing processes listed in Table 1 in which it is not removed.

Dietary exposure estimation

3.1.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the seven 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, 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. 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, 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 0.382 mg TOS/kg bw per day in infants at the 95th percentile.

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 3.

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 two food manufacturing processes from the exposure assessment was based on > 99% of TOS removal. This is not expected to have an impact on the overall estimate derived.

Margin of exposure

3.2

Since toxicological tests were not considered necessary by the Panel because the production strain qualified for the qualified presumption of safety (QPS) approach and no issues of concerns arose from the manufacturing process of the food enzyme (EFSA CEP Panel, 2024), a margin of exposure was not calculated.

CONCLUSION

4

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

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of alpha‐amylase from Bacillus licheniformis AE‐TA in accordance with the Regulation (EC) No 1331/2008. October 2022. Submitted by Amano Enzymes Inc.

Additional information. April 2024. Submitted by Amano Enzymes Inc.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionIUBMBInternational Union of Biochemistry and Molecular BiologyQPSqualified presumption of safetyRMraw materialTOStotal organic solids

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‐2023‐00305

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, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Claude Lambré, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Inger‐Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details