Safety evaluation of an extension of use of the food enzyme bacillolysin from the non‐genetically modified Bacillus amyloliquefaciens strain AE‐NP

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 confirms the safety of the food enzyme bacillolysin for extended use in fifteen food manufacturing processes.

Findings

Dietary exposure to the food enzyme–TOS was estimated at up to 35.251 mg/kg body weight per day.

The enzyme was deemed safe for use in the revised conditions across European populations.

Abstract

The food enzyme bacillolysin (EC 3.4.24.28) is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐NP 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 thirteen food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of fifteen food manufacturing processes. As the food enzyme–total organic solids (TOS) are removed in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining thirteen processes. Dietary exposure was calculated to be up to 35.251 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 recommended 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	45.4	45.4
Production of cereal‐based products other than baked	Cereals, flour	227.2
	Cereals for low‐protein cereal products	18,172.7	18,172.7
Production of brewed products	Cereals	227.2	227.2
Production of distilled alcohol	Cereals	90.9	90.9
Processing of dairy products
Production of flavouring preparation from dairy products	Dairy products	45.4	45.4
Production of modified milk proteins	Milk proteins	227.2	227.2	Other uses
		13,629.5	13,629.5	Infant formulae
Processing of meat and fish products
Production of modified meat and fish products	Meat and fish products	45.4	45.4
Production of protein hydrolysates from meat and fish proteins	Meat and fish proteins	227.2	227.2
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseed, nuts	9.1	9.1
Production of edible oils from plant and algae	Algal cells	1088.1	1088.1
Production of tea and other herbal and fruit infusions	Tea leaves, herbs, fruits	27.3	27.3
Production of plant extracts	Plant material	27.3
Production of protein hydrolysates from plants and fungi	Plant proteins	227.2	227.2	Other uses
		13,629.5	13,629.5	Infant formulae
Processing of eggs and egg products	Eggs	45.4	45.4
Processing of yeast and yeast products	Yeast	45.4	45.4

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)	0.312–9.565 (12)	0.501–5.153 (15)	0.412–0.724 (19)	0.151–0.541 (21)	0.178–0.436 (22)	0.130–0.291 (23)
Min–max 95th percentile (number of surveys)	1.339–35.251 (11)	0.926–16.203 (14)	0.769–1.466 (19)	0.313–1.054 (20)	0.411–1.373 (22)	0.320–0.707 (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
For yeast processing, although only yeast extract is produced, the food categories chosen for calculation cover also those containing mannoproteins resulting from the treatment of yeast cell walls.	+
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions in disaggregation FoodEx categories	+/−
Use of technical factors in the exposure model	+/−
The calculation excluded low‐protein cereals from the other cereal‐based products	−
Exclusion of two processes from the exposure assessment: – Production of distilled alcohol – Production of edible oils from plant and algae	−

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.

Bacillolysin (protease) from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐NP) 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 30 September 2022, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the conditions of the use of the food enzyme Bacillolysin (protease) from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐NP).

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: Bacillolysin (protease) from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐NP), 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 food enzyme bacillolysin from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐NP).

Additional information, requested from the applicant during the assessment process on 20 September 2023, was received on 19 October 2023.

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 08 March to 29 March 2024.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureBacillolysinSystematic name–Synonyms Bacillus metalloendopeptidase; Bacillus subtilis neutral proteinaseIUBMB NoEC3.4.24.28CAS No9080‐56‐2EINECS No232‐991‐2

Bacillolysins catalyse the hydrolysis of peptide bonds of proteins with broad specificity, releasing peptides and amino acids.

All aspects concerning the safety of this food enzyme were evaluated in March 2024, when used in thirteen food manufacturing processes (EFSA CEP Panel, 2024). Following a request to update the intended uses (adding two food manufacturing processes), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in fifteen 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 fifteen food manufacturing processes at the recommended use levels summarised in Table 1.

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

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

In the production of cereal‐based products other than baked, the food enzyme is added to cereals after the milling step.8 The proteolytic reaction of the food enzyme partially cleaves the peptide bonds in the gluten network, improving the rheology of the dough. The food enzyme–TOS remain in the final products.

In the processing of plant extracts, the food enzyme is added to plant extracts9 to improve the sensory properties of the final food ingredients,10 in which the food enzyme–TOS remain.

Based on the thermostability evaluated previously (EFSA CEP Panel, 2024) and the downstream processing steps applied in the food manufacturing processes, it is expected that this bacillolysin will be inactivated or removed in the food manufacturing processes listed in Table 1.

Dietary exposure estimation

3.1.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the thirteen 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 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 35.251 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 bacillolysin produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐NP does not give rise to safety concerns under the revised intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of Bacillolysin from Bacillus amyloliquefaciens AE‐NP in accordance with the Regulation (EC) No 1331/2008. September 2022. Submitted by Amano Enzymes Inc.

Additional information. October 2023. 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‐2023‐00004

COPYRIGHT FOR NON‐EFSA CONTENT

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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