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

TL;DR

This paper evaluates the safety of expanding 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 glutaminase under revised usage conditions across 18 food processes.

Findings

Dietary exposure to the enzyme's total organic solids was estimated at up to 0.678 mg/kg body weight per day.

The enzyme was deemed safe for use in 18 food manufacturing processes after an updated safety evaluation.

Exposure calculations excluded two processes where the enzyme solids were removed from the final product.

Abstract

The food enzyme glutaminase (l‐glutamine amidohydrolase; EC 3.5.1.2) is produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐GT 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 five food manufacturing processes. Subsequently, the applicant requested to extend its use to thirteen additional processes and to revise the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of eighteen 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 sixteen processes. Dietary exposure was calculated to be up to 0.678 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 dairy products
Production of cheese	Milk	5.1
Production of flavouring preparations from dairy products	Dairy products (cream, cheese, butter etc.) 8	5.1	33
Production of modified milk proteins d	Milk proteins	25.5
Processing of eggs and egg products	Eggs	5.1	203
Processing of meat and fish products
Production of modified meat and fish products	Meat and fish	5.1
Production of protein hydrolysates from meat and fish proteins	Animal proteins	25.5	33
Processing of cereals and other grains
Production of baked products	Flour	5.1
Production of brewed products	Cereals	25.5
Production of distilled alcohol	Cereals	10.2
Production of cereal‐based products other than baked	Cereals	5.1
Processing of fruits and vegetables
Production of fruit and vegetable products other than juices	Fruit and vegetable	25.5
Processing of plant‐ and fungal‐derived products
Production of protein hydrolysates from plants and fungi d	Plant proteins	25.5	33
Production of meat analogues e	Textured vegetable proteins	5.1
Production of edible oils from plant and algae	Algal cells	122.3
Production of plant‐based analogues of milk and milk products	Plant‐based drink	5.1
Production of tea and other herbal and fruit infusions	Tea leaves	5.1
Production of coffee extracts	Coffee beans	25.5
Processing of yeast and yeast products	Yeast extract, autolysed yeast	20.4	590

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)	0.040–0.374 (12)	0.072–0.271 (15)	0.062–0.258 (19)	0.030–0.140 (21)	0.023–0.082 (22)	0.018–0.095 (23)
Min–max 95th percentile (number of surveys)	0.084–0.678 (11)	0.115–0.653 (14)	0.100–0.500 (19)	0.066–0.294 (20)	0.052–0.183 (22)	0.041–0.186 (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
Yeast processing: food categories chosen for calculation are broader than yeast extract and include also those relevant to yeast autolysates and yeast cell wall, despite that the food enzyme under application is not used to treat the yeast cell wall.	+
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	+/−
Exclusion of two processes from the exposure estimation: ‐ 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.

Glutaminase from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐GT) 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 8 November 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: Glutaminase from a non‐genetically modified strain of Bacillus amyloliquefaciens (strain AE‐GT), 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 glutaminase from a non‐genetically modified Bacillus amyloliquefaciens (strain AE‐GT).

Additional information, requested from the applicant during the assessment process on 25 September 2023 and 20 December 2023, was received on 24 October 2023 and 18 January 2024, respectively (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 16 February to 08 March 2024.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureGlutaminaseSystematic name l‐glutamine amidohydrolaseSynonymsGlutaminase I; l‐glutaminase; glutamine aminohydrolaseIUBMB NoEC 3.5.1.2CAS No9001‐47‐2EINECS No618‐332‐5

Glutaminases catalyse the hydrolysis of the carboxamide group of l‐glutamine, releasing l‐glutamic acid and ammonia.

All aspects concerning the safety of this food enzyme, when used in five food manufacturing processes, were evaluated in January 2024 (EFSA CEP Panel, 2024). Following a request to update the intended uses (adding thirteen food manufacturing processes and revising use levels), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in eighteen 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 eighteen 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 in the current assessment when compared to the previously reported levels. The applicant ascribes these changes to modifications of the current food manufacturing processes.10

In the majority of the additional food manufacturing processes, the hydrolysis catalysed by the food enzyme enriches the sensory properties of the final foods.11

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

In the production of cheese, the food enzyme is added to milk during the formation of the curd.12 The food enzyme–TOS remain in the final foods.

In the production of modified milk proteins, the food enzyme is added to protein concentrates after the solubilisation step.13 The food enzyme–TOS remain in the final foods.

In the production of modified meat and fish products, the food enzyme is added to cut or minced meat and fish,14 or to meat broth for the production of extracts.15 The food enzyme–TOS remain in the final foods.

In the production of baked products and in the production of cereal‐based products other than baked, the food enzyme is added to flour during dough making.16 The food enzyme–TOS remain in the final foods.

In the production of brewed products, the food enzyme is added during the mashing step for beer production.17 In the production of fermented beverages like sake or rice wine, it may be added during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps.18 The food enzyme–TOS remain in the final foods.

In the production of distilled alcohol, the food enzyme is added to cereals during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps.19 The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of fruit and vegetable products other than juices, the food enzyme is added to the mash after the heat treatment.20 The food enzyme–TOS remain in the final foods.

In the production of meat analogues, the food enzyme is added to textured vegetable proteins.21 The food enzyme–TOS remain in the final foods.

In the production of edible oils from plant and algae, the food enzyme is added to algal cells during the cell hydrolysis step.22 The food enzyme–TOS are removed in the final processed foods by deodorisation (EFSA CEP Panel, 2023).

In the production of plant‐based analogues of milk and milk products, the food enzyme is added to the plant‐based drinks.23 The food enzyme–TOS remain in the final foods.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaves at the beginning of the food manufacturing process.24 The food enzyme–TOS remain in the final products.

In the production of coffee extracts, the food enzyme could be added to the green coffee beans before roasting or after roasting, and to the ground coffee before extraction.25 The food enzyme can also be added directly to the coffee extract.26 The food enzyme–TOS remain in the final products.

Based on the thermostability evaluated previously (EFSA CEP Panel, 2024) and the downstream processing steps applied in the food processes, it is expected that the food enzyme will be inactivated or removed in all 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 sixteen 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) together with the information provided in Appendix C. 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 0.678 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 estimation was based on >99% of TOS removal. This is not expected to 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 glutaminase produced with the non‐genetically modified Bacillus amyloliquefaciens strain AE‐GT does not give rise to safety concerns under the revised intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

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

Additional information. October 2023. Submitted by Amano Enzymes Inc.

Additional information. January 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‐2023‐00356

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