Safety evaluation of a second extension of use of the food enzyme triacylglycerol lipase from the non‐genetically modified Rhizopus arrhizus strain AE‐TL(B)

TL;DR

This paper evaluates the safety of extending the use of a food enzyme in infant oil production and confirms it remains safe.

Contribution

The study provides an updated safety evaluation for a revised use of triacylglycerol lipase in infant nutrition.

Findings

Dietary exposure to the enzyme was estimated at up to 0.33 mg TOS/kg body weight per day.

The margin of exposure was calculated to be at least 5939, indicating no safety concerns.

EFSA concluded the enzyme is safe under the revised intended conditions of use.

Abstract

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐TL(B) by Amano Enzymes Inc. Two safety evaluations of this food enzyme were made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in six food manufacturing processes. Subsequently, the applicant has requested to extend its use to the production of 2‐palmitic acid‐enriched vegetable oil intended for infants aged 0–3 months, to withdraw one food manufacturing process and to revise one use level. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of five food manufacturing processes. Dietary exposure to the food enzyme‐total organic solids (TOS) was estimated to be up to 0.33 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1960 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 5939. Based on the new data, the revised margin of exposure and the previous 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	Raw material (RM)	Recommended use level (mg TOS/kg RM)
		Current evaluation a		Second evaluation a , b	First evaluation a , c
Processing of dairy products
Production of cheese	Milk	0.0014–0.14		0.14
Production of flavouring preparations from dairy products	Cheese, cream, butter and other dairy materials	0.07–6.7		6.7	187–311
Processing of cereals and other grains
Production of baked products	Flour	0.002–0.2		0.2
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products	Cereals, legumes, oilseeds, nuts, etc.	1.0–99.8		99.8
Processing of fats and oils
Production of modified fats and oils by interesterification	Edible vegetable oils or edible vegetable oil fractions, free fatty acids made from edible vegetable oil	0.4–43.7	All other uses	38.3	12.5
	Vegetable oil	0.4–43.7	Infant formula for infants aged 0–3 months 8
Production of free fatty acids by hydrolysis	Edible fats and oils			3.3

TABLE 2. Updated dietary exposure to the food enzyme–TOS in the standard six population groups for all the involved food manufacturing processes.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.001–0.019 (14)	0.006–0.054 (17)	0.010–0.025 (21)	0.005–0.013 (23)	0.003–0.009 (23)	0.003–0.008 (25)
Min–max 95th percentile (number of surveys)	0.002–0.069 (13)	0.018–0.090 (16)	0.024–0.068 (21)	0.012–0.037 (22)	0.011–0.029 (23)	0.008–0.030 (24)

TABLE 3. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate in six population groups for all the involved food manufacturing processes.

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	+/−

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate in infants aged 0–3 months for the production of modified fats and oils by interesterification.

Sources of uncertainties	Direction of impact
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
The highest consumption of infant formula reported for the period of 14–27 days of life was used to calculate the dietary exposure in infants from 0 to 3 months of life (EFSA Scientific Committee, 2017)	+
The maximum energy density in infant formula was considered in the calculation (Codex Stan 72‐1981, Commission Delegated Regulation (EU) 2016/127)	+
The maximum fat content in infant formula was considered in the calculation (Codex Stan 72‐1981, Commission Delegated Regulation (EU) 2016/127)	+
The maximum use level of 2‐palmitic acid‐enriched vegetable oil over total fat content in infant formula was considered in the calculation	+
Assumption that all the infant formulae for infants aged 0–3 months contain the 2‐palmitic acid‐enriched vegetable oil	+

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definitions 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 1331/20082 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 on food enzymes.

Triacylglycerol lipase produced from non‐genetically modified Rhizopus arrhizus (strain AE‐TL(B)) 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 18 September 2024, a new application was introduced by the applicant “Amano Enzymes Inc.” for an extension of the condition of use of the food enzyme Triacylglycerol lipase produced from non‐genetically modified Rhizopus arrhizus (strain AE‐TL(B)).

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 the following food enzyme: extension of the condition of use of triacylglycerol lipase produced from non‐genetically modified Rhizopus arrhizus (strain AE‐TL(B)), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation 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 triacylglycerol lipase from a non‐genetically modified Rhizopus arrhizus strain AE‐TL(B).

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 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, 2023a) have been followed for the evaluation.

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 24 April to 15 May 2025.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureTriacylglycerol lipaseSystematic nameTriacylglycerol acylhydrolaseSynonymsLipase; triglyceride lipaseIUBMB NoEC 3.1.1.3CAS No9001‐62‐1EINECS No232‐619‐9

Triacylglycerol lipases catalyse, in the presence of water, the hydrolysis of the ester linkages in triacylglycerols, resulting in the generation of glycerol, free fatty acids, diacylglycerols and monoacylglycerols. At very low concentrations of water, interesterification, i.e. the exchange of free fatty acids between two or more triacylglycerols, may occur.

All aspects concerning the safety of this food enzyme, when used in two food manufacturing processes, were evaluated in June 2023 (EFSA CEP Panel, 2023b). The intended uses were then updated to include four additional food manufacturing processes and to revise the use levels in July 2024 (EFSA FEZ Panel, 2024).

Following a further request to update the intended uses (extending the use of the production of modified fats and oils by interesterification to infants aged 0–3 months, withdrawing one food manufacturing process and revising one use level), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in five food manufacturing processes.

Dietary exposure

3.1

The current dietary exposure supersedes Section 3.1 of the previous evaluation (EFSA FEZ Panel, 2024).

Revised intended use of the food enzyme

3.1.1

The food enzyme is intended to be used in five 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 applicant withdrew one intended use of this food enzyme (the production of free fatty acids by hydrolysis).9

For the production of modified fats and oils by interesterification, the use of the food enzyme is extended to the production of 2‐palmitic acid‐enriched vegetable oil used as an ingredient in infant formula intended for infants aged 0–3 months.10

The food enzyme has a temperature optimum around 35–40°C (pH 7) and a pH optimum around pH 6–7 (37°C). After a pre‐incubation of the food enzyme for 30 min at different temperatures, the triacylglycerol lipase activity was stable up to 50°C and was not detected above 70°C (EFSA CEP Panel, 2023b). Based on these data, the Panel concluded that this triacylglycerol lipase is inactivated in the majority of the food manufacturing processes listed in Table 1. However, the food enzyme may remain in its active form in cheeses, depending on the processing conditions. It may also remain in its active form in baked products, depending on the heat treatment conditions, as a kinetic model developed to predict enzyme inactivation during bread making suggested that food enzymes may not be fully inactivated during baking (Zhang et al., 2017).

Dietary exposure estimation

3.1.2

Two sets of calculations were made. One to cover the population groups with age between 4 months and elderly. The other for infants aged 0–3 months for the production of modified fats and oils by interesterification used in infant formula.

Dietary exposure estimation in six population groups for all the involved food manufacturing processes

3.1.2.1

The first set calculated chronic exposure to the food enzyme–TOS using the FEIM webtool11 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).

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 the 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 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix B). The highest dietary exposure was estimated to be 0.09 mg TOS/kg bw per day in toddlers at the 95th percentile.

Dietary exposure estimation in infants aged 0–3 months for the production of modified fats and oils by interesterification

3.1.2.2

The second set calculated the chronic exposure to the food enzyme–TOS in infants aged 0–3 months for the production of modified fats and oils by interesterification (e.g. 2‐palmitic acid‐enriched vegetable oil), when used as an ingredient in infant formulae. It was considered that:

• 1 kg of vegetable oil is needed to produce 1 kg of vegetable oil enriched in 2‐palmitic acid;12
• the highest consumption reported for the period of 14–27 days of life, which corresponds to a value of 260 mL/kg bw per day, can be used in the exposure assessment of substances present in food intended for infants below 16 weeks of age. This reflects the highest relative consumption on a body weight basis and also covers the potential high consumption rates of preterm infants on enteral (formula) feeding (EFSA Scientific Committee, 2017);
• infant formula has an energy between 60 and 70 kcal per 100 mL (Codex Stan 72‐1981,13 Commission Delegated Regulation (EU) 2016/12714);
• infant formula has a total fat content between 4.4 and 6.0 g/100 kcal (Codex Stan 72‐1981; Commission Delegated Regulation (EU) 2016/127);
• the maximum use level of 2‐palmitic acid‐enriched vegetable oil in infant formula is 70% of the total fat content.15

By multiplying these parameters with the maximum recommended use level, the highest dietary exposure was estimated to be 0.33 mg TOS/kg bw per day in infants aged 0–3 months.

Uncertainty analysis

3.1.3

Uncertainty analysis of the dietary exposure estimation in six population groups for all the involved food manufacturing processes

3.1.3.1

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties of the dietary exposure estimate in the standard six population groups for all the involved food manufacturing processes have been considered and are summarised in Table 3.

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.

Uncertainty analysis of the dietary exposure estimation in infants aged 0–3 months for the production of modified fats and oils by interesterification

3.1.3.2

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties of the dietary exposure estimate in infants aged 0–3 months for the production of modified fats and oils by interesterification used in infant formula have been considered and are summarised in Table 4.

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.

Margin of exposure

3.2

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1960 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure of at least 22,791 (EFSA FEZ Panel, 2024).

For the proposed uses, a comparison of the NOAEL with the exposure estimates of 0.001–0.054 mg TOS/kg bw per day at the mean and from 0.002 to 0.090 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 21,778 in six population groups.

For the production of modified fats and oils by interesterification used in infant formulae, a comparison of the NOAEL with the exposure estimate of 0.33 mg TOS/kg bw per day resulted in a margin of exposure of at least 5939 in infants aged 0–3 months.

CONCLUSION

4

Based on the revised margin of exposure and the previous evaluation, the Panel concluded that the food enzyme triacylglycerol lipase produced with the non‐genetically modified Rhizopus arrhizus strain AE‐TL(B) does not give rise to safety concerns under the revised intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for an extension of use of the food enzyme triacylglycerol lipase from a non‐genetically modified Rhizopus arrhizus strain AE‐TL(B). September 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 UnionFEZEFSA Panel on Food EnzymesIUBMBInternational Union of Biochemistry and Molecular BiologyNOAELno observed adverse effect levelRMraw materialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00664

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, Henk Van Loveren, Laurence Vernis and Holger Zorn.

Supporting information

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