Safety evaluation of the food enzyme triacylglycerol lipase from the genetically modified Komagatella phaffii strain LALL‐LI2

TL;DR

This paper evaluates the safety of a genetically modified enzyme used in food processing and concludes it is safe under intended use.

Contribution

The study provides a safety evaluation of a triacylglycerol lipase enzyme from a genetically modified yeast strain.

Findings

The enzyme is free from viable cells and DNA of the production organism.

Exposure estimates in European populations are below safety thresholds.

No homology to known allergens was found, though a low risk of allergic reactions cannot be excluded.

Abstract

The food enzyme triacylglycerol lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) is produced with the genetically modified Komagatella phaffii strain LALL‐LI2 by Danstar Ferment AG. The production strain met the requirements for the qualified presumption of safety (QPS). The food enzyme was considered free from viable cells of the production organism and its DNA. It is intended to be used in the processing of cereals and other grains for the production of baked products. Dietary exposure was estimated to be up to 0.071 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concerns resulting from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the triacylglycerol lipase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3
Triacylglycerol lipase activity	LBLU/g a	5795	6664	13,454
Protein	%	1.1	1.2	2.2
Ash	%	0.6	1.1	1.8
Water	%	97.2	96.7	94.5
Total organic solids (TOS) b	%	2.2	2.2	3.7
Activity/TOS ratio	LBLU /mg TOS	263.4	302.9	363.6

TABLE 2. Intended use and recommended use level of the food enzyme as provided by the applicant. 27

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b , c
Processing of cereals and other grains
Production of baked products	Flour	3.2–6.4

TABLE 3. Summary of the estimated dietary exposure to 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)	0.000–0.015 (12)	0.001–0.036 (15)	0.000–0.036 (19)	0.000–0.018 (21)	0.005–0.012 (22)	0.006–0.011 (23)
Min–max 95th percentile (number of surveys)	0.000–0.043 (11)	0.003–0.067 (14)	0.002–0.071 (19)	0.001–0.036 (20)	0.012–0.031 (22)	0.012–0.021 (22)

TABLE 4. 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	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

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 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 EU 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.

On 18 January 2024, a new application was introduced by the applicant “DANSTAR FERMENT AG” for the authorisation of the food enzyme Triacylglycerol lipase from a genetically modified Komagataella phaffii (strain LALL‐LI2).

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: Triacylglycerol lipase from a genetically modified Komagataella phaffii (strain LALL‐LI2), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes, and food flavourings.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme triacylglycerol lipase from Komagataella phaffii strain LALL‐LI2.

Additional information was requested to the applicant during the assessment process on 18 October 2024 and received on 17 December 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 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, 2023) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20023 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 4 to 25 December 2024.4 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.

The food enzyme under assessment is intended to be used in the processing of cereals and other grains for the production of baked products as defined in the EFSA guidance (EFSA CEP Panel, 2023).

Source of the food enzyme

3.1

The enzyme is produced with the genetically modified yeast Komagatella phaffii strain LALL‐LI2, which is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany) with deposition number DSM 34898.5 The production strain was identified as K. phaffii by sequence analysis of the D1/D2 region of the large ribosomal subunit coding sequence, obtained from whole genome sequence (WGS) data.

The species K. phaffii is included in the list of organisms for which the qualified presumption of safety (QPS) approach may be applied for production purposes only (implying the absence of viable cells of the production organisms in the final product; EFSA, 2007; EFSA BIOHAZ Panel, 2022).6 In addition, the genetic modifications should not give rise to safety concerns. The genetic modifications are considered safe (see Section 3.1.4) and the product is free from viable cells (see Section 3.3.4). Therefore, the production strain meets the requirements to qualify for the QPS approach and is considered safe.

Characteristics of the parental microorganisms

3.1.1

The parental strain, which is also the recipient, is the ■■■■■ strain K. phaffii ■■■■■.

Characteristics of introduced sequences

3.1.2

The sequence encoding the triacylglycerol lipase derives from the ascomycetous fungus Fusarium oxysporum. It was preceded by ■■■■■ which replaced the original signal peptide of the triacylglycerol lipase. This construction was placed under the control of the promoter and terminator sequences of the ■■■■■ genes, respectively.

■■■■■.

Description of the genetic modification

3.1.3

The purpose of the genetic modification was to enable the production strain to synthesise a triacylglycerol lipase from Fusarium oxysporum.

For this, ■■■■■

The presence and the location of the insertion cassettes and the deletion of the ■■■■■ genes were confirmed by WGS analysis. WGS analysis further confirmed the absence of off‐target genetic modifications.7

Safety aspects of the genetic modification

3.1.4

The technical dossier contains all necessary information on the recipient microorganism, the donor organism and the genetic modification process.

The production strain K. phaffii LALL‐LI2 differs from the recipient strain in its ability to produce triacylglycerol lipase from Fusarium oxysporum ■■■■■. No plasmid vectors or antimicrobial resistance (AMR) genes were used during the genetic modification. A search of the WGS in two databases confirmed the absence of AMR genes in the genome of the production strain.

No issues of concern arising from the genetic modifications were identified by the Panel.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,8 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current good manufacturing practice.

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch or fed‐batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by centrifugation or filtration. The supernatant or filtrate containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.9 The applicant provided information on the identity of the substances used to control the fermentation and in the subsequent downstream processing of the food enzyme.10

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The triacylglycerol lipase is a single polypeptide chain of 331 amino acids.11 The molecular mass of the mature protein, calculated from the amino acid sequence, is 35 kDa.12 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.13 A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about 25 kDa. The discrepancy between apparent and expected molecular mass was attributed by the applicant to the presence of a site‐specific protease of the production organism that truncates the protein to a size of approximately 25 kDa. No other enzyme activities were reported.

The applicant's in‐house determination of triacylglycerol lipase activity is based on the hydrolysis of tributyrin (reaction conditions: pH 7, 21°C, 5 min). The released butyric acid is quantified by titration with sodium hydroxide. The enzyme activity is expressed in Lallemand Baking Lipase Units (LBLU)/g. One LBLU is defined as the quantity of enzyme that releases 1 μmol of butyric acid per min under the reaction conditions.14

To determine the pH and temperature optimum and the thermostability profile of the enzyme, a different activity assay was used substituting tributyrin by DGGR (1,2‐di‐O‐lauryl‐rac‐glycero‐3‐glutaric acid 6‐methylresorufin ester) as a substrate. The food enzyme has a temperature optimum around 33–37°C (pH 5, 10 min) and a pH optimum around pH 5–7 (30°C, 10 min).^9^ Thermostability was tested after pre‐incubation of the food enzyme for 15 min at different temperatures. The enzyme activity decreased from 30°C, showing no residual activity above 60°C.15

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three pilot‐scale batches (Table 1).16 The mean total organic solids (TOS) of the three batches was 2.7% and the mean enzyme activity/TOS ratio was 310 LBLU/mg TOS.

Purity

3.3.3

The lead content in the three pilot batches was below 0.1 mg/kg,17 ^,^ 18 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

The food enzyme complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).19 No antimicrobial activity was detected in any of the tested batches.20

The Panel considered that the information provided on the purity of the food enzyme was sufficient.

Viable cells and DNA of the production strain

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. Ten millilitres of a 1/10 dilution was centrifuged and the pellets were suspended in the residual supernatant, plated onto selective agar plates and incubated at 30°C for 2 days.21 No colonies were produced. A positive control was included.22

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction analysis of three batches in triplicate. No DNA was detected with primers that would amplify a 926‐bp fragment that is specific for the triacylglycerol lipase encoding gene,23 with a limit of detection of 1 ng spiked DNA/g food enzyme.24

Toxicological data

3.4

As the production strain qualifies for the QPS approach of safety assessment and no issues of concern arising from the production process of the food enzyme were identified (see Sections 3.1, 3.2 and 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

Allergenicity

3.4.1

The allergenicity assessment considered only the food enzyme and not additives, carriers, or other excipients that may be used in the final formulation.

The potential allergenicity of the triacylglycerol lipase produced with the Komagataella phaffi strain LALL‐LI2 was assessed by comparing its amino acid sequence with those of known allergens as described in the EFSA GMO Scientific Opinion (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found in the AllergenOnline database.25

No reports on oral or respiratory sensitisation or elicitation reactions of the triacylglycerol lipase under assessment have been published.26

Respiratory allergy following occupational exposure to triacylglycerol lipase has been reported (Brant et al., 2004). Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Adverse reactions upon dietary exposure to triacylglycerol lipases in individuals sensitised through the respiratory route have not been reported.

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the triacylglycerol lipase under assessment.

Yeast extract, a known source of allergens, is present in the culture medium. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

The Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in one food manufacturing process at the recommended use level summarised in Table 2.

TABLE 2: Intended use and recommended use level of the food enzyme as provided by the applicant. 27

In the production of baked products, the food enzyme is added to flour during dough making.28 The triacylglycerol lipase hydrolyses fats and oils in flour to achieve emulsifying effects and enhance dough development. The food enzyme‐TOS remains in the baked products.

Based on data provided on thermostability (see Section 3.3.1), the Panel considered that the food enzyme is inactivated during the production of baked products.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme‐TOS was calculated using the FEIM webtool29 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved the selection of relevant food categories and the application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 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 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 0.071 mg TOS/kg body weight (bw) per day in children at the 95th percentile.

Uncertainty analysis

3.6

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 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

3.7

Since no toxicological assessment was considered necessary by the Panel, a margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme triacylglycerol lipase produced with the genetically modified Komagatella phaffii strain LALL‐LI2 does not give rise to safety concerns under the intended conditions of use.

The Panel considered the food enzyme free from viable cells of the production organism and recombinant DNA.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the authorisation of triacylglycerol lipase from a genetically modified Komagataella phaffii strain LALL‐LI2. April 2024. Submitted by DANSTAR FERMENT AG.

Additional information. December 2024. Submitted by DANSTAR FERMENT AG.ABBREVIATIONSAMRAntimicrobial resistanceCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food EnzymesGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonMOEmargin of exposurePCRpolymerase chain reactionQPSqualified presumption of safetyTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00201

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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 LM Solano, Monika Sramkova, Henk Van Loveren, Laurence Vernis, and Holger Zorn.

LEGAL NOTICE

The scientific output published implements EFSA's decision on the confidentiality requests submitted on specific items. As certain items have been awarded confidential status by EFSA, they are consequently withheld from public disclosure by redaction.

Supporting information

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