Safety evaluation of the food enzyme phospholipase D from the non‐genetically modified Streptomyces netropsis strain DSM 40093

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified bacteria for use in modifying phospholipids from lecithin.

Contribution

The novelty lies in the safety assessment of phospholipase D from Streptomyces netropsis for food use.

Findings

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

No homology was found between the enzyme and known allergens.

The enzyme is considered safe under intended use conditions.

Abstract

The food enzyme phospholipase D (phosphatidylcholine phosphatidohydrolase; EC 3.1.4.4) is produced with the non‐genetically modified microorganism Streptomyces netropsis strain DSM 40093 by Chemi S.p.A. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in the production of modified phospholipids from lecithin. Since residual amounts of total organic solids of the food enzyme are removed by repeated washing steps, dietary exposure was not calculated and the toxicity testing was considered unnecessary. A search for the homology of the amino acid sequence of the phospholipase D 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 preparation.

Parameters	Unit	Batches
		1	2	3
Phospholipase D activity	U/g a	3550	4105	3583
Protein	%	2.3	2.2	2.1
Ash	%	74.6	76.1	71.0
Water	%	2.3	1.8	6.1
Total organic solids (TOS) b	%	23.1	22.1	22.9
Activity/ TOS ratio	U/mg TOS	15.4	18.6	15.6

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 25

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM)
Production of modified phospholipids a
Phosphatidylserine (PS)	Lecithins	800–1600
N‐acyl phosphatidyl‐ethanolamine (NAPE)	Lecithins	3000–6000
Phosphatidic acid (PA)	Lecithins	950–1900

TABLE 3. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model assumptions and factors
Exclusion of one process from the exposure estimation: – Production of modified phospholipids	–

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides a 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 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. Five applications have been introduced by the companies “Intertek Scientific & Regulatory Consultancy” for the authorisation of the food enzymes Catalase from Aspergillus niger (strain CTS 2093), Glucose oxidase from Penicillium chrysogenum (strain PGO 19‐162), Tannase from Aspergillus oryzae (strain TAN 206) and Glucoamylase from Rhyzopus oryzae (strain CU634‐1775), and “RDA Scientific Consultants GmbH” for the authorisation of the food enzyme Phospholipase D from Streptomyces netropsis (DSZM No. 40093). Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Catalase from Aspergillus niger (strain CTS 2093), Glucose oxidase from Penicillium chrysogenum (strain PGO 19‐162), Tannase from Aspergillus oryzae (strain TAN 206), Glucoamylase from Rhyzopus oryzae (strain CU634‐1775) and Phospholipase D from Streptomyces netropsis (DSZM No. 40093) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme Phospholipase D from Streptomyces netropsis (DSZM No. 40093).

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Phospholipase D from Streptomyces netropsis strain DSM 40093.

Additional information requested from the applicant during the assessment process on 22 September 2022 and 15 December 2023 was received on 30 September 2023 and 13 September 2024, respectively (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 22 September 2022, the applicant requested a clarification teleconference on 20 December 2022, after which the applicant provided additional data on 30 September 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, 2009a) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009b) as well as the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) have been followed for the evaluation of the application. Additional information was requested in accordance with the updated ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the guidance on the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

ASSESSMENT

3

IUBMB nomenclaturePhospholipase DSystematic namePhosphatidylcholine phosphatidohydrolaseSynonymsLipophosphodiesterase II; lecithinase D; choline phosphataseIUBMB NoEC 3.1.4.4CAS No9001‐87‐0EINECS No232‐639‐8

Phospholipases D catalyse the hydrolysis of the phosphodiester linkage in phosphatidylcholine, resulting in the release of phosphatidic acid and choline. In the presence of serine, they also catalyse the transphosphatidation from phosphatidylcholine to phosphatidylserine.

The food enzyme under assessment is intended to be used in the production of modified phospholipids from lecithins.

Source of the food enzyme

3.1

The phospholipase D is produced with the non‐genetically modified bacterium Streptomyces netropsis strain DSM 40093, which is deposited at the German Collection of Microorganisms and Cell Cultures (DSMZ, Germany).4 The strain was isolated from soil.5

The production strain was identified as S. netropsis by whole genome sequence (WGS) analysis showing an ANI of 99.6% with the type strain ■■■■■6 The WGS of the production strain was screened for the presence of antimicrobial resistance genes against two regularly updated databases, and no genes of concern were found. No virulence genes or toxin‐encoding genes were identified in the genome of the production strain.

Production of the food enzyme

3.2

The food enzyme is manufactured by a third party who supplies a liquid product to the applicant. The applicant then formulates the food enzyme to produce a dried preparation.

All stages of the manufacturing process are done according to the Food Hygiene Regulation (EC) No 852/2004,7 with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with Good Manufacturing Practice.8

The production strain is grown as a pure culture using a typical industrial medium in a ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the cells of the production strain are ■■■■■ treated and separated from the fermentation broth by filtration. The filtrate containing the enzyme is 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. The enzyme is precipitated with ■■■■■ in the presence of ■■■■■. The solids are recovered by centrifugation, dried and then granulated to produce the food enzyme preparation.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 phospholipase D is a single polypeptide chain of ■■■■■ amino acids.11 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.12 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A consistent protein pattern was observed across all batches. The gels showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme.13

No other enzymatic activities were reported.

The applicant's in‐house determination of phospholipase D activity is based on the hydrolysis of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (reaction conditions: ■■■■■). The release of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphoric acid (DOPA) was measured chromatographically.14 The enzyme activity is expressed in (U)/g. One U is defined as the amount of DOPA (1 μmol) formed in 1 h.15

The food enzyme has a temperature optimum around 60°C (■■■■■) and a pH optimum around pH 5 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 180 min at different temperatures. The food enzyme retained 75% of its activity at 80°C, the maximum temperature tested.16

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches intended for commercialisation (Table 1).17 The mean total organic solids (TOS) were 22.7% and the mean enzyme activity/TOS ratio was 16.5 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 1 mg/kg, which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). In addition, arsenic, cadmium and mercury contents were below the limits of detection (LoDs) of the employed methods.18 ^,^ 19

The food enzyme preparation 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).20 No antimicrobial activity was detected in any of the tested batches.21

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

Viable cells 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. The analysis was based on the capacity of Streptomyces to form characteristic spheres visible by eye when grown in liquid culture. ■■■■■ ■■■■■ No sphere was observed in the test samples. A positive control was used. The absence of mycelia in the test samples was also confirmed by phase contrast microscopy.22

Toxicological data

3.4

No toxicological tests were provided by the applicant. The Panel considered that toxicological tests are unnecessary because the food enzyme is intended to be used in a food manufacturing process with negligible exposure (see Section 3.5).

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 phospholipase D produced with the Streptomyces netropsis strain DSM 40093 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 Allermatch and AllergenOnline databases.23

No reports on oral or respiratory sensitisation or elicitation reactions of the phospholipase D under assessment have been published. In addition, no allergic reactions upon dietary exposure to any phospholipase D have been reported in the literature.24

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

■■■■■ and ■■■■■, known sources of allergens, are present in the culture medium. During the fermentation process, these products 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 negligible exposure to the food enzyme (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 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 the production of modified phospholipids from lecithins at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 25

In the production of phosphatidylserine (PS) and N‐acyl phosphatidyl‐ethanolamine (NAPE), the food enzyme is added to fatty acid‐free lecithin together with ■■■■■ or ■■■■■, respectively, and other reagents.26 After the synthesis of PS, PA and NAPE, the downstream processing includes repeated washing steps with different solvents that are capable to remove the phospholipase D from the finished phospholipid products, which are finally precipitated with ■■■■■ or ■■■■■.27

In the production of phosphatidic acid (PA), the food enzyme is added to an aqueous buffer together with ■■■■■. The synthesised PA is washed ■■■■■ with water and dried.28

Using the enzymatic activity as the proxy, the applicant made a theoretical calculation of the enzyme removal efficiency by the downstream processes: 85% or 99.98% of enzymatic activity could be removed from the PS in the downstream process without or with the initial washing step, respectively. Washing with water could remove 99.94% of the enzymatic activity in the PA.29 To establish the extent of this removal, the applicant measured the amount of food enzyme present in the modified phospholipids with a silver‐stained SDS‐PAGE analysis, showing a removal higher than 99% for the three modified phospholipids.30 The experimental data confirmed the theoretical calculations. The Panel considered these data sufficient to confirm that > 99% of the food enzyme‐TOS is removed during the production of modified phospholipids (PS, NAPE and PA).

All three types of modified phospholipids are used as ingredients in food supplements or in fortified foods.31

Dietary exposure estimation

3.5.2

As the Panel accepted the evidence provided as sufficient to conclude that the residual amounts of food enzyme‐TOS in the modified phospholipids are negligible, a dietary exposure was not calculated.

Uncertainty analysis

3.5.3

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 exclusion of one food manufacturing process from the exposure estimation was based on > 99% of TOS removal.

Margin of exposure

3.6

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

CONCLUSIONS

4

Based on the data provided and evidence of the removal of TOS during the production of modified phospholipids, the Panel concluded that the food enzyme Phospholipase D produced with the non‐genetically modified Streptomyces netropsis strain DSM 40093 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Phospholipase D from Streptomyces netropsis (DSZM No. 40093). September 2016. Submitted by Chemi S.P.A.

Additional information. September 2023. Submitted by Chemi S.P.A.

Additional information. September 2024. Submitted by Chemi S.P.A.

ABBREVIATIONSCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonSDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsWGSwhole genome sequencingWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2016‐00536

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, Holger Zorn

NOTE

The full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality will be received from the European Commission.