Safety evaluation of the food enzyme lysozyme from hens' eggs

TL;DR

This study evaluates the safety of lysozyme from hens' eggs used as a food enzyme and concludes it is safe under intended conditions, though it may cause allergic reactions in egg-allergic individuals.

Contribution

The novelty lies in the safety assessment of lysozyme from hens' eggs as a food enzyme under specific usage conditions.

Findings

The dietary exposure to lysozyme is up to 6.943 mg/kg body weight per day in European populations.

Exposure levels are comparable to egg intake for most age groups but lower for children.

Lysozyme is an allergen and may cause allergic reactions in egg-allergic individuals.

Abstract

The food enzyme lysozyme (peptidoglycan N‐acetylmuramoylhydrolase; EC 3.2.1.17) is produced from hens' eggs by E.P.S. S.P.A. Egg Powder Specialists. No issue of concern was identified from the food enzyme manufacturing process. It is intended to be used in three food manufacturing processes. The dietary exposure was estimated to be up to 6.943 mg total organic solids/kg body weight per day in European populations. This exposure is lower than the intake of the corresponding fraction from eggs for infants, toddlers and children, and is comparable for adolescents, adults and the elderly. Lysozyme from egg is an allergen. The Panel considered that there is a risk of allergic reactions upon dietary exposure to this food enzyme for egg allergic individuals. Based on the data provided, the origin of the food enzyme and an exposure to the food enzyme comparable to the intake from eggs, the Panel concluded that the food enzyme lysozyme does not give rise to safety concerns, under the intended conditions of use.

Tables

TABLE 1. Composition of the food enzyme in liquid form.

Parameters	Unit	Batches
		7	8	9
Lysozyme activity	FIP U/g a	9,300,000	9,200,000	9,300,000
Protein	%	20.9	20.7	20.9
Ash	%	0.1	0.1	0.1
Water	%	78.2	78.3	78.4
Total organic solids (TOS) b	%	21.7	21.6	21.5
Activity/TOS ratio	FIP U/mg TOS	42,857	42,593	43,256

TABLE 2. Composition of the food enzyme in spray‐dried (granular and powder) form.

Parameters	Unit	Batches (granular form)			Batches (powder form)
		1	2	3	4	5	6
Lysozyme activity	FIP U/g a	42,431,000	42,862,000	42,487,000	41,853,000	42,302,000	42,276,000
Protein	%	96.5	97.1	99.2	99.0	97.3	95.5
Ash	%	0.3	0.3	0.3	0.3	0.3	0.1
Water	%	4.2	3.4	4.4	2.9	2.8	3.0
Total organic solids (TOS) b	%	95.5	96.3	95.3	96.8	96.9	96.9
Activity/TOS ratio	FIP U/mg TOS	44,430	44,509	44,582	43,237	43,655	43,628

TABLE 3. Intended uses and recommended use levels 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
Processing of dairy products
Production of cheese	Milk	24–39
Processing of cereals and other grains
Production of brewed products c	Cereals	230–1380
	Beer	48–289
Processing of fruits and vegetables
Production of wine and wine vinegar d	Grapes	34–337
	Wine	48–482

TABLE 4. 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	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.022–0.272 (14)	0.086–0.418 (17)	0.069–0.236 (21)	0.043–0.301 (23)	0.283–1.821 (23)	0.131–2.000 (25)
Min–max 95th percentile (number of surveys)	0.106–0.881 (13)	0.209–1.256 (16)	0.186–0.733 (21)	0.116–1.377 (22)	1.405–6.943 (23)	0.774–5.368 (24)

TABLE 5. Summary of estimated dietary exposure to the SMT‐Equivalent, resulting from the consumption of egg white in six population groups.

Population group	Estimated exposure (mg/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)	0.036–1.019 (14)	0.511–3.515 (17)	0.950–2.500 (21)	0.427–1.390 (23)	0.301–0.902 (23)	0.281–1.017 (25)
Min–max 95th percentile (number of surveys)	0–6.653 (13)	2.636–11.463 (16)	2.634–6.954 (21)	1.387–4.233 (22)	1.198–3.127 (23)	1.115–2.566 (24)

TABLE 6. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
	Exposure to FE‐TOS	Exposure to SMT‐Equivalent
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	+	NA
For the production of wine and wine vinegar, the applicant excluded wine vinegar as a product to be treated by this food enzyme. 39 However, for the calculation, EFSA used the default list of food groups for this process, which includes wine and wine vinegar	+	NA
For the production of brewed products, the applicant excluded pasteurised and filtrated beer as a product to be treated by this food enzyme. 40 However, for the calculation, EFSA used the default list of food groups for this process, which includes all types of beer	+	NA
Whenever two or more use levels are applied to different raw materials for the same food manufacturing process, the higher use level was chosen to be used in the calculation	+	NA
Use of recipe fractions to disaggregate FoodEx categories	+/−	+/−
Use of technical factors in the exposure model	+/−	+/−
Selection of FoodEx categories likely to contain egg for the intake assessment of SMT‐Equivalent, based on the national food descriptors	NA	+/−
The applied food enzyme yield factor was the mean value	NA	+/−

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 15 March 2024, a new application was introduced by the applicant “E.P.S. S.P.A. Egg Powder Specialists” for the authorisation of the food enzyme Lysozyme from albumen of hens' eggs.

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: Lysozyme from albumen of hens' eggs, in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings.3

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme lysozyme from hen's egg.

Additional information, requested from the applicant during the assessment process on 24 March 2025, was received on 3 April 2025 (see ‘ Documentation as 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 178/20024 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 28 March to 18 April 2025.5 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureLysozymeSystematic namePeptidoglycan N‐acetylmuramoyl hydrolaseSynonymsMuramidase, mucopeptide N‐acetylmuramoylhydrolase, N‐acetylmuramide glycanohydrolaseIUBMB NoEC 3.2.1.17CAS No12650‐88‐3EINECS No235‐747‐3

Lysozymes catalyse the hydrolysis of β‐(1,4)‐linkages between N‐acetylmuramic acid and N‐acetyl‐d‐glucosamine residues in peptidoglycans of bacterial cell walls.

The food enzyme under assessment is intended to be used in three food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): 1) processing of dairy products for the production of cheese; 2) processing of cereals and other grains for the production of brewed products; 3) processing of fruits and vegetables for the production of wine and wine vinegar.

Source of the food enzyme

3.1

The lysozyme is extracted from the eggs of hens (Gallus gallus domesticus).6 These eggs fulfil all requirements of European legislation covering the trade of eggs and egg products intended for human consumption.7

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 Good Manufacturing Practice.9

The egg white is passed through a cation exchange column to preferably bind lysozyme. The column is then washed with water to remove albumin fractions. The lysozyme is then eluted from the column with a sodium chloride solution, purified, concentrated by ultrafiltration and precipitated by pH adjustment. The enzyme recovered by centrifugation is mixed with hydrochloric acid, further purified, concentrated as lysozyme hydrochloride, filtrated and pasteurised. The lysozyme may be used in a liquid form or spray‐dried for use in a granular or powder form.10 The applicant provided full information on the identity of all substances used in the manufacturing process of the food enzyme.11 Around ■■■■■ of liquid hens' egg white is needed to produce 1 kg of dry lysozyme,12 corresponding to a yield factor of ■■■■■.

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 lysozyme is a single polypeptide chain of 129 amino acids.13 The molecular mass of the mature protein, calculated from the amino acid sequence, is 14.3 kDa (Cagielska‐Radziejewska et al., 2008; Canfield, 1963).14

The applicant's in‐house determination of lysozyme activity is based on the reduction in optical density of a Micrococcus luteus ■■■■■ suspension (reaction conditions: pH 7.0, 25°C, ■■■■■) measured spectrophotometrically at 450 nm. The enzyme activity is expressed in Fédération Internationale Pharmaceutique Units (FIP U)/g. One FIP U is defined as the amount of lysozyme that causes a decrease in absorbance of 0.001 per minute under the assay conditions.15

The food enzyme has a temperature optimum around 40°C ■■■■■ and a pH optimum around pH 7.5 ■■■■■. Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures up to 85°C (pH 7.0). Enzyme activity decreased above 60°C, showing 16.6% of residual activity at 85°C.16

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for nine batches intended for commercialisation: three in liquid form (Table 1), three in granular form and three in powder form (Table 2).17 The mean total organic solids (TOS) of the three batches in liquid, granular and powder form were 21.6%, 95.7% and 96.9%, respectively. The mean enzyme activity/TOS ratio of the three batches in liquid, granular and powder form were 42,902 FIP U/mg TOS, 44,507 FIP U/mg TOS and 43,507 FIP U/mg TOS, respectively.

The applicant produces the food enzyme in a way to meet the specifications for lysozyme authorised as a food additive (E 1105) under Regulation (EU) No 231/2012.18 The specifications require a minimum lysozyme hydrochloride content of 950 mg/g on a dry matter basis. The analytical data provided demonstrated compliance with the specifications.

Purity

3.3.3

The lead content in the nine commercial batches was below 0.125 mg/kg,19 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 and mercury concentrations were below the limits of quantification (LoQs) of the employed methods.20 ^,^ 21

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).22 Furthermore, the presence of Campylobacter sp. was not detected in 10 g of three food enzyme batches.23 Moulds and yeasts were present in the nine batches in concentrations below 100 cfu/g.24

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

Toxicological data

3.4

According to the Commission Implementing Regulation (EU) No 562/2012,25 an application for the safety evaluation of a food enzyme does not need to include toxicological data if the food enzyme is obtained from edible parts of an animal intended or reasonably expected to be ingested by humans.

According to the EFSA ‘Scientific Guidance for the submission of dossiers on Food Enzymes’, for food enzymes derived from plants and animals that are consumed by the European population, two criteria must be met: (i) no hazard is introduced through the manufacturing process and (ii) when it can be demonstrated that the dietary exposure to the food enzyme–TOS is within the same order of magnitude as the dietary intake of the fraction of the plant or animal material comparable to the food enzyme–TOS (EFSA CEP Panel, 2021).

The Panel considered that these requirements are fulfilled, because:

• the safety of lysozyme has already been evaluated by JECFA (1992) and it is authorised as a food additive in the EU;
• the manufacturing process of the food enzyme does not introduce substances that raise safety concerns;
• the compositional and purity data provided were considered sufficient;
• eggs, the source of lysozyme, are extensively consumed throughout the EU and elsewhere in the world;
• the dietary exposure to the food enzyme–TOS under the intended conditions of use was within the same magnitude compared to the intake from eggs (see Section 3.5).

The Panel considered that sufficient information has been provided on the animal source, its history of consumption as well as the manufacturing process. Therefore, the need for toxicological data is waived.

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.

Egg allergy is one of the most frequent food allergies; egg is listed in the Regulation (EU) No 1169/2011.26 Lysozyme (Gal d 4) is a component of egg white, which contributes to the clinical response in egg allergic individuals (Bianchi, 1982; Hoffman, 1983; JECFA, 1992). Allergic reactions in food prepared with lysozyme, for example in cheese and wine, have been demonstrated (Kirschner et al., 2009; Marseglia et al., 2013).

In conclusion, the Panel considered that under the intended conditions of use, there is a risk of allergic reactions upon dietary exposure to this food enzyme for egg allergic individuals.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in three food manufacturing processes at the recommended use levels summarised in Table 3.

TABLE 3: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 27

In the production of cheese, the food enzyme is added to milk during coagulation.30 It is used to prevent a phenomenon referred to as ‘late blowing’, which is caused mainly by the growth of Clostridium tyrobutyricum.31 The food enzyme–TOS remain in the cheeses.

In the production of brewed products, the food enzyme may be used to inhibit the spoilage by lactic acid bacteria.32 ^,^ 33 The food enzyme–TOS remain in the brewed products.

In the production of wine, the food enzyme may be added to grapes before crushing or after alcoholic fermentation. It may also be added to wines during filtration and bottling.34 It is used to control the malolactic fermentation.35 This could reduce the need for high levels of sulfur dioxide. The food enzyme–TOS remain in the wine.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing steps applied in the respective food manufacturing processes, the Panel considered that this lysozyme may remain in its active form in all the food manufacturing processes listed in Table 3, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

Following the EFSA Guidance Document on food enzymes (EFSA CEP Panel, 2021), a comparison was made between the chronic exposures:

• dietary exposure to the food enzyme–TOS, resulting from the intended uses as proposed by the applicant (herein referred as ‘FE‐TOS’) and
• dietary exposure to a fraction in hens' egg comparable to the food enzyme–TOS resulting from the consumption of eggs or egg derived foods (herein referred to as source material–TOS equivalent, ‘SMT‐Equivalent’).

In both cases, chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool36 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).

Estimated dietary exposure to the food enzyme–TOS

3.5.2.1

Table 4 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 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 6.943 mg TOS/kg bw per day in adults at the 95th percentile.

Estimated dietary exposure to the SMT‐Equivalent

3.5.2.2

The chronic dietary exposure to the SMT‐Equivalent was calculated in three steps: firstly, the intake of hens' egg from all dietary sources was calculated by applying recipe and conversion fractions reported in Appendix C. Secondly, this intake of egg was converted to the intake of egg white, using a factor of 60% to account for the amount of egg white in an egg.37 Thirdly, the intake of egg white was converted into a fraction comparable to the food enzyme–TOS, by applying a yield factor (■■■■■)38 provided by the applicant to take into account the yield of the food enzyme–TOS from egg (Section 3.2).

Table 5 provides an overview of the estimated exposure to the SMT‐Equivalent. Detailed mean and 95th percentile exposure to the SMT‐Equivalent 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 3 and 4. 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 11.463 mg/kg bw per day in toddlers at the 95th percentile.

Comparison of the two exposure estimates

3.5.2.3

The intake of the SMT‐Equivalent (Table 5) is one order of magnitude greater than the dietary exposure to the food enzyme–TOS (Table 4) for infants, toddlers and children, and is comparable for adolescents, adults and the elderly.

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 6.

Both estimates are derived using the same food consumption data and exposure model and, hence, share a number of uncertainties, which do not have an effect on the comparison of the two estimates.

Margin of exposure

3.6

A margin of exposure was not calculated, because the Panel considered toxicological tests unnecessary. This was because (i) the food enzyme is obtained from hen's egg, a food consumed by the European population; (ii) the dietary exposure to the food enzyme–TOS is within the same magnitude or below the dietary intake of the comparable fraction of egg white; (iii) no hazard is introduced through the food enzyme manufacturing process.

CONCLUSIONS

4

Based on the data provided, the origin of the food enzyme being egg white and a dietary exposure to the food enzyme comparable to its source in regular diet, the Panel concluded that the food enzyme lysozyme does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorization of lysozyme from hens' egg. March 2024. Submitted by E.P.S. S.P.A. Egg Powder Specialists.

Additional information. April 2025. Submitted by E.P.S. S.P.A. Egg Powder Specialists.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsFEfood enzymeFEZEFSA Panel on Food EnzymesIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLoQlimit of quantificationSMTsource material‐TOSTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2024‐00309

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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.

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 and to the SMT‐Equivalent in details