Safety evaluation of the food enzyme protein‐glutamine glutaminase from the non‐genetically modified Chryseobacterium proteolyticum strain AE‐PG

Abstract

The food enzyme protein‐glutamine glutaminase (protein‐L‐glutamine amidohydrolase; EC 3.5.1.44) is produced with the non‐genetically modified Chryseobacterium proteolyticum strain AE‐PG by Amano Enzyme Inc. The food enzyme was considered free from viable cells and DNA of the production organism. It is intended to be used in three food manufacturing processes. Dietary exposure was estimated to be up to 0.089 mg total organic solids (TOS)/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90‐day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 93 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 1045. A search for the homology of the amino acid sequence of the protein‐glutamine glutaminase to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure 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.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ 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 1332/2008¹ 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 1331/2008 ² 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.

1.1. Background and Terms of Reference as provided by the requestor

1.1.1. Background as provided by the European Commission

Only food enzymes included in the European Union (EU) Community 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 1332/2008¹ on food enzymes.

Five applications have been introduced by the company ‘Amano Enzyme Inc.’ for the authorisation of the food enzymes Beta‐amylase from Bacillus flexus (strain AE‐BAF), Triacylglycerol lipase from Mucor javanicus (strain AE‐LM), Beta‐glucanase from Cellulosimicrobium cellulans (strain AE‐TN), Laccase from Trametes hirusta (strain AE‐OR) and Protein‐glutaminase from Chryseobacterium proteolyticum (strain AE‐PG).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008,² 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.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Beta‐amylase from Bacillus flexus (strain AE‐BAF), Triacylglycerol lipase from Mucor javanicus (strain AE‐LM), Beta‐glucanase from Cellulosimicrobium cellulans (strain AE‐TN), Laccase from Trametes hirusta (strain AE‐OR) and Protein‐glutaminase from Chryseobacterium proteolyticum (strain AE‐PG) in accordance with Article 17.3 of Regulation (EC) No 1332/2008¹ on food enzymes.

1.2. Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme protein‐glutamine glutaminase from the non‐genetically modified C. proteolyticum strain AE‐PG.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme protein‐glutamine glutaminase from the non‐genetically modified C. proteolyticum (strain AE‐PG).

Additional information, requested from the applicant during the assessment phase on 27 March 2023 and 10 February 2025, were received on 7 November 2024 and 28 August 2025 (see ‘Documentation provided to EFSA’).

Following the request for additional data sent by EFSA on 27 March 2023, the applicant requested a clarification teleconference on 17 November 2023, after which the applicant provided additional data on 7 November 2024.

2.2. Methodologies

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 ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA CEF Panel, 2009) 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).

3. ASSESSMENT ⁴

IUBMB nomenclature	Protein‐glutamine glutaminase
Systematic name	Protein‐L‐glutamine amidohydrolase
Synonyms	Peptidoglutaminase II; glutaminyl‐peptide glutaminase; peptidylglutaminase II
IUBMB No	EC 3.5.1.44
CAS No	62213‐11‐0
EINECS No	669‐835‐1

Protein‐glutamine glutaminases catalyse the hydrolysis of protein‐bound glutaminyl residues to glutamyl residues, releasing ammonia.

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): processing of dairy products for the production of (1) cheese and (2) fermented dairy products; (3) processing of plant‐ and fungal‐derived products for the production of plant‐based analogues of milk and milk products.

3.1. Source of the food enzyme ⁵

The protein‐glutamine glutaminase is produced with the non‐genetically modified bacterium C. proteolyticum strain AE‐PG, which is deposited at the Biological Resource Center, National Institute of Technology and Evaluation (NBRC, Japan), with the deposition number ■■■■■. ⁶

C. proteolyticum is a proposed new Chryseobacterium species for which the type strain is ■■■■■ (Yamaguchi & Yokoe, 2000). A phylogenetic analysis based on the ■■■■■ genes was provided and showed that the production strain clustered together with the type strain ■■■■■ and the reference strain ■■■■■. In addition, the production strain showed an average nucleotide identity (ANI) of ■■■■■% to the reference strain ■■■■■. Phylogenomic analysis based on ■■■■■ further clustered the production strain with the reference strain ■■■■■. Even though C. proteolyticum is not yet a valid name with standing in prokaryotic nomenclature, the production strain is considered to belong to this species in this assessment.

The production strain was derived from a soil isolate by ■■■■■.

The genome of the production strain was screened for the presence of AMR genes and one was found. Because only one genome of C. proteolyticum is available in databases, the intrinsic nature of this gene could not be firmly established. Nevertheless, a potential risk associated with the presence of this gene can be disregarded because the absence of DNA and viable cells of the production organism in the food enzyme is demonstrated (see Section 3.3.4).

3.2. Production of the food enzyme

The food enzyme is manufactured 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 submerged batch fermentation system ⁹ with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration. The 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. ¹⁰ 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. ¹¹

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.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The protein‐glutamine glutaminase is a single polypeptide chain of ■■■■■ amino acids. ¹² The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹³ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. ¹⁴ A consistent protein pattern was observed across all batches. The gel showed a single protein band migrating between the ■■■■■ and ■■■■■ kDa markers, ¹⁵ consistent with the expected molecular mass of the enzyme.

No other enzyme activities were reported. ¹⁶

The applicant's in‐house determination of protein‐glutamine glutaminase activity is based on hydrolysis of ■■■■■ (reaction conditions: pH ■■■■■, ■■■■■°C, ■■■■■ min). The release of ammonia is measured spectrophotometrically. The enzyme activity is expressed in Units (U)/g. One U is defined as the amount of enzyme that releases 1 μmoL of ammonia per minute under the conditions of the assay. ¹⁷

The food enzyme has a temperature optimum around 60°C (pH ■■■■■) and a pH optimum between pH 5.0 and 7.0 (■■■■■°C). ¹⁸ Thermostability was tested by pre‐incubation of the food enzyme for 10 and 60 min at different temperatures (pH ■■■■■). The food enzyme activity decreased above 50°C, showing no residual activity at 70°C after 60 min and at 80°C after 10 min of incubation. ¹⁹

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation and two batches produced for the toxicological tests (Table 1). ²⁰ The mean total organic solids (TOS) of the three batches intended for commercialisation was 1.7% and the mean enzyme activity/TOS ratio was 65.3 U/mg TOS.

TABLE 1.

Composition of the food enzyme. ²¹

Parameters	Unit	Batches
		1	2	3	4 a	5 b
Protein‐glutamine glutaminase activity	U/mL c	758	629	683	3390	549
Protein	%	0.6	0.6	0.6	11.8	NA e
Ash	%	< 0.1	0.2	0.2	< 0.1	0.1
Water	%	96.3	99.1	99.0	86.5	99.0
Total organic solids (TOS) d	%	3.7	0.7	0.8	13.5	0.9
Activity/TOS ratio	U/mg TOS	20.5	89.9	85.4	25.1	61.0

^a Batch used for the genotoxicity studies.

^b Batch used for the repeated dose 90‐day oral toxicity study in rats.

^c U: Unit (see Section 3.3.1).

^d TOS calculated as 100% – % water – % ash.

^e NA: not analysed.

3.3.3. Purity ²²

The lead content in all batches was below 5 mg/kg ^{23 , 24} 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). ²⁵ No antimicrobial activity was detected in any of the tested batches. ²⁶

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

3.3.4. Viable cells and DNA of the production strain ²⁷

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■. No colonies from the production strain were detected. A positive control was included. ²⁸

The absence of recombinant DNA in the food enzyme was demonstrated by polymerase chain reaction, in three batches analysed in triplicate. No DNA was detected with primers that would amplify ■■■■■, with a limit of detection of 10 ng spiked DNA/g food enzyme. ²⁹

3.4. Toxicological data ³⁰

A battery of toxicological tests including a bacterial reverse mutation test (Ames test), an in vitro mammalian cell micronucleus test and a repeated dose 90‐day oral toxicity study in rats has been provided.

The batches 4 and 5 (Table 1) used in these studies have similar activity/TOS ratio to the commercial batches and thus are considered suitable as test items.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation test (Ames test) was performed according to the Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 2020) and following Good Laboratory Practice (GLP). ³¹ Four strains of S. Typhimurium (TA98, TA100, TA1535 and TA1537) and E. coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the pre‐incubation method.

Based on the results from the range‐finding experiment, two main experiments were carried out in triplicate, using five concentrations of the food enzyme of 313, 625, 1250, 2500 and 5000 μg TOS/plate.

No cytotoxicity was observed at any concentration of the test substance. Upon treatment with the food enzyme, there was no biologically relevant increase in the number of revertant colonies above the control values in any strain tested, with or without S9‐mix.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme protein‐glutamine glutaminase did not induce gene mutations under the test conditions applied in this study.

3.4.1.2. In vitro mammalian cell micronucleus test

The in vitro mammalian cell micronucleus test was carried out according to the OECD Test Guideline 487 (OECD, 2023) and following GLP. ³² A range‐finding test and a main experiment were performed with duplicate cultures of human lymphoblastoid TK6 cells. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In a range‐finding test, cells were exposed to the food enzyme at ten concentrations from 9.77 to 5000 μg TOS/mL in a short‐term treatment (4‐h exposure and 20‐h recovery period) either with or without S9‐mix and in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix. Precipitates in the culture medium were observed at ≥ 625 μg TOS/mL in all treatment conditions. The 50% cell‐growth inhibition concentration (IC₅₀) was 388 μg TOS/mL in the short‐term treatment without S9‐mix, 524 μg TOS/mL in the short‐term treatment with S9‐mix and 385 μg TOS/mL in the continuous treatment without S9‐mix.

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for the frequency of mononucleated micronucleated cells at concentrations of 225, 300, 375 and 450 μg TOS/mL in a short‐term treatment without S9‐mix, at concentrations of 450, 525 and 600 μg TOS/mL in a short‐term treatment with S9‐mix and at concentrations of 150, 225, 300 and 375 μg TOS/mL in a long‐term treatment without S9‐mix.

Precipitates in the culture medium were observed at ≥525 μg TOS/mL. The cell‐growth inhibition rate at the highest concentrations scored was 59%, 52% and 53% in the short‐term treatment without S9‐mix and with S9‐mix and in the continuous treatment without S9‐mix, respectively. The frequency of mononucleated micronucleated cells was not statistically significantly different to the negative controls at all concentrations tested.

The study was considered reliable without restrictions and the results of high relevance.

The Panel concluded that the food enzyme protein‐glutamine glutaminase did not induce an increase in the frequency of mononucleated micronucleated cells under the test conditions applied in this study.

3.4.2. Repeated dose 90‐day oral toxicity study in rodents ³³

The repeated dose 90‐day oral toxicity study followed Japanese guidelines (Notification No. 29 and No. 655, Ministry of Health and Welfare (MHW), 1996, 1999) and Japanese GLP (Notification No. 424, MHW, 1997). ³⁴ The study was in accordance with OECD Test Guideline 408 (OECD, 1998) with the following deviations: sensory activity examination, functional observations (motor activity and grip strength) and a histopathological examination of medulla/pons and spinal cord at three levels: cervical, midthoracic and lumbar (only thoracic level was examined in this study) were not performed. The Panel considered that these deviations are minor and do not impact the evaluation of the study.

Groups of 12 male and 12 female Sprague–Dawley (Crl:CD(SD)) IGS SPF rats received the food enzyme by gavage in doses of 23, 47 and 93 mg TOS/kg bw per day. ³⁵ Controls received the vehicle (water for injection).

No mortality was observed.

Haematological investigation revealed a statistically significant decrease in mean corpuscular volume (MCV) in mid‐dose males (−3%), a decrease in fibrinogen (Fg) in high‐dose males (−34%) and in high‐dose females (−25%), an increase in haemoglobin in high‐dose females (+4%), an increase in prothrombin time (PT) in high‐dose females (+6%), an increase in the percentage of lymphocytes in mid‐ and high‐dose males (+7%, +8%) and a decrease in the percentage of segmented neutrophils in high‐dose males (−31%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (except for Fg), there was no dose–response relationship (MCV), the changes were small (% of lymphocytes), there were no changes in other relevant parameters (other clotting parameters, red and white blood cell counts), there were no histopathological changes in the relevant organs and the changes were within the historical control values (all parameters, except for Fg in males and PT in females). ³⁶

Clinical chemistry investigation revealed a statistically significant increase in total cholesterol (TChol) in mid‐ and high‐dose males (+28%, +21%), an increase in phospholipids (PL) in mid‐ and high‐dose males (+21%, +19%), a decrease in lactate dehydrogenase (LDH) in high‐dose females (−27%), a decrease in chloride concentration (Cl) in low‐ and mid‐dose males (−2% both), a decrease in total protein (TP) (−6%) and in albumin (Alb) (−6%) in low‐dose females, an increase in glucose (Glc) in mid‐dose males (+11%) and an increase in alkaline phosphatase (ALP) activity in mid‐dose females (+45%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there was no dose–response relationship (TChol, PL, Cl, TP, Alb, Glc, ALP), the changes were small (LDH, TP, ALP), there were no changes in other relevant parameters (globulin, A/G ratio, other liver enzymes, other electrolytes), there were no histopathological changes in the liver and kidneys and the changes were within the historical control values (all parameters). ³⁷

The urinalysis revealed a statistically significant increase in the 1‐day excretion of sodium (Na) (+42%) and Cl (+44%) in high‐dose females, and an increase in the incidence of occult blood in the urine in high‐dose males (7/12 vs. 3/12 in the control). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all parameters), there were no histopathological changes in the kidneys and the changes were within the historical control values (1‐day excretion of Na and Cl). ³⁸

Statistically significant changes detected in organ weights were an increase in absolute thymus weight (+33%) and in relative liver weight (+7%) in mid‐dose males. The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), there was no dose–response relationship (both parameters), there were no histopathological changes in these organs and the changes were within the historical control values (both parameters). ³⁹

The microscopic examination revealed minimal (6/12, 7/12, 9/12 vs. 5/12 in the control) to mild (0/12, 1/12, 1/12 vs. 1/12 in the control) focal myocarditis in low‐, mid‐ and high‐dose males and minimal focal myocarditis in low‐, mid‐ and high‐dose females (1/12, 2/12, 3/12 vs. 1/12 in the control). The Panel interpreted the diagnostic term used by the applicant as referring to the focal infiltrates of inflammatory cells, based on their presence in the control animals and their focal distribution. Focal infiltrates of inflammatory cells are commonly seen in the hearts of rodents and may be associated with naturally occurring rodent progressive cardiomyopathy (PCM) or xenobiotic‐induced disease. Rodent PCM is a spontaneous, chronic and progressive myocardial condition that affects many strains of rats and mice with varying incidence and severity and with higher prevalence in male rodents than females (Berridge et al., 2016; Chanut et al., 2013). The Panel considered that, based on the dose‐related increased incidence, a test item relationship could not be ruled out. Nevertheless, the Panel regarded the cardiac finding as not adverse based on the focal distribution and minimal to mild severity.

Moreover, minimal (6/12 vs. 5/12 in the control) to mild (3/12 vs. 1/12 in the control) interstitial cell infiltration in prostate was observed in high‐dose males. Inflammatory changes of the secondary sex organs with varying incidence and severity are a common background finding in rats and mice (Creasy et al., 2012). The Panel considered the prostatic findings observed in high‐dose males as not toxicologically relevant based on the small increase in incidence and similar grade of severity compared to controls.

No other statistically significant or toxicologically relevant differences from controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 93 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity ⁴⁰

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 protein‐glutamine glutaminase from the C. proteolyticum strain AE‐PG 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 Allergen Database for Food Safety. ⁴¹

No reports on oral or respiratory sensitisation or elicitation reactions of the protein‐glutamine glutaminase under assessment have been published. ⁴² In addition, no reactions upon dietary exposure to any protein‐glutamine glutaminase have been reported in the literature.

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 protein‐glutamine glutaminase under assessment.

■■■■■ and ■■■■■, products from soy and fish that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ⁴³ ) are used as raw materials. In addition, ■■■■■ and ■■■■■, known sources of allergens, are also 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 due to the raw materials 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 conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in three food manufacturing processes 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. ⁴⁴

Food manufacturing process	Raw material (RM)	Recommended use level (mg TOS/kg RM) a
Processing of dairy products
Production of cheese 45	Milk	0.037–0.37
Production of fermented dairy products 46	Milk	0.037–0.37
Processing of plant‐ and fungal‐derived products
Production of plant‐based analogues of milk and milk products 47	Cereals, legumes, oilseeds, nuts etc.	13.1–131

Abbreviation: TOS, total organic solids.

^a The numbers in bold were used for calculation.

In all the food manufacturing processes, the use of this food enzyme enhances the sensory properties of the final foods. ⁴⁸

In the production of cheese, the food enzyme is added to milk during the formation of the curd. ⁴⁹ The food enzyme–TOS remain in the cheeses.

In the production of fermented dairy products, the food enzyme could be added during the incubation or before the fermentation steps. ⁵⁰ The food enzyme–TOS remain in the fermented dairy products.

In the production of plant‐based analogues of milk and milk products, the food enzyme could be added to a slurry of plant materials during the incubation step in combination with other enzymes. ⁵¹ The food enzyme–TOS remain in the final foods.

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 the food enzyme is inactivated during the production of plant‐based analogues of milk and milk products but may remain in its active form in cheeses and in fermented dairy products, depending on the processing conditions.

3.5.2. Dietary exposure estimation

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool ⁵² 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).

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 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 0.089 mg TOS/kg bw per day in toddlers at the 95th percentile.

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)	0.001–0.011 (12)	0.003–0.046 (15)	0.002–0.014 (19)	0.001–0.004 (21)	0.001–0.004 (22)	0.001–0.004 (23)
Min–max 95th percentile (number of surveys)	0.003–0.079 (11)	0.007–0.089 (14)	0.004–0.069 (19)	0.002–0.006 (20)	0.002–0.030 (22)	0.002–0.029 (22)

3.5.3. Uncertainty analysis

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.

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	+/−

Note: +: uncertainty with potential to cause overestimation of exposure. –: uncertainty with potential to cause underestimation of exposure.

Abbreviation: TOS, total organic solids.

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.

3.6. Margin of exposure

A comparison of the NOAEL (93 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.001–0.046 mg TOS/kg bw per day at the mean and from 0.002 to 0.089 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 1045.

4. CONCLUSION

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme protein‐glutamine glutaminase produced with the non‐genetically modified C. proteolyticum strain AE‐PG does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Technical dossier `Application for authorisation of Protein‐glutaminase from Chryseobacterium proteolyticum AE‐PG in accordance with Regulation (EC) No 1331/2008′. 26 February 2015. Submitted by Amano Enzyme Inc.

Additional information. 7 November 2024. Submitted by Amano Enzyme Inc.

Additional information. 28 August 2025. Submitted by Amano Enzyme Inc.

ABBREVIATIONS

A/G

albumin/globulin ratio

Alb

albumin

ALP

alkaline phosphatase

AMR

antimicrobial resistance

ANI

average nucleotide identity

■■■■■

■■■■■

bw

body weight

CAS

Chemical Abstracts Service

Cl

chloride

DNA

deoxyribonucleic acid

EFSA CEF Panel

EFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing Aids

EFSA CEP Panel

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EFSA FEZ Panel

EFSA Panel on Food Enzymes

EFSA GMO Panel

EFSA Panel on Genetically Modified Organisms

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

FEIM

Food Enzyme Intake Model

Fg

fibrinogen

FoodEx

standardised food classification and description system

G

globulin

Glc

glucose

GLP

Good Laboratory Practice

GM

genetically modified

GMO

genetically modified organism

IC₅₀

50% cell‐growth inhibition concentration

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LDH

lactate dehydrogenase

LoQ

limit of quantification

MCV

mean corpuscular volume

MHW

Ministry of Health and Welfare

NA

not analysed

Na

sodium

NBRC

Biological Resource Center, National Institute of Technology and Evaluation

NOAEL

no observed adverse effect level

non‐GM

non‐genetically modified

OECD

Organisation for Economic Co‐operation and Development

PCM

progressive cardiomyopathy

PL

phospholipids

PT

prothrombin time

rDNA

ribosomal deoxyribonucleic acid

RM

raw material

SPF

specific pathogen free

TChol

total cholesterol

TK6

human lymphoblastoid cells

TOS

total organic solids

TP

total protein

U

unit

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2015‐00695

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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.

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.

Supporting information

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

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

Appendix A can be found in the online version of this output (in the ‘Supporting Information’ section). The file contains two sheets, corresponding to two tables.

Table 1: Average and 95th percentile exposure to the food enzyme–TOS per age class, country and survey.

Table 2: Contribution of food categories to the dietary exposure to the food enzyme–TOS per age class, country and survey.

APPENDIX B. Population groups considered for the exposure assessment

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, the Netherlands, Portugal, Republic of North Macedonia,* Serbia,* Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, the Netherlands, Portugal, Republic of North Macedonia,* Serbia,* Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina,* Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* the Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Bosnia and Herzegovina,* Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* the Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* the Netherlands, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden

^*Consumption data from these pre‐accession countries are not reported in Table 3 of this opinion, however, they are included in Appendix B for testing purpose.

^a The terms ‘children’ and ‘the elderly’ correspond, respectively, to ‘other children’ and the merge of ‘elderly’ and ‘very elderly’ in the Guidance of EFSA on the ‘Use of the EFSA Comprehensive European Food Consumption Database in Exposure Assessment’ (EFSA, 2011).

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Van Loveren, H. , Vernis, L. , Kovalkovičová, N. , Andryszkiewicz, M. , Cavanna, D. , Lunardi, S. , & Liu, Y. (2025). Safety evaluation of the food enzyme protein‐glutamine glutaminase from the non‐genetically modified Chryseobacterium proteolyticum strain AE‐PG . EFSA Journal, 23(12), e9775. 10.2903/j.efsa.2025.9775