Safety evaluation of a food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase activities from the non‐genetically modified Aspergillus niger strain CCTCC M 2023236

Abstract

The food enzyme containing endo‐polygalacturonase (1,4‐α‐d‐galacturonan glycanohydrolase; EC 3.2.1.15), pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) and pectin lyase (1,4‐6‐O‐methyl‐α‐d‐galacturonan lyase; EC 4.2.2.10) activities is produced with the non‐genetically modified Aspergillus niger strain CCTCC M 2023236 by Suntaq International Limited. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in six food manufacturing processes. Dietary exposure was estimated to be up to 1.221 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 2000 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 1638. A search for the homology of the three amino acid sequences to known allergens was made and matches with 14 respiratory allergens were found. The Panel considered that under the intended conditions of use, a risk of allergic reactions upon dietary exposure cannot be excluded. 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 1332/2008 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 1331/2008 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 Association of Manufacturers and Formulators of Enzyme Products (AMFEP), and by the companies “DSM Food Specialties B.V” and “Novozymes A/S” for the authorisation of the food enzymes Pectinase, Poly‐galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A₂ from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo‐1,4‐β‐xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM‐SO), respectively.

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008 ⁴ , the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contains 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 safety assessments on the food enzymes Pectinase, Poly‐galacturonase, Pectin esterase, Pectin lyase and Arabanase from Aspergillus niger, Phospholipase A2 from a genetically modified strain of Aspergillus niger (strain PLA), Pectinesterase from a genetically modified strain of Aspergillus niger (strain PME), Endo‐1,4‐B‐xylanase from a genetically modified strain of Aspergillus niger (strain XEA) and Maltogenic amylase produced by a genetically modified strain of Bacillus subtilis (strain NZYM‐SO) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 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 enzymes Pectinase, Polygalacturonase, Pectin esterase, Pectin lyase and Arabanase from A. niger submitted by AMFEP.

The application was submitted initially as a joint dossier ⁵ and identified as the EFSA‐Q‐2015‐00042. During a meeting between EFSA, the European Commission and AMFEP, ⁶ it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00227 and concerns the food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase activities produced with the A. niger strain CCTCC M 2023236 and submitted by Suntaq International Limited.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the pectinase complex containing three claimed activities produced by a non‐genetically modified A. niger CCTCC M 2023236.

Additional information, requested from the applicant during the assessment process on 9 December 2024, was received on 29 April 2025 (see ‘Documentation provided to EFSA’).

Following the reception of additional data, EFSA requested a clarification teleconference on 23 February 2026, after which the applicant provided Spontaneous information on 25 February 2026 (see ‘Documentation provided to EFSA’).

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, 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).

3. ASSESSMENT

The food enzyme under application contains three declared activities:

IUBMB nomenclature	Endo‐polygalacturonase
Systematic name	1,4‐α‐d‐galacturonan glycanohydrolase
Synonyms	Pectinase; pectin hydrolase; endo‐d‐galacturonase
IUBMB No	EC 3.2.1.15
CAS No	9032‐75‐1
EINECS No	232–885‐6

Endo‐polygalacturonases catalyse the random hydrolysis of α‐1,4 glycosidic bonds between galacturonic acid residues in polygalacturonans, resulting in their progressive depolymerisation.

IUBMB nomenclature	Pectinesterase
Systematic name	Pectin pectylhydrolase
Synonyms	Pectin methylesterase; pectin demethoxylase
IUBMB No	EC 3.1.1.11
CAS No	9025‐98‐3
EINECS No	232–807‐0

Pectinesterases catalyse the de‐esterification of pectin, resulting in the generation of pectic acid and methanol.

IUBMB nomenclature	Pectin lyase
Systematic name	1,4‐6‐O‐methyl‐α‐d‐galacturonan lyase
Synonyms	Pectin trans‐eliminase; polymethylgalacturonic transeliminase; pectin methyltranseliminase
IUBMB No	4.2.2.10
CAS No	9033‐35‐6
EINECS No	232‐894‐5

Pectin lyases catalyse the β‐eliminative cleavage of 1,4‐α‐d‐galactosiduronic linkages in galacturonans, resulting in the generation of oligosaccharides with 4‐deoxy‐6‐O‐methyl‐α‐d‐galact‐4‐enuronosyl groups at their non‐reducing ends.

The food enzyme under assessment is intended to be used in six food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of fruits and vegetables for the production of (1) juices, (2) fruit and vegetable products other than juices, (3) wine and wine vinegar and (4) alcoholic beverages other than grape wine; processing of plant‐ and fungal‐derived products for the production of (5) tea and other herbal and fruit infusions and (6) plant extracts.

3.1. Source of the food enzyme

The food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase activities is produced with the non‐genetically modified filamentous fungus A. niger strain CCTCC M 2023236 (AN‐SHPC‐108), which is deposited at the China Centre for Type Culture Collection (CCTCCC, China) with deposition number ■■■■■. ⁷ The production strain was identified as A. niger by whole genome sequencing analysis showing an average nucleotide identity value of >99% with the reference genome of ■■■■■. ⁸ The production strain A. niger CCTCC M 2023236 was derived from the parental strain ■■■■■. ⁹

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ¹⁰ with food safety procedures based on Hazard Analysis and Critical Control Points ¹¹ and in accordance with Good Manufacturing Practice. ¹²

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 solid biomass is removed from the fermentation broth by filtration. The filtrate containing the enzyme is then further purified and concentrated, including a filtration step in which the enzyme protein is retained, while most of the low molecular mass material passes the membrane and is discarded. ¹³ 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. ¹⁴

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 endo‐polygalacturonase has ■■■■■ amino acids ¹⁵ and the molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. ¹⁶ The pectinesterase has ■■■■■ amino acids ¹⁷ and a calculated molecular mass of ■■■■■ kDa. ¹⁸ The pectin lyase has ■■■■■ amino acids ¹⁹ and a calculated molecular mass of ■■■■■ kDa. ²⁰ The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. ²¹ A consistent protein pattern was observed across all batches. The gel showed bands migrating between the marker proteins of ■■■■■ and ■■■■■ kDa in all batches, consistent with the expected masses of the enzymes. ²² Arabinofuranosidase activity was reported by the applicant. No other enzyme activities were reported.

The determination of endo‐polygalacturonase activity is based on the ■■■■■ (reaction conditions ■■■■■). The enzyme activity is determined by measuring the release of ■■■■■ spectrophotometrically and expressed in Units (U)/g or (U)/mL. One U is defined as the amount of enzyme that produces 1 mg of galacturonic acid equivalent in 1 h under the conditions of the assay. ²³

The determination of pectinesterase activity is based on the hydrolysis of ester bonds of pectin (reaction conditions pH 3.5, 50°C). The enzyme activity is determined by titration of the free carboxylic acid groups formed during the reaction and expressed in Units (U)/g or (U)/mL. One U is defined as the amount of enzyme that releases 1 μmol of acid per min under the assay conditions. ²⁴

The determination of pectin lyase activity is based on ■■■■■ (reaction conditions ■■■■■). ■■■■■ measured spectrophotometrically at ■■■■■ The enzyme activity is expressed in Units (U)/g or (U)/mL. One U is defined as the amount of enzyme that releases 1 μmol of galacturonic acid equivalent with a double bond at its non‐reducing end per minute under the assay conditions. ²⁵

The endo‐polygalacturonase has a temperature optimum around 50°C (■■■■■) and a pH optimum around 3.5 (■■■■■). ²⁶ The pectin lyase and pectinesterase have temperature optima around 60°C (■■■■■) and pH optima around 4.5 (■■■■■). ²⁷

Thermostability was tested by pre‐incubation of the food enzyme for 15 min at different temperatures. The enzyme activities decreased above 40°C showing no residual activities at 70°C. ²⁸

3.3.2. Chemical parameters

Data were provided for six batches of food enzyme intended for commercialisation: three in dry form (batch 1–3) and three in liquid form (batch 4–6). Batch 3 was used for the toxicological tests (Table 1). ²⁹ The mean total organic solids (TOS) of the three dried food enzyme batches was 92.2% and that of the three liquid batches was 16.7%. The mean enzyme activity/TOS ratio was 395.1 U/mg TOS (endo‐polygalacturonase), 27 U/mg TOS (pectin lyase) and 16.2 U/mg TOS (pectinesterase) for the dried batches; and 585 U/mg TOS (endo‐polygalacturonase), 4.1 U/mg TOS (pectin lyase) and 24.6 U/mg TOS (pectinesterase) for the liquid batches.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		Dry form			Liquid form
		1	2	3 a	4	5	6
Endo‐polygalacturonase activity	U/g b	320,736	322,418	450,366	97,518	98,705	96,796
Pectin lyase activity	U/g c	2275	2308	3294	685	680	712
Pectinesterase activity	U/g d	13,086	12,978	18,605	4088	4235	4003
Protein	%	43.7	43.7	57.4	9.96	9.93	9.82
Ash	%	2.0	2.0	2.1	0.4	0.4	0.5
Water	%	5.9	5.7	5.6	82.9	82.7	83.0
Total organic solids (TOS) e	%	92.1	92.3	92.3	16.7	16.9	16.5
Endo‐polygalacturonase activity/mg TOS ratio	U/mg TOS	348.2	349.3	487.9	583.9	584.5	586.5
Pectin lyase activity/mg TOS ratio	U/mg TOS	2.5	2.5	3.6	4.1	4.0	4.3
Pectinesterase activity/mg TOS ratio	U/mg TOS	14.2	14.1	20.2	24.5	25.1	24.3

^a Batch used for the toxicological studies.

^b Unit: U/g (see Section 3.3.1).

^c Unit: U/g (see Section 3.3.1).

^d Unit: U/g (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the six batches was below 0.2 mg/kg ³⁰ 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, the mercury content was below the limit of quantification (LoQ) of the employed method. For arsenic and cadmium, the average concentrations determined in the six batches were 0.54 mg/kg and 0.26 mg/kg, respectively. ^{31 , 32} The Panel considered these concentrations as not of concern.

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). ³³ In addition, all batches were examined for a presence of moulds and yeasts and none were detected. ³⁴ No antimicrobial activity was detected in any of the tested batches. ³⁵

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). Secondary metabolites may also originate from the raw materials used in the fermentation process. The presence of aflatoxins (B1, B2, G1 and G2), deoxynivalenol (DON), fumonisins (B1 and B2), ochratoxin A, sterigmatocystin, T2‐toxin, HT2‐toxin and zearalenone was examined in all food enzyme batches and was below the LoQs of the applied analytical methods, except for DON. Using the highest estimated dietary exposure of 1.221 mg TOS/kg bw per day as the reference (see Section 3.5.2), European consumers could be exposed to DON up to 0.106 ng/kg bw per day. As this estimate is below the tolerable daily intake (TDI) for DON (1 μg/kg bw per day, EFSA CONTAM Panel, 2017), the Panel considered this concentration as of no concern. ^{36 , 37} Since the production strain is not able to produce DON, the occurrence of DON in the food enzyme is likely to have originated from a contamination of the raw material. Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

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

3.3.4. Viable cells of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated in six independent batches analysed in triplicate. One gram of product was incubated in non‐selective agar medium at 30°C for 10 days. From this, colonies with morphology similar to the positive control were inoculated on differential agar plates and further incubated for 3 days. No colonies of the production strain were detected. ³⁸

3.4. Toxicological data

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 batch 3 (Table 1) used in these studies is a batch intended for commercialisation and has similar enzyme activity/TOS ratio as the other batches, and thus is considered suitable as a test item.

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). ³⁹ Five strains of S. Typhimurium (TA98, TA100, TA102, TA1535 and TA1537) were used with or without metabolic activation (S9‐mix). A preliminary test and two experiments were carried out in duplicate or triplicate respectively, applying the pre‐incubation and standard plate incorporation methods.

Based on the results from the preliminary test, the main experiments were carried out using five concentrations of the food enzyme of 50, 158, 500, 1582 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 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 OECD Test Guideline 487 (OECD, 2016) and following GLP. ⁴⁰ A dose range finding test and a main experiment were performed with duplicate cultures of Chinese hamster ovary cell line. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

Based on the results of the range finding test, in the main experiment, cells were exposed to the food enzyme and scored for the frequency of bi‐nucleated cells with micronuclei (MNBN) at concentrations of 1250, 2500 and 5000 μg TOS/mL in a short‐term treatment (4‐h exposure and 20‐h recovery period) either with or without S9‐mix, or in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

In the long‐term treatment without S9‐mix, cytotoxicity of 24% (based on cytokinesis‐block proliferation index) was observed at 5000 μg TOS/mL. The frequency of MNBN was not statistically significantly different to the negative controls at any concentration tested.

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

The Panel concluded that the food enzyme did not induce an increase in the frequency of MNBN under the test conditions applied in this study.

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

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018) ⁴¹ with the following deviation: urea was not measured. The Panel considered that this deviation is minor and does not impact the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley (Crl:CD(SD)) rats received the food enzyme by gavage in doses of 500, 1000 or 2000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (ultrapure water).

No mortality was observed.

Feed consumption was statistically significantly increased in week 13 of administration in mid‐dose females (+34%). The Panel considered the change as not toxicologically relevant, as it was only recorded at a single time interval, it was only observed in one sex, there was no dose–response relationship and there was no statistically significant change in the body weight and/or the body weight gain.

In the functional observations, a statistically significant increase in mean number of rearing was observed in high‐dose males (+56%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex.

Haematological investigations revealed a statistically significant decrease in white blood cell count (WBC) in mid‐dose males (−27%), a decrease in absolute lymphocyte counts (Lymph) in mid‐ and high‐dose males (−31% and −24%), and a decrease in absolute neutrophil (Neu) and monocyte (Mon) counts in high‐dose females (−55% and −46%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (all), there was no dose–response relationship (WBC and Lymph), there were no changes in other relevant parameters (other white blood cell parameters) and there were no histopathological changes in lymphohematopoietic organs or tissues.

Clinical chemistry investigations revealed a statistically significant increase in creatinine (Crea) in low‐ and mid‐dose females (+30% and +33%), an increase in triglycerides (TGL) in high‐dose males (+75%), an increase in glucose (Glc) in high‐dose females (+19%), a decrease in aspartate aminotransferase (AST) in mid‐dose males (−21%) and an increase in high‐dose females (+27%), a decrease in calcium concentration (Ca) in mid‐ and high‐dose males and in high‐dose females (−6%, −5% and −6%) and an increase in globulin (Glo) in low‐dose males (+8%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (Crea, TGL, Glc, Glob), there was no consistency between changes in males and females (AST), there was no dose–response relationship (Crea, AST in males, Ca in males, Glo), the change was small (AST), there were no changes in other relevant parameters (clinical markers of kidney or liver damage) and there were no histopathological changes in liver or kidney.

Statistically significant changes in hormone levels included an increase in triiodothyronine (T3) levels in high‐dose females (+180%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there were no changes in other relevant parameters (thyrotropin and thyroxine) and there were no histopathological changes in the thyroid or pituitary glands.

Statistically significant changes detected in absolute organ weights were a decrease in adrenal gland in mid‐ and high‐dose males (−21% and −20%), a decrease in brain and testes in high‐dose males (−5% and −9%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (adrenal gland, brain), the changes were small (brain and testes) and there were no histopathological changes in these organs.

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

The Panel identified a no observed adverse effect level (NOAEL) of 2000 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

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 food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase produced with A. niger strain CCTCC M 2023236 was assessed by comparing their amino acid sequences 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, matches with 12 respiratory allergens for endo‐polygalacturonase, two respiratory allergens for pectinesterase and no match for pectin lyase were found in the AllergenOnline database. ⁴²

The matches (36.3%–43.2% sequence identity) were allergenic polygalacturonases and pectin methylesterases from pollen of different species: olive tree (Olea europaea), Johnson grass (Sorghum halepense), Timothy grass (Phleum pratense), maize (Zea mays), Mountain cedar (Juniper ashei), Bahia grass (Paspalum notatum), London and oriental plane tree (Platanus acerifolia and P. orientalis), Japanese cedar (Cryptomeria japonica), Japanese cypress (Chamaecyparis obtusa), Easter lily (Lilium longiflorum) and saltwort (Salsola kali), all known as respiratory allergens.

Polygalacturonases are relevant allergens in tree, grass and weed pollen. The pollen food allergy syndrome, particularly in cedar and juniper pollen‐allergic patients, was noted upon consumption of e.g. banana and tomato (Bonds et al., 2019; Poncet et al., 2020). A tomato polygalacturonase was identified as cross‐reactive food allergen (Ghiani et al., 2016). Notably, no match of this tomato allergen with the food enzyme under assessment was found. The pollen food allergy syndrome is typically restricted to the buccal cavity and rarely leads to severe allergy symptoms (Sarkar et al., 2018).

No reports on oral or respiratory sensitisation or elicitation reactions of the food enzyme under assessment have been published.

The Panel considered that the results of the sequence homology search and the available literature indicate a risk of allergic reactions for pollen‐allergic individuals upon dietary exposure to the food enzyme under assessment.

The production strain belongs to the Aspergillus genus, which is known to cause respiratory allergy (Kurup et al., 2000; Shen & Han, 1998; Vermani et al., 2015). Allergic reactions upon dietary exposure have been observed, but are rare (Xing et al., 2022). The biomass is removed during the production process; however, allergenic proteins of the production strain can be released into the culture medium from which the food enzyme is obtained.

■■■■■ 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.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins are present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5), 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, particularly in pollen‐allergic individuals, cannot be excluded.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in six 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. ⁴³

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	14.1
Production of fruit and vegetable products other than juices	Fruit and vegetables	56.3
Production of wine and wine vinegar	Grapes	13.1
Production of alcoholic beverages other than grape wine	Fruit	14.1
Processing of plant‐ and fungal‐derived products
Production of tea and other herbal and fruit infusions	Tea leaves and other plants	187.6
Production of plant extracts	Plant materials	187.6

^a The name has been harmonised by EFSA in accordance with the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023).

^b The numbers in bold were used for calculation.

The food enzyme is used to treat plant materials to degrade pectin. The disruption of the structural polymers reduces viscosity, thus improving the processability and increasing the yield of the plant products. This also facilitates the release of colour and flavouring compounds.

In the production of juices, the food enzyme may be added to fruit and vegetables before maceration or to the raw juices and during depectinisation. ⁴⁴ The food enzyme–TOS remain in the juices.

In the production of fruit and vegetable products other than juices, the food enzyme is added to the crushed pulp. ⁴⁵ The food enzyme–TOS remain in the final foods.

In the production of wine and wine vinegar, the food enzyme may be added during crushing. It can also be added during maceration, fermentation and clarification. ⁴⁶ The food enzyme–TOS remain in wine and wine vinegars.

In the production of alcoholic beverages other than grape wine, the food enzyme may be added to fruits such as apple and pear after milling or to the fruit must before fermentation. ⁴⁷ The food enzyme–TOS remain in the final alcoholic beverages.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to the mash of plant material (tea leaves, ginger, herbs). ⁴⁸ The food enzyme–TOS remain in the final products.

In the production of plant extracts, the food enzyme is added to plant materials (e.g. ginger, ginseng). ⁴⁹ The food enzyme–TOS remain in the plant extracts.

Based on data provided on thermostability (see Section 3.3.1) and the downstream processing within the respective food manufacturing processes, the Panel considered that the food enzyme is inactivated during the production of fruit and vegetable products other than juices. However, it may remain in its active form in the other food manufacturing processes listed in Table 2, depending on the heat treatment conditions.

3.5.2. Dietary exposure estimation

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool ⁵⁰ 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 1.221 mg TOS/kg bw per day in toddlers at the 95th percentile.

TABLE 3.

Summary of the estimated dietary exposure to the 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)	0.030–0.684 (12)	0.104–0.538 (15)	0.085–0.316 (19)	0.022–0.190 (21)	0.023–0.140 (22)	0.017–0.110 (23)
Min–max 95th percentile (number of surveys)	0.132–0.978 (11)	0.361–1.221 (14)	0.256–0.758 (19)	0.084–0.539 (20)	0.082–0.402 (22)	0.076–0.307 (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
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/−
Use of technical factors in the exposure model	+/−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

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 (2000 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.017–0.684 mg TOS/kg bw per day at the mean and from 0.076 to 1.221 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 1638.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase activities produced with the non‐genetically modified A. niger strain CCTCC M 2023236 does not give rise to safety concerns under the intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Request for the authorisation of pectinase produced by Aspergillus niger CCTCC M 2023236 for use as a food processing aid from Suntaq International Limited, EC Mandate M 2015–0016. January 2015, updated March 2023. Submitted by Suntaq International Limited.

Spontaneous submission. February 2026. Submitted Suntaq International Limited.

ABBREVIATIONS

AST

aspartate aminotransferase

bw

body weight

Ca

calcium concentration

CAS

Chemical Abstracts Service

CCTCCC

China Centre for Type Culture Collection, China

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

Crea

creatinine

DON

deoxynivalenol

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

Glc

glucose

Glo

globulin

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kiloDalton

LOD

limit of detection

LoQ

limit of quantification

MNBN

bi‐nucleated cells with micronuclei

MOE

margin of exposure

Mon

monocyte

Neu

absolute neutrophil

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Co‐operation and Development

SDS‐PAGE

sodium dodecyl sulfate‐polyacrylamide gel electrophoresis

T3

triiodothyronine

TDI

tolerable daily intake

TGL

triglycerides

TOS

total organic solids

WBC

white blood cell count

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00227

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 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.

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*, 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. , Van Loveren, H. , Vernis, L. , Criado, A. , Cavanna, D. , Andryszkiewicz, M. , Gomes, A. , & Liu, Y. (2026). Safety evaluation of a food enzyme containing endo‐polygalacturonase, pectinesterase and pectin lyase activities from the non‐genetically modified Aspergillus niger strain CCTCC M 2023236. EFSA Journal, 24(4), e10019. 10.2903/j.efsa.2026.10019