Safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR‐962

Abstract

The food enzyme pectinesterase (pectin pectylhydrolase; EC 3.1.1.11) is produced with the genetically modified Aspergillus oryzae strain AR‐962 by AB Enzymes GmbH. The genetic modifications did not give rise to safety concerns. The food enzyme was free from viable cells of the production organism and its DNA. It is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation. Since residual amounts of total organic solids are removed by repeated washing or distillation, dietary exposure to the food enzyme total organic solids (TOS) from the production of flavouring extracts and coffee demucilation was considered not necessary. For the remaining three food processes, dietary exposure to the food enzyme‐TOS was estimated to be up to 0.647 mg TOS/kg 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 1,000 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 1,546. A search for the similarity of the amino acid sequence to those of known allergens was made and two matches with pollen allergens were found. The Panel considered that, under the intended conditions of use the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen allergens, 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 micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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 European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

The “Guidance on submission of a dossier on food enzymes for safety evaluation” (EFSA, 2009a) lays down the administrative, technical and toxicological data required.

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 1332/2008 on food enzymes.

An application has been introduced by the applicant “AB Enzymes GmbH” for the authorization of food enzyme Pectin esterase form a genetically modified Aspergillus oryzae (strain AR‐962).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 ³ implementing Regulation (EC) No 1331/2008, the Commission has verified that the application falls 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 assessment on the following food enzyme: Pectin esterase form a genetically modified Aspergillus oryzae (strain AR‐962) in accordance with Article 29 of Regulation (EC) No 178/2002 ⁴ and Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

2. Data and methodologies

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme pectin esterase from a genetically modified A. oryzae (strain AR‐962). The dossier was updated on 14 December 2020.

Additional information was requested from the applicant during the assessment process on 21 June 2021, on 21 October 2021 and on 25 March 2022, and was consequently provided (see ‘Documentation provided to EFSA’).

Following the reception of additional data by EFSA on 21 September 2021, EFSA requested a clarification teleconference on 5 October 2021, after which the applicant provided additional data on 18 February 2022.

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, 2009b) as well as in the ‘Statement on characterisation of microorganisms used for the production of food enzymes’ (EFSA CEP Panel, 2019) and following the relevant existing guidance documents of EFSA Scientific Committees.

The current ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) has been followed for the evaluation of the application with the exception of the exposure assessment, which was carried out in accordance with the methodology described in the CEF Panel ‘Statement on the exposure assessment of food enzymes’ (EFSA CEF Panel, 2016).

3. Assessment

IUBMB nomenclature	Pectinesterase
Systematic name	Pectin pectylhydrolase
Synonyms	Pectin methylesterase, pectin methoxylase, 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. The food enzyme under assessment is intended to be used in five food manufacturing processes: fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice, production of wine and wine vinegar, production of plant extracts as flavouring preparations and coffee demucilation.

3.1. Source of the food enzyme

The pectinesterase is produced with the genetically modified filamentous fungus Aspergillus oryzae strain AR‐962, which is deposited at the Westerdijk Fungal Biodiversity Institute (the Netherlands), with the deposit number ■■■■■. ⁵ The AR‐962 strain was identified as Aspergillus oryzae ■■■■■. ⁶

3.1.1. Characteristics of the parental and recipient microorganisms

The parental strain is ■■■■■ ^{8 , 9}

3.1.2. Characteristics of introduced sequences

The sequence encoding the pectinesterase ■■■■■. ¹⁰

■■■■■. ¹¹

3.1.3. Description of the genetic modification process

The purpose of genetic modification was to enable the production strain to synthesise pectinesterase ■■■■■

■■■■■. ¹²

3.1.4. Safety aspects of the genetic modification

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

The production strain A. oryzae strain AR‐962 differs from the recipient strain in its capacity to produce the pectinesterase ■■■■■

■■■■■ ¹² ■■■■■.

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

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004 ¹³ , with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice. ¹⁴

The production strain is grown as a pure culture using a typical industrial medium in a submerged, ■■■■■ fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by filtration, leaving a filtrate containing the food enzyme. 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. ¹⁵ 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. ¹⁶

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 mature pectinesterase is a single polypeptide chain of ■■■■■ amino acids. ¹⁷ The molecular mass of the mature protein, calculated from the amino acid sequence, was ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. The gel showed several major protein bands migrating between the ■■■■■ and ■■■■■ kDa marker bands, corresponding to different forms of the glycosylated pectin esterase. ¹⁸ The food enzyme was tested for cellulase, protease and amylase activities. Cellulase and protease activities were detected. ¹⁹ No other enzyme activities were reported.

The in‐house determination of pectinesterase activity is based on hydrolysis of citrus pectin (reaction conditions: pH 4.5, 30°C, 5 min). The enzymatic activity is determined by measuring the released free carboxylic groups that are titrated with sodium hydroxide. Pectinesterase activity is expressed in Pectin Esterase Activity Units/g (PE/g). One unit is defined as the amount of the enzyme that will release 1 μmol of acid groups per minute. ²⁰

The food enzyme has a temperature optimum around ■■■■■ and a pH optimum around pH ■■■■■ Thermostability was tested after a pre‐incubation of the food enzyme at 85°C for different durations (pH 4.5). Pectinesterase activity decreased by more than 99% after 1 min of pre‐incubation and it was not detected after 2 min of pre‐incubation. ²¹

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches of the food enzyme, one of which (batch 1) was used for toxicological tests (Table 1). ²² The mean total organic solids (TOS) of the three food enzyme batches was 10.5% and the mean enzyme activity/TOS ratio was 196.9 PE/mg TOS.

Table 1.

Composition of the food enzyme

Parameters	Unit	Batches
		1 (a)	2	3
Pectinesterase activity	PE/mg (b)	23.7	14.7	22.3
Protein	%	8.5	5.1	9.1
Ash	%	0.5	0.3	0.4
Water	%	87.0	93.1	87.1
Total organic solids (TOS) (c)	%	12.5	6.6	12.5
Pectinesterase activity/TOS	PE/mg TOS	189.6	222.7	178.4

^(a)Batch used for the toxicological studies.

^(b)PE: Pectin esterase Unit (see Section 3.3.1).

^(c)TOS calculated as 100% − % water − % ash.

3.3.3. Purity

The lead content in the three 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). In addition, the levels of arsenic, cadmium and mercury were below the limits of detection (LoD) of the employed methods.

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). ²⁴ No antimicrobial activity was detected in any of the tested batches. ²⁴

Strains of Aspergillus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The presence of aflatoxins: B1, B2, G1, G2, fumonisins: B1, B2, ochratoxin A and sterigmatocystin was examined in the three food enzyme batches and all were below the limit of quantification (LoQ) of the applied methods. ^{24 , 25} Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme TOS.

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

3.3.4. Viable cells and DNA of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated ■■■■■. ²⁶ No colonies were produced. ²⁷

The absence of recombinant DNA in the food enzyme was demonstrated ■■■■■. ²⁸

3.4. Toxicological data

A battery of toxicological tests, including a bacterial gene mutation assay (Ames test), an in vitro mammalian cell micronucleus assay and a repeated dose 90‐day oral toxicity study in rats, has been provided. The batch 1 (Table 1) used in these studies is one of the commercial batches with similar protein pattern and purity as other batches used for commercialisation and is considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation assay (Ames test) was performed according to Organisation for Economic Co‐operation and Development (OECD) Test Guideline 471 (OECD, 1997) and following Good Laboratory Practice (GLP). ²⁹

Five strains of Salmonella Typhimurium (TA98, TA100, TA1535, TA1537 and TA102) were used in the presence or absence of metabolic activation (S9‐mix), applying the standard plate incorporation method (Experiment I) and preincubation method (Experiment II). The experiments were carried out in triplicate using six concentrations of the food enzyme (31.6, 100, 316, 1,000, 2,500 and 5,000 μg TOS/plate).

A toxic effect, evident as a reduction in the background lawn, occurred in S. Typhimurium TA98 in the absence of S9‐mix at 2,500 and 5,000 μg TOS/plate in the second experiment. A significant increase in revertant colony numbers (2.15‐fold vs. the control value) was recorded in S. Typhimurium TA1537 in the presence of S9‐mix at 5,000 μg TOS/plate in the first experiment, but this was not reproduced in the second experiment. The Panel considered this finding not to be biologically relevant. Upon treatment with the food enzyme there was no significant increase in revertant colony numbers above the control values in any other strain with or without S9‐mix.

The Panel concluded that the food enzyme did not induce gene mutations under the test conditions employed in this study.

3.4.1.2. In vitro mammalian cell micronucleus assay

The in vitro micronucleus test was carried out according to OECD Draft Guideline 487 (OECD, 2016) and following GLP. ³⁰

A pre‐experiment for cytotoxicity was performed at concentrations ranging from 7.8–5,000 μg TOS/mL. Based on these results, two separate experiments were performed in duplicate cultures of human peripheral whole blood lymphocytes. In the first experiment, the cells were exposed to the food enzyme at 500, 600 and 700 μg TOS/mL, and at 500, 700 and 900 μg TOS/mL in the short‐term treatment (4 h followed by 40 h recovery period) without and with metabolic activation (S9‐mix), respectively. In the second experiment, the cells were exposed to the food enzyme at 50, 100 and 150 μg TOS/mL in the continuous treatment (44 h) in the absence of S9‐mix.

The reduction of the cytokinesis‐block‐proliferation index (CPBI) was applied to evaluate cytotoxicity. In the first experiment, cytotoxicity was observed at 600 μg TOS/mL (48%) and 700 μg TOS/mL (53%) (in the absence of S9‐mix) and at 700 μg TOS/mL (39%) and 900 μg TOS/mL (58%) (in the presence of S9‐mix). In the second experiment, a cytotoxicity of 58% was detected at 150 μg TOS/mL (without S9‐mix). The frequency of bi‐nucleated cells with micronuclei (MNBN) was comparable to the negative controls at all concentrations tested.

The Panel concluded that the food enzyme pectinesterase did not induce an increase in the frequency of MNBN in cultured human peripheral blood lymphocytes, under the test conditions employed in this study.

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

The repeated dose 90‐day oral toxicity study was planned to be performed in accordance with OECD Test Guideline 408 (OECD, 2018) and following GLP. ³¹ The Panel noted the following deviations from the intended study plan: functional observations and ophthalmological examination were not performed in 5 males from low‐, mid‐ and high‐dose groups, in 5 control females and in all females from low‐, mid‐ and high‐dose groups, and 5 females from the control, low‐, mid‐ and high‐dose groups were not fasted overnight before blood sampling for haematology and clinical chemistry analyses. That Panel considered that these deviations did not impact on the evaluation of the study. Groups of 10 male and 10 female Wistar (Crl:WI(Han)) rats received by gavage the food enzyme in 100, 300 and 1,000 mg TOS/kg body weight (bw) per day. Controls received the vehicle (water).

No mortality was observed.

The body weight was statistically significantly decreased in low‐dose females on days 22, 29, 36 and 50 (−7%, −7%, −6%, −6% and −7%, respectively). The body weight gain was statistically significantly increased in low‐ and high‐dose males (+98% and 95%, respectively) in week 10. The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically (both parameters), they were only observed in one sex (both parameters), the changes were small (body weight), there was no dose–response relationship (both parameters) and the changes were without a statistically significant effect on the final body weight and body weight gain.

During weekly detailed clinical observations, a statistically significant decrease in a score for a sleeping animal (−30%) and an increase in a score for an animal moving in the cage in low‐dose males were recorded in week 11. In the treated female groups, several statistically significant differences to controls were recorded: a statistically significantly increased score for a sleeping animal in mid‐ and high‐dose groups (+67% in both groups) in week 1, an increased score for a response to handling in the mid‐dose group (+13%) in week 1, an increased score for a sleeping animal (+100%) and a decreased score for an animal moving in the cage (−100%) in the high‐dose group in week 2, a decreased score for a sleeping animal (−50%) and an increased score for an animal moving in the cage in the high‐dose group in week 5, an increased score for a response to handling (+38%) in the high‐dose group in week 10, a decreased score for a sleeping animal (−60%) and an increased score for an animal moving in the cage in the low‐dose group in week 11. The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically (all parameters), they were only observed in one sex (except for a decrease in sleeping and increase in movements in low‐dose males and low‐dose females in week 11) and there was no dose–response relationship (a decrease in sleeping and an increase in moving at the low dose in both sexes in week 11, a response to handling in week 1).

In the functional observations in week 13, a statistically significant decreased score for a sleeping animal (−60%) and increased scores for defecation and for grooming in high‐dose males, increased scores for rearing supported in the mid‐and high‐dose males and an increased score for a response to handling (+10%) in low‐dose males were reported. The Panel considered the changes as not toxicologically relevant as they were not observed during the detailed weekly clinical observations (grooming, response to handling), there was no dose–response relationship (response to handling, rearing supported) and they were only recorded sporadically (decrease for sleeping).

The haematological investigations revealed a statistically significant decrease in reticulocytes (−19%) in low‐dose males, an increase in red blood cells (RBC, +9%) and haemoglobin (Hb, +7%), and haematocrit (HCT, +6%) in low‐dose females, a decrease in mean corpuscular volume (MCV) in low‐ and mid‐dose females (−3 and −4%, respectively), an increased mean corpuscular haemoglobin concentration (MCHC) in mid‐ and high‐dose females (+2% in both groups), and an increased activated partial thromboplastin time (APTT) in high‐dose females (+19.6%). 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 (reticulocytes, RBC, Hb, HCT, MCV), the changes were small (RBC, Hb, HCT, MCV, MCHC) and there were no changes in other relevant parameters (for APTT in prothrombin time).

The clinical chemistry investigation revealed a statistically significant decrease in alanine aminotransferase (ALT) in all treated males (−22%, −24% and −25% at low‐, mid‐ and high‐doses, respectively) and in high‐dose females (−28%), a decreased sodium (Na) concentration in all treated males (−3%, −5% and −3% at low‐, mid‐ and high‐doses, respectively), a decreased total protein (tProt) concentration in mid‐dose males (−6%), a decrease in aspartate‐aminotransferase (AST, −24%) in high‐dose females, an increase in total bilirubin in all treated females (+31%, +27% and +38% at low‐, mid‐ and high‐doses, respectively), a decrease in concentrations of creatinine in mid‐ and high‐dose females (−18% and‐18%, respectively), of urea in all treated females (−15%, −21% and −24% at low‐, mid‐ and high‐doses, respectively) and an increase in low density lipoprotein (LDL) in mid‐dose females (+70%). Potassium (K) was dose‐dependently increased in all treated males (+8%, +19% and +26% at low‐, mid‐ and high‐doses, respectively) and females (+34, +35 and +63% at low‐, mid‐ and high‐doses, respectively), with statistical significance in females only. The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters except for ALT), there was no dose–response relationship (Na, tProt, total bilirubin, creatinine, LDL), the changes were small (ALT, AST, tProt) and there were no histopathological changes in the kidneys (for K).

The urinalysis revealed a statistically significant increase in the incidence of glucose positive samples in high‐dose males (6/10 vs. 0/10 in controls). 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 (no increase in blood glucose) and there were no histopathological changes in the kidneys.

No other statistically significant or biologically relevant differences to controls were reported.

The Panel identified a no observed adverse effect level (NOAEL) of 1,000 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

The allergenicity assessment considered only the food enzyme and not any carrier or other excipient, which may be used in the final formulation.

The potential allergenicity of the pectinesterase produced with the genetically modified A. oryzae strain AR‐962 was assessed by comparing its amino acid sequence with those of known allergens according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms’ (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, two matches were found. The matching allergens were Sal k 1 pectin methylesterase from Russian thistle (Salsola kali) and Ole e 11 pectinesterase from olive tree (Olea europaea). ³²

No information is available on oral and respiratory sensitisation or elicitation reactions of this pectinesterase.

Pectinesterases present in plant tissues and pollen are reported for their role in allergenicity: the allergen Ole e 11, a pectinesterase from Olive tree (Olea europaea) was identified as a source of allergy (Salamanca et al., 2010), as well as Sal k 1, a pectinesterase from Russian thistle (Salsola kali) (Barderas et al., 2007). The Panel noted that oral allergy syndrome, i.e., allergic reactions mainly in the mouth, and seldomly leading to anaphylaxis, is associated with sensitisation to olive and Russian thistle pollen.

■■■■■, a product that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³³ ) is used as raw material. In addition, ■■■■■, known sources of allergens, are also present in the media fed to the microorganisms. However, during the fermentation process, these products will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the fungal biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that no potentially allergenic residues are present in the food enzyme.

The Panel considered that, under the intended conditions of use the risk of allergic reactions by dietary exposure, particularly in individuals sensitised to pollen allergens, 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 five food 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 ^(c)

Food manufacturing process (a)		Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) (b)
Fruit and vegetable processing for juice production		Fruits and vegetables	2
Fruit and vegetable processing for products other than juice	Puree	Fruits and vegetables	13
	Fruit firming		26
Production of wine and wine vinegar		Grapes	1
Production of plant extracts as flavouring preparations (d)		Fruit and vegetables	2
Coffee demucilation		Coffee cherry	0.5

^(a)The description has been harmonised by EFSA according to the ‘EC working document describing the food processes in which food enzymes are intended to be used’ – not yet published at the time of adoption of this opinion.

^(b)The numbers in bold were used for calculations.

^(c)Technical dossier/2nd submission/pg. 30.

^(d)Additional data September 2021/Answer 6.

In fruit and vegetable processing, the function of pectinesterase is to aid the depolymerisation of pectin in different raw materials at various points in the production process. For juice production, the food enzyme can be added during the peeling and crushing; to the crush mash of fruits/vegetables (with or without peels) and/or to the pressed juice before clarification and filtration. ³⁴ The disruption of the gel structure reduces the viscosity, thus improving the pressing ability of the pulp and consequently increasing the yield of fruit juices. The enzymatic treatment can reduce haze and enhance colour and aroma. ³⁵ The food enzyme‐TOS remains in the juices.

In puree production, the pectinesterase is added to the crushed pulp before pasteurisation. ³⁶ The enzymatic treatment reduces viscosity and improves consistency of puree. Treatment with pectinesterase can also improve the firmness of jams, canned and frozen fruit and vegetables products. ³⁶ The food enzyme‐TOS remains in these products.

In wine and wine vinegar production, pectinesterase is often added together with other cell wall hydrolytic enzymes during crushing. It can be added also during maceration and clarification steps. Such enzymatic treatment eases pressing and facilitates the extraction of aromatic compounds. ³⁷ The food enzyme‐TOS may remain in wine and wine vinegar.

To produce essential oils, fruit components rich in oil are treated with the pectinesterase to assist the release of aromatic compounds from the raw material. ³⁸ It is expected that the food enzyme TOS partitions with the water phase, therefore, it is not carried into the oil phase. The aroma concentrates are primarily used in the reconstitution of juices. Samples of the apple aroma concentrate and orange essential oil and additional samples obtained by trichloroacetic acid precipitation were separated by SDS‐PAGE and stained with Coomassie Blue. No proteins of the food enzyme were detected. ^{39 , 40} The Panel accepted this evidence as sufficient to support the lack of TOS transfer into the essential oils.

In coffee processing, pectinesterase is added to green coffee berry during pulping and fermentation to degrade the mucilage. ⁴¹ The food enzyme‐TOS is removed during the subsequent washing steps (EFSA CEP Panel, 2021b).

Based on data provided on thermostability (see Section 3.3.1), the pectinesterase would be inactivated by heat in most of the food processes, but may remain active in wine and wine vinegar, and in juices, depending on the pasteurisation conditions.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021a), a dietary exposure was calculated only for food manufacturing processes where the food enzyme‐TOS remains in the final foods, i.e., fruit and vegetable processing for juice production, fruit and vegetable processing for products other than juice and production of wine and wine vinegar.

Chronic exposure to the food enzyme‐TOS was calculated by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021a). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2021b). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only 1 day per subject were excluded and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).

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 43 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 22 European countries (Appendix B). The highest dietary exposure was estimated to be 0.647 mg TOS/kg bw per day in infants at the 95th percentile.

Table 3.

Summary of 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)	0.009–0.467 (12)	0.031–0.219 (15)	0.021–0.223 (19)	0.004–0.118 (21)	0.003–0.058 (22)	0.003–0.066 (23)
Min–max 95th percentile (number of surveys)	0.037–0.647 (11)	0.084–0.566 (14)	0.070–0.466 (19)	0.016–0.262 (20)	0.013–0.146 (22)	0.013–0.149 (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 was 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	+/−
Exclusion of other processes from the exposure assessment –Production of plant extract as flavouring preparations –Coffee demucilation

+: Uncertainty with potential to cause overestimation of exposure.

–: Uncertainty with potential to cause underestimation of exposure.

The conservative approach applied to the exposure estimate to food enzyme‐TOS, in particular assumptions made on the occurrence and use levels of this specific food enzyme, is likely to have led to a considerable overestimation of the exposure.

The exclusion of two food manufacturing processes from the exposure assessment was based on > 99% of TOS removal during these processes and is not expected to have an impact on the overall estimate derived.

3.6. Margin of exposure

A comparison of the NOAEL (1,000 mg TOS/kg bw per day) from the 90‐day rat study with the derived exposure estimates of 0.003–0.467 mg TOS/kg bw per day at the mean and from 0.013–0.647 mg TOS/kg bw per day at the 95th percentile, resulted in margin of exposure of at least 1,546.

4. Conclusions

Based on the data provided, the removal of TOS during the production of plant extracts as flavouring preparations and coffee demucilation, and the derived margin of exposure for the remaining three food manufacturing processes, the Panel concluded that the food enzyme pectinesterase produced with the genetically modified Aspergillus oryzae strain AR‐962 does not give rise to safety concerns under the intended conditions of use.

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

5. Documentation as provided to EFSA

Application for authorisation of Pectinesterase from a genetically modified Aspergillus oryzae (strain AR‐962). December 2020. Submitted by AB Enzymes GmbH.

Additional information. September 2021. Submitted by AB Enzymes GmbH.

Additional information. February 2022. Submitted by AB Enzymes GmbH.

Additional information. June 2022. Submitted by AB Enzymes GmbH.

Abbreviations

bw

body weight

CAS

Chemical Abstracts Service

CBPI

cytokinesis‐block‐proliferation index

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

European Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organisation of the United Nations

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

OECD

Organisation for Economic Cooperation and Development

PCR

polymerase chain reaction

TOS

total organic solids

WHO

World Health Organisation

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

Information provided in this appendix is shown in an excel file (downloadable https://efsa.onlinelibrary.wiley.com/doi/10.2903/j.efsa.2023.7832#support‐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, Netherlands, Portugal, 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, Netherlands, Portugal, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden
Adults	From 18 years up to and including 64 years of age	Austria, Belgium, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Netherlands, Portugal, Romania, 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, Netherlands, Portugal, Romania, Slovenia, Spain, Sweden

(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).

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

Suggested citation: EFSA CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré C, Barat Baviera JM, Bolognesi C, Cocconcelli PS, Crebelli R, Gott DM, Grob K, Lampi E, Mengelers M, Mortensen A, Rivière G, Steffensen I‐L, Tlustos C, Van Loveren H, Vernis L, Zorn H, Glandorf B, Herman L, Aguilera J, Andryszkiewicz M, Kovalkovicova N, Liu Y, de Sousa RF and Chesson A, 2023. Scientific Opinion on the safety evaluation of the food enzyme pectinesterase from the genetically modified Aspergillus oryzae strain AR‐962. EFSA Journal 2023;21(2):7832, 17 pp. 10.2903/j.efsa.2023.7832