Safety evaluation of the food enzyme arabinan endo‐1,5‐α‐L‐arabinanase from the non‐genetically modified Aspergillus aculeatinus strain CBS 148915

TL;DR

This study evaluates the safety of a food enzyme produced by a non-genetically modified fungus and concludes it is safe for use in most food manufacturing processes.

Contribution

The study provides a comprehensive safety evaluation of arabinan endo-1,5-α-L-arabinanase from Aspergillus aculeatinus for food use.

Findings

Genotoxicity tests showed no safety concerns.

The enzyme's amino acid sequence does not match known allergens.

The enzyme is considered safe for 11 of 12 proposed food manufacturing uses.

Abstract

The food enzyme arabinan endo‐1,5‐α‐L‐arabinanase (5‐α‐L‐arabinan 5‐α‐L‐arabinanohydrolase; EC 3.2.1.99) is produced with the non‐genetically modified Aspergillus aculeatinus strain CBS 148915 by Solyve. The food enzyme was considered free from viable cells of the production organism. The applicant proposed the use of the food enzyme in 12 food manufacturing processes. Since the use of the enzyme in one proposed use is not allowed in the European Union and residual amounts of food enzyme–total organic solids (TOS) are removed in four processes, dietary exposure was calculated for the remaining seven food manufacturing processes. It was estimated to be up to 1.499 mg 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 301 mg TOS/kg bw per day, the highest dose tested, which when compared with the estimated dietary exposure, results in a margin of exposure of at least 201. A search for the homology of the amino acid sequence of the arabinan endo‐1,5‐α‐L‐arabinanase 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 in 11 food manufacturing processes (excluding the use in the production of juices).

Tables

TABLE 1. Composition of the food enzyme preparation. 23

Parameters	Unit	Batches
		1	2	3
				Without excipient a	With excipient b
Arabinan endo‐1,5‐α‐L‐arabinanase activity	U/g c	345	478	870	446
Protein	%	3.2	3.4	9.4	4.8
Ash	%	0.12	0.13	0.18	0.13
Water	%	45.2	44.5	91.0	44.7
■■■■■ (excipient)	%	50.0	50.0	0	50.0
Total organic solids (TOS) d	%	4.7	5.4	8.8	5.2
Activity/TOS ratio	U/mg TOS	7.3	8.9	9.9	8.6

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 40

Food manufacturing process	Raw material (RM)	Maximal recommended use level (mg TOS/kg RM) a
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	20 b
Production of fruit and vegetable products other than juices	Fruit and vegetables	20
Production of wine and wine vinegars	Grapes	75
Production of distilled alcoholic beverages c	Fruit	170
Production of alcoholic beverages other than grape wine	Fruit	128
Processing of plant‐ and fungal‐derived products
Production of refined and unrefined sugar	Sugar beet	170
Production of edible oils from plant and algae	Oilseeds, olive	170
Production of green coffee beans by demucilation	Coffee cherry	85
Production of coffee extracts	Coffee bean	85
Production of coffee substitutes	Cereals	85
Production of tea and other herbal and fruit infusions	Tea leaves and other plants	170
Production of plant extracts (as flavouring preparations)	Plants	170

TABLE 3. Summary of the estimated dietary exposure to food enzyme–TOS in six population groups for use in 12 food manufacturing processes as proposed by the applicant.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.021–0.283 (14)	0.099–0.831 (17)	0.098–0.693 (21)	0.030–0.249 (23)	0.064–0.261 (23)	0.047–0.336 (25)
Min–max 95th percentile (number of surveys)	0.083–0.573 (13)	0.317–0.936 (16)	0.302–1.794 (21)	0.108–0.606 (22)	0.200–0.747 (23)	0.122–1.052 (24)

TABLE 4. Summary of the estimated dietary exposure to food enzyme–TOS in six population groups for use in 11 food manufacturing processes (production of fruit juices excluded).

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.016–0.266 (14)	0.024–0.345 (17)	0.018–0.421 (21)	0.010–0.198 (23)	0.040–0.189 (23)	0.034–0.316 (25)
Min–max 95th percentile (number of surveys)	0.042–0.538 (13)	0.070–0.593 (16)	0.050–1.499 (21)	0.034–0.506 (22)	0.141–0.599 (23)	0.090–1.018 (24)

TABLE 5. 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	+
Assumption that the food enzyme‐TOS are fully transferred into wines and in alcoholic beverages other than grape wine	+
Minor FoodEx categories found to only sporadically contain molasses were excluded from the exposure assessment	−
‘Brown sugar’ produced through use of cane molasses or caramelised sugar syrup was excluded, due to it being a niche product on the European market	−
The transfer of food enzyme‐TOS into cane and beet molasses/syrups was assumed to be 100%	+
No distinction was made between beet molasses and cane syrups used as ingredients in foods	+/−
Use of technical factors in the exposure model	+/−
Exclusion of four processes from the exposure assessment: – Production of distilled alcoholic beverages – Production of edible oils from plant and algae – Production of green coffee beans by demucilation – Production of plant extracts	−

Full text

INTRODUCTION

1

Article 3 of the Regulation (EC) No 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or microorganisms or products thereof, including a product obtained by a fermentation process using microorganisms: (i) containing one or more enzymes capable of catalysing a specific biochemical reaction and (ii) added to food for a technological purpose at any stage of the manufacturing, processing, preparation, treatment, packaging, transport or storage of foods.

‘Food enzyme preparation’ means a formulation consisting of one or more food enzymes in which substances such as food additives and/or other food ingredients are incorporated to facilitate their storage, sale, standardisation, dilution or dissolution.

Before January 2009, food enzymes other than those used as food additives were not regulated or were regulated as processing aids under the legislation of the Member States. On 20 January 2009, Regulation (EC) No 1332/2008^1^ on food enzymes came into force. This Regulation applies to enzymes that are added to food to perform a technological function in the manufacture, processing, preparation, treatment, packaging, transport or storage of such food, including enzymes used as processing aids. Regulation (EC) No 1331/20082 established the European Union (EU) procedures for the safety assessment and the authorisation procedure of food additives, food enzymes and food flavourings. The use of a food enzyme shall be authorised only if it is demonstrated that:

• it does not pose a safety concern to the health of the consumer at the level of use proposed;
• there is a reasonable technological need;
• its use does not mislead the consumer. All food enzymes currently on the EU market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

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

Background and Terms of Reference as provided by the requestor

1.1

Background as provided by the European Commission

1.1.1

Only food enzymes included in the 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 1332/2008^1^ on food enzymes.

Five applications have been introduced by the companies 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 234/20113 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.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes 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) in accordance with Article 17.3 of Regulation (EC) No 1332/2008^1^ on food enzymes.

Interpretation of the Terms of Reference

1.2

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of the food enzyme pectinase, poly‐galacturonase, pectin esterase, pectin lyase and arabanase from Aspergillus niger, submitted by AMFEP.

The application was submitted initially as a joint dossier4 and identified as the EFSA‐Q‐2015‐00038, EFSA‐Q‐2015‐00039, EFSA‐Q‐2015‐00040, EFSA‐Q‐2015‐00041, EFSA‐Q‐2015‐00042. EFSA‐Q‐2015‐00042 specifically concerns the request for EFSA to perform a scientific risk assessment on the food enzyme arabanase. Agreement to split the joint dossiers into individual data packages was made between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).5

The current opinion addresses one data package originating from the former joint dossier. This data package is identified as EFSA‐Q‐2023‐00241 and concerns the food enzyme arabanase produced from the non‐genetically modified Aspergillus aculeatinus strain CBS 148915 and submitted by Solyve.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme arabanase from the non‐genetically modified Aspergillus aculeatinus strain CBS 148915. The data package was submitted on 31 March 2023.

Additional information, requested from the applicant during the assessment phase on 25 March 2024, was received on 25 June 2024 (see Documentation provided to EFSA).

Methodologies

2.2

The assessment was conducted in line with the principles described in the EFSA ‘Guidance on transparency in the scientific aspects of risk assessment’ (EFSA, 2009) and following the relevant guidance documents of the EFSA Scientific Committee.

The ‘Guidance on the submission of a dossier on food enzymes for safety evaluation’ (EFSA CEP 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).

ASSESSMENT

6

3

IUBMB nomenclatureArabinan endo‐1,5‐α‐L‐arabinanaseSystematic name5‐α‐L‐arabinan 5‐α‐L‐arabinanohydrolaseSynonymsArabinanase; arabanase; arase; endo‐arabinaseIUBMB NoEC 3.2.1.99CAS No75432‐96‐1EINECS No–

Arabinan endo‐1,5‐α‐L‐arabinanases catalyse the hydrolysis of (1‐5)‐α‐arabinofuranosidic linkages in arabinans resulting in the generation of arabinose oligomers of shorter length.

The food enzyme under assessment is intended to be used in 12 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 vinegars, (4) distilled alcoholic beverages and (5) alcoholic beverages other than grape wine; processing of plant‐ and fungal‐derived products for the production of (6) refined and unrefined sugar, (7) edible oils from plant and algae, (8) green coffee beans by demucilation, (9) coffee extracts, (10) coffee substitutes, (11) tea and other herbal and fruit infusions and (12) plant extracts as flavouring preparations. Only 11 uses were considered in the present assessment, excluding the use of this food enzyme for the production of juices.

Source of the food enzyme

7

3.1

The arabinan endo‐1,5‐α‐L‐arabinanase is produced with the non‐genetically modified filamentous fungus Aspergillus aculeatinus strain CBS 148915 (ARA1‐GLY), which is deposited at the Westerdijk Fungal Biodiversity Institute Culture collection (The Netherlands) with the deposition number CBS 148915.8 The production strain was identified as A. aculeatinus ■■■■■ was also performed, confirming the taxonomic identification of the production strain.9

A search for genes involved in the biosynthesis of secondary metabolites and virulence factors was performed. Applying a threshold of 80% identity and 70% coverage, no virulence factors of concern were identified. ■■■■■ gene clusters involved in the biosynthesis of secondary metabolites were found, among which only roquefortine was considered of concern. This is further discussed in Section 3.3.3.10

Production of the food enzyme

3.2

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

The production strain is grown as a pure culture using a typical industrial medium in ■■■■■ with conventional process controls in place. After completion of the fermentation, the enzyme is ■■■■■ and ■■■■■ containing the enzyme is then further purified and concentrated, including ■■■■■ in which enzyme protein is retained, while most of the low molecular mass material passes the filtration 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.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The arabinan endo‐1,5‐α‐L‐arabinanase is a single polypeptide chain of ■■■■■ amino acids.15 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.16 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.17 A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about ■■■■■ kDa, consistent with the expected mass of the enzyme. Proteomic analysis of the food enzyme showed that it contains α‐L‐arabinofuranosidase, rhamnogalacturonan acetylesterase and rhamnogalacturonan lyase enzymes.

No other enzyme activities were reported.18

The applicant's in‐house determination of arabinan endo‐1,5‐α‐L‐arabinanase activity is based on the hydrolysis of azurine‐cross‐linked‐debranched arabinan (reaction conditions: pH 4.0, 30°C, 20 min). The release of dyed arabinose is measured spectrophotometrically at 590 nm. The enzyme activity is expressed in Units (U)/g. One Unit is the amount of enzyme required to hydrolyse the substrate to release soluble oligomers corresponding to an absorbance of 7.0 units.19

The food enzyme has a temperature optimum around 60°C (pH 4.0) and a pH optimum around pH 3.5 (30°C).20 Thermostability was tested after pre‐incubation of the food enzyme for 10 min at different temperatures (pH 3.2). The enzyme showed a residual activity of 90% at 50°C and 12.9% at 65°C.21

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme preparation were provided for three batches intended for commercialisation. Batch 3 was analysed with and without excipient. The one without excipient was used for the genotoxicity studies, and the one with excipient was used for the repeated dose 90‐day oral toxicity study (Table 1).22 The mean total organic solids (TOS) of the three food enzyme preparation batches was 5.1%, and the mean enzyme activity/TOS ratio was 8.3 U/mg TOS.

TABLE 1: Composition of the food enzyme preparation. 23

Purity

3.3.3

The lead content in all the batches was below 0.13 mg/kg,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, cadmium and mercury concentrations were below the limits of quantification (LoQ) of the employed methods. For arsenic, the average concentration determined in the commercial batches was 0.02 mg/kg.25 ^,^ 26 The Panel considered this concentration as not of concern.

The food enzyme preparation complies with the microbiological criteria for total coliforms, Escherichia coli and Salmonella, as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).27 No antimicrobial activity was detected in any of the tested batches.28

Strains of Aspergillus species, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites (Frisvad et al., 2018). The genome of the production strain was shown to contain a gene cluster involved in the synthesis of roquefortine (see Section 3.1). The presence of aflatoxins B1, B2, G1 and G2, fumonisins B1 and B2, ochratoxin A, HT2 toxin, T‐2 toxin, zearalenone and deoxynivalenol (DON) was examined in three food enzyme batches and was below the LoQ of the applied methods.29 ^,^ 30 Adverse effects caused by the possible presence of other secondary metabolites, including roquefortine, are addressed by the toxicological examination of the food enzyme.

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

Viable cells

31

3.3.4

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. ■■■■■. No colonies of the production strain were detected. A positive control was included.32

Toxicological data

33

3.4

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) has a similar activity/mg TOS ratio as the batches used for commercialisation and thus is considered suitable as a test item.

Genotoxicity

3.4.1

Bacterial reverse mutation test

3.4.1.1

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).34

Five strains of Salmonella Typhimurium (TA98, TA100, TA102, TA1535 and TA1537) were used with or without metabolic activation (S9‐mix). Two experiments were carried out in triplicate, applying the standard plate incorporation method (the first experiment) and the pre‐incubation method (the second experiment) without and with S9‐mix.

The first experiment was carried out using seven concentrations of the food enzyme of 5, 16, 50, 160, 500, 1600 and 5000 μg TOS/plate. The second experiment was carried out using six concentrations of the food enzyme of 160, 300, 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 arabinan endo‐1,5‐α‐L‐arabinanase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.4.1.2

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.35 An experiment was performed with duplicate cultures of human peripheral whole blood lymphocytes. The cell cultures were treated with the food enzyme with or without metabolic activation (S9‐mix).

In a range‐finding test, no cytotoxicity (based on the replication index) above 50% was seen at any concentration tested up to 5000 μg TOS/mL with and without S9‐mix.

In the main experiment, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 250, 400, 500, 1000, 2500, 4000 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery period) without S9‐mix, at concentrations of 500, 1000 and 5000 μg TOS/mL in a short‐term treatment with S9‐mix, and in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

No cytotoxicity was seen either in the short‐term treatment with and without S9‐mix or in the long‐term treatment. The frequency of MNBN was statistically significantly different from the negative controls at a concentration of 1000 μg TOS/mL in the short‐term treatment without S9‐mix and at a concentration of 500 μg TOS/mL in the short‐term treatment with S9‐mix. However, no concentration‐related response was observed, and the values were within the 95th percentile range of the historical negative control values, and therefore, the changes were not considered to be of biological relevance. In the long‐term treatment without S9‐mix, the frequency of MNBN was not statistically significantly different from 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 arabinan endo‐1,5‐α‐L‐arabinanase did not induce an increase in the frequency of MNBN under the test conditions applied in this study.

Repeated dose 90‐day oral toxicity study in rodents

3.4.2

The repeated dose 90‐day oral toxicity study was performed under GLP and according to the OECD Test Guideline 408 (OECD, 2018).36

Groups of 10 male and 10 female Sprague–Dawley SD rats received the food enzyme by gavage in doses of 75, 150 and 301 mg TOS/kg bw per day. Controls received the vehicle (water‐glycerol, 1:1, w/w).

No mortality was observed.

The feed consumption was statistically significantly decreased on day 22 of administration in mid‐ and high‐dose males (−4% and −5%) and on day 92 in high‐dose males (−5%). The Panel considered the changes as not toxicologically relevant, as they were only recorded at single time intervals, they were only observed in one sex, the changes were small and there was no statistically significant change in the final feed consumption, body weight and body weight gain.

In the functional observations, on day 27, a statistically significant increase in rearing in open field measurement was reported in mid‐dose females (+22%). On day 80, a decrease in the landing foot splay measurements of distance between ink blots (LAN 1, the first measurement: ‐40%; LAN 2, the second measurement: ‐34%; LAM, average measurement: −37%) and an increase in the averaged reading of the animal grips on the horizontal bar with forelimbs (+11%) were observed in high‐dose males. The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically (all parameters), they were only observed in one sex (all parameters) and there were no other signs of neurotoxicity.

Clinical chemistry investigations revealed a statistically significant decrease in chloride concentration in mid‐ and high‐dose males (−2% each) and an increase in sodium concentration in high‐dose females (+1%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (both parameters), the changes were small (both parameters), there were no changes in other relevant parameters (other electrolytes) and there were no histopathological changes in the kidneys.

The urinalysis revealed a statistically significant increase in diuresis in low‐ and mid‐dose males (+48% and +60%). The Panel considered the change as not toxicologically relevant, as it was only observed in one sex, there was no dose–response relationship, there were no changes in other relevant parameters and there were no histopathological changes in kidneys.

Statistically significant changes detected in organ weights were a decrease in relative heart weight in high‐dose males (−7%) and relative kidney weight in mid‐dose males (−7%). 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 (relative kidney weight) and there were no changes in absolute weights or histopathological changes in heart and kidneys.

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

The Panel identified a no observed adverse effect level (NOAEL) of 301 mg TOS/kg bw per day, the highest dose tested.

Allergenicity

3.4.3

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 arabinan endo‐1,5‐α‐L‐arabinanase produced with the Aspergillus aculeatinus strain CBS 148915 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 AllergenOnline database.37

No reports on oral and respiratory sensitisation or elicitation reactions of the arabinan endo‐1,5‐α‐L‐arabinanase under assessment have been published.38 No allergenic reactions upon dietary exposure to any endo‐1,5‐α‐L‐arabinanase have been reported in the literature.

The Panel considered that the results of the sequence homology search and the available literature search do not indicate a risk of allergic reactions upon dietary exposure to the arabinan endo‐1,5‐α‐L‐arabinanase 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 ■■■■■,39 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 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, when used for the production of distilled alcohols, the Panel considered that a risk of allergic reactions upon dietary exposure can be excluded. For the remaining intended uses, the risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The applicant proposed to use this food enzyme to treat plant materials in 12 food manufacturing processes at the recommended use levels shown in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 40

The action of arabinan endo‐1,5‐α‐L‐arabinanase is to degrade arabinans in the cell wall. The disruption of the structural polymers reduces viscosity, thus increasing the yield of the plant products and facilitating the release of colour and flavouring compounds.

For the production of juices and other fruit and vegetable products, the food enzyme is added to fruit or vegetables during cutting or crushing.41 The food enzyme–TOS remains in the final foods.

During the production of juice concentrates from fruit and vegetable, flavouring preparations recovered by distillation are also obtained,42 which may be used to restore flavour in concentrated fruit juices, or purees and jams. The distillation step is expected to remove the food enzyme–TOS from these flavouring preparations. To substantiate the extent of this removal, the applicant measured the nitrogen content in the flavour condensate obtained from an orange juice treated with a polygalacturonase (as a proxy for arabinan endo‐1,5‐α‐L‐arabinanase), and no nitrogen was detected.43 The Panel considered the technical information and experimental data sufficient to demonstrate the removal (> 99%) of the food enzyme–TOS in these types of flavouring preparations.

In the production of wine and wine vinegars, the food enzyme could be added to grapes during pressing, maceration, fermentation and clarification steps.44 The subsequent downstream processing steps applied, that may include an ultrafiltration step and the addition of bentonite, could theoretically remove the food enzyme–TOS from the wines. In order to substantiate such removal, the applicant measured, as a proxy, the amount of proteins in white, red and rosé wine samples treated with ultrafiltration membrane or bentonite. The protein content was measured applying the Bradford method before and after the downstream processing steps. After ultrafiltration or treatment with bentonite, the results showed a residual amount of proteins lower than the limit of detection (LoD) of the method for white and rosé wines.45 ^,^ 46 However, the LoD of the method corresponds to a concentration of the food enzyme 200 times higher than that used in wine making.47 Therefore, the Panel considered the selected analytical method not sensitive enough to prove the removal of the food enzyme–TOS in wine making. The Panel also noted that bentonite is not always used in wine production and, when used, it selectively removes the protein fraction of TOS, but the effect on the non‐protein fraction of TOS is not known. Therefore, the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS is transferred into the wines.

In the production of distilled alcoholic beverages, the food enzyme is added to fruits during maceration.48 The food enzyme–TOS is not carried over into the distillates (EFSA CEP Panel, 2023).

In the production of alcoholic beverages other than grape wine, the food enzyme is added to fruits such as apples and pears during maceration or to the pressed juice after pasteurisation.49 The subsequent downstream processing steps applied, that include an ultrafiltration step, could theoretically remove the food enzyme–TOS from the alcoholic beverages. In order to substantiate such removal, the applicant measured, as a proxy, the amount of proteins in cider samples treated using an ultrafiltration membrane and/or bentonite. The protein content was measured applying the Bradford method before and after the downstream processing steps. After ultrafiltration and treatment with bentonite, the results showed a residual amount of proteins lower than the LoD of the method for cider.50 ^,^ 51 However, the LoD of the method corresponds to a concentration of the food enzyme 200 times higher than that used in the production of cider.52 Therefore, the Panel considered the selected analytical method not sensitive enough to prove the removal of the food enzyme–TOS in alcoholic beverages other than grape wine. The Panel also noted that, when used, bentonite selectively removes the protein fraction of TOS but the effect on the non‐protein fraction of TOS is not known. In addition, the production flowchart and the technical dossiers do not mention bentonite in the purification steps of this food manufacturing process.53 ^,^ 54 On the basis of these considerations, the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS are transferred into the alcoholic beverages other than grape wine.

In the production of refined and unrefined sugars, the food enzyme is added to sugar beet pulp.55 The food enzyme–TOS is not carried into the refined sugar due to the crystallisation, but remains in the molasses (EFSA CEP Panel, 2023).

In the production of edible oils from plant and algae, the food enzyme is used to treat olives56 or oilseeds57 before pressing to manufacture refined oils (including refined olive oils58) and olive pomace oils.59 The food enzyme–TOS is removed from the refined oils in the refining process (EFSA CEP Panel, 2023).

In the production of green coffee beans by demucilation, the food enzyme is added to coffee cherries before fermentation60 to remove the mucilaginous coat. After the separation, the remaining residual food enzyme–TOS is removed from the green coffee beans by repeated washing (EFSA CEP Panel, 2023).

In production of coffee extracts, the food enzyme is used to treat the roasted coffee beans after milling.61 The food enzyme–TOS remain in these extracts.

In the production of coffee substitutes, the food enzyme is added to cereals after roasting and milling.62 The food enzyme–TOS remains in the coffee substitutes.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaves or to herbs before fermentation.63 The food enzyme–TOS remains in the final products.

In the production of plant extracts, the food enzyme is added to the plant materials during maceration.64 The subsequent downstream processing steps applied, which include a distillation step to obtain flavouring preparations, are expected to remove the food enzyme‐TOS from the extracts. To substantiate this removal, the applicant measured the nitrogen content in the flavour condensate obtained from an orange juice treated with a polygalacturonase (as a proxy for arabinan endo‐1,5‐α‐L‐arabinanase), and no nitrogen was detected.65 The Panel considered the technical information and experimental data sufficient to demonstrate the removal of the food enzyme–TOS in these types of plant extracts.

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 in the majority of the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remains. However, it may remain in its active form in wine and juices, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated firstly according to the uses proposed by the applicant for the food manufacturing processes where the food enzyme–TOS remains in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool66 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 the contribution from each FoodEx category to the total dietary exposure are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 51 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 27 European countries (Appendix B). The highest dietary exposure was estimated to be 1.794 mg TOS/kg bw per day in children at the 95th percentile. Fruit juices are among the four top contributing foods for children.

The Panel noted that, according to the Directive 2012/12/EU,67 the use of arabinan endo‐1,5‐α‐L‐arabinanase is not permitted in the treatment of fruits for juice production. The Panel made another estimation by excluding the use of this food enzyme for juice production. The results are reported in Table 4.

Uncertainty analysis

3.5.3

In accordance with the guidance provided in the EFSA opinion related to uncertainties in dietary exposure assessment (EFSA, 2006), the following sources of uncertainties have been considered and are summarised in Table 5.

The conservative approach applied to estimate the 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.

The exclusion of four food manufacturing processes from the exposure estimation was based on > 99% of TOS removal. This is not expected to impact the overall estimate derived.

Margin of exposure

3.6

When the food enzyme is used in 12 food manufacturing processes as proposed by the applicant, a comparison of the NOAEL (301 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.021–0.831 mg TOS/kg bw per day at the mean and from 0.083 to 1.794 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure (MoE) for infants, toddlers, children, adolescents, adults and the elderly of 525, 321, 168, 497, 403 and 286, respectively. The calculated MoE would indicate the safety concern for the children.

When the food enzyme is used in 11 food manufacturing processes (production of juices excluded – a scenario taking into account Directive 2012/12/EU68 where the use of arabinan endo‐1,5‐α‐L‐arabinanase is not permitted in the treatment of fruits for juice production), the MoE is calculated for 11 food manufacturing processes. A comparison of the NOAEL (301 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.001–0.421 mg TOS/kg bw per day at the mean and from 0.034 to 1.499 mg TOS/kg bw per day at the 95th percentile resulted in a MoE for infants, toddlers, children, adolescents, adults and the elderly, of 560, 508, 201, 595, 503 and 296, respectively.

CONCLUSIONS

4

Based on the data provided, the removal of TOS during four food manufacturing processes and the derived margin of exposure for the remaining seven processes, the Panel concluded that the food enzyme arabinan endo‐1,5‐α‐L‐arabinanase produced with the non‐genetically modified Aspergillus aculeatinus strain CBS 148915 does not give rise to safety concerns under the intended conditions of use in 11 food manufacturing processes (excluding the use in the production of juices).

REMARK

5

The term ‘olive oil’ is defined in the Regulation (EU) No 1308/201369 as ‘composed of refined olive oils and virgin olive oils’. The term ‘virgin olive oils’ means ‘oils obtained from the fruit of the olive tree solely by mechanical or other physical means under conditions that do not lead to alterations in the oil, which have not undergone any treatment other than washing, decantation, centrifugation or filtration, to the exclusion of oils obtained using solvents or using adjuvants having a chemical or biochemical action, or by re‐esterification process and any mixture with oils of other kinds’. In accordance with the law, the use of enzymes is not permitted in the production of virgin olive oils in the European Union. Therefore, the use of this food enzyme in the production of virgin olive oils is excluded from this assessment.

DOCUMENTATION AS PROVIDED TO EFSA

6

Technical dossier (joint dossier). 21 November 2014. Submitted by Solyve. The data package was updated on 31 March 2023.

Additional information. 25 June 2024. Submitted by Solyve.

ABBREVIATIONSAMFEPAssociation of Manufacturers and Formulators of Enzyme Productsbwbody weightCASChemical Abstracts ServiceCEFEFSA Panel on Food Contact Materials, Enzymes, Flavourings and Processing AidsCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsDONdeoxynivalenolEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsFEIMFood Enzyme Intake ModelFEZEFSA Panel on Food EnzymesFoodExstandardised food classification and description systemGLPGood Laboratory PracticeGMgenetically modifiedGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLAMaverage measurement of distance between ink blotsLAN1the first measurement of distance between ink blotsLAN2the second measurement of distance between ink blotsLoDlimit of detectionLoQlimit of quantificationMNBNbi‐nucleated cells with micronucleiMoEmargin of exposureNOAELno observed adverse effect levelnon‐GMnon‐genetically modifiedOECDOrganisation for Economic Co‐operation and DevelopmentRMraw materialTOStotal organic solidsUUnit■■■■■■■■■■WHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00241

COPYRIGHT FOR NON‐EFSA CONTENT

EFSA may include images or other content for which it does not hold copyright. In such cases, EFSA indicates the copyright holder and users should seek permission to reproduce the content from the original source.

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