Safety evaluation of the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase from the non‐genetically modified Aspergillus tubingensis strain CBS 138353

Abstract

The food enzyme containing endo‐1,4‐β‐xylanase (4‐β‐D‐xylan xylanohydrolase; EC 3.2.1.8) and endo‐1,3(4)‐β‐glucanase (3‐(1‐3;1‐4)‐β‐D‐glucan 3(4)‐glucanohydrolase; EC 3.2.1.6) is produced with the non‐genetically modified Aspergillus tubingensis strain CBS 138353 by Solyve. The food enzyme was considered free from viable cells of the production organism. It is intended to be used in 11 food manufacturing processes. Since residual amounts of food enzyme–total organic solids (TOS) are removed during three processes, dietary exposure was calculated for the remaining eight food manufacturing processes. It was estimated to be up to 2.635 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 1230 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 467. A search for the homology of the amino acid sequences of the food enzyme to known allergens was made and no match was found. The Panel considered that a risk of allergic reactions upon dietary exposure to the food enzyme cannot be excluded, but that the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides a 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 1332/2008 on food enzymes.

Four applications have been introduced by the companies “Puratos NV sa.”, “Novozymes A/S.”, “Meito Sangyo Co., Ltd” and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzymes Inulinase from a genetically modified strain of Aspergillus Oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger respectively.

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 four applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Inulinase from a genetically modified strain of Aspergillus oryzae (strain MUCL 44346), Trypsin from porcine pancreatic glands, Triacylglycerol lipase from Candida cylindracea, and Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger 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 food enzyme Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger submitted by AMFEP.

The application was submitted initially as a joint dossier ⁴ and identified as the EFSA‐Q‐2015‐00340, EFSA‐Q‐2018‐01035 and EFSA‐Q‐2018‐01034. During a meeting between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP), ⁵ it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses two data packages originating from the former joint dossier. These data packages are identified as EFSA‐Q‐2023‐00230 and EFSA‐Q‐2023‐00235 and concern the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase produced from the non‐genetically modified Aspergillus tubingensis strain CBS 138353 and submitted by Solyve. Although the original mandate refers to the food enzyme as produced from Aspergillus niger, the new data package identified the production microorganism as Aspergillus tubingensis.

The feasibility to combine the assessment of two EFSA question numbers in one single opinion is based on the following clarification from the applicant: (i) the production strain of the endo‐1,4‐β‐xylanase (EFSA‐Q‐2023‐00230) and the endo‐1,3(4)‐β‐glucanase (EFSA‐Q‐2023‐00235) is identical; (ii) the fermentation and purification process are identical for both enzymes and (iii) the toxicological studies and the batches of the food enzyme presented in the two initial applications are the same. ⁶

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted two dossiers in support of the application for authorisation of the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase from the non‐genetically modified Aspergillus tubingensis strain CBS 138353.

Additional information was requested from the applicant during the assessment phase on 21 August 2023 and 25 March 2024 and received on 11 September 2023 and 25 June 2024 (see Section 6).

Following the reception of additional data by EFSA on 11 September 2023, EFSA requested a clarification teleconference on 17 October 2023.

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 contains two declared activities:

IUBMB nomenclature	Endo‐1,4‐β‐xylanase
Systematic name	4‐β‐D‐xylan xylanohydrolase
Synonyms	Xylanase; endo‐(1,4)‐D‐β‐xylanase
IUBMB No	EC 3.2.1.8
CAS No	9025‐57‐4
EINECS No	232‐800‐2

Endo‐1,4‐β xylanases catalyse the random hydrolysis of 1,4‐β‐D‐xylosidic linkages in xylans (including arabinoxylans), resulting in the generation of (1,4)‐β‐D‐xylan oligosaccharides of different lengths.

IUBMB nomenclature	Endo‐1,3(4)‐β‐glucanase
Systematic name	3‐(1,3;1,4)‐β‐D‐glucan 3(4)‐glucanohydrolase
Synonyms	Endo‐1,3‐β‐D‐glucanase; laminarinase; β‐1,3‐glucanase
IUBMB No	EC 3.2.1.6
CAS No	62213‐14‐3
EINECS No	263‐462‐4

Endo‐1,3(4)‐β‐glucanases catalyse the hydrolysis of 1,3‐ and 1,4‐β‐glycosidic linkages in mixed‐linked β‐D‐glucans resulting in the generation of partially hydrolysed β‐D‐glucans.

The food enzyme under assessment is intended to be used in 11 food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): processing of cereals and other grains for the production of (1) flour, (2) starch and gluten fractions, (3) baked products, (4) cereal‐based products other than baked, (5) brewed products and (6) distilled alcohol; processing of fruits and vegetables for the production of (7) juices, (8) fruit and vegetable products other than juices and (9) wine and wine vinegars; (10) processing of plant‐ and fungal‐derived products for the production of plant extracts as flavouring preparations and (11) processing of yeast and yeast products.

3.1. Source of the food enzyme

The food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase is produced with the non‐genetically modified filamentous fungus Aspergillus tubingensis strain CBS 138353 (AXA5), which is deposited as A. neoniger at the Westerdijk Fungal Biodiversity Institute Culture collection (The Netherlands), with the deposition number CBS 138353. ⁷ The production strain was identified as A. tubingensis by phylogenomic analysis. ⁸

A search for genes involved in the biosynthesis of secondary metabolites in the genome of the production strain was provided and no matches of concern were found.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004, ⁹ with food safety procedures based on Hazard Analysis and Critical Control Points and in accordance with good manufacturing practice. ¹⁰

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 enzyme is extracted with ■■■■■ and the solid biomass is removed by centrifugation. The supernatant containing the enzyme is then further purified and concentrated, including an ultrafiltration step in which the enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded. ¹¹ 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. ¹²

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‐1,4‐β‐xylanase consists of two isoforms of ■■■■■ and ■■■■■ amino acids, respectively. ¹³ The molecular mass of the mature proteins, calculated from the amino acid sequences, is ■■■■■ and ■■■■■ kDa. The endo‐1,3(4)‐β‐glucanase is a single polypeptide chain of ■■■■■ amino acids. ¹⁴ The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa. The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. A consistent protein pattern was observed across all batches. ¹⁵ α‐L‐Arabinofuranosidase, α‐glucosidase, β‐glucosidase and β‐galactosidase activities were reported by the applicant also to be present in the food enzyme. No other enzyme activities were reported. ¹⁶

The applicant's in‐house determination of endo‐1,4‐β‐xylanase activity is based on the hydrolysis of azo‐dyed oat‐spelt xylan (reaction conditions: pH 2.75, 30°C, 10 min) and the release of dyed oligosaccharides is measured spectrophotometrically at 590 nm. The enzyme activity is expressed in acid xylanase units (AXAU)/mL. One AXAU is defined as the quantity of enzyme that releases oligosaccharides so that the absorbance is increased by 0.2 units under the conditions of the assay. ¹⁷

The applicant's in‐house determination of endo‐1,3(4)‐β‐glucanase activity is based on the hydrolysis of azo‐dyed barley glucan (reaction conditions: pH 4.8, 30°C, 20 min) and the release of dyed oligoglucans is measured spectrophotometrically at 590 nm. The enzyme activity is expressed in β‐glucanase units (AGLU)/mL. One AGLU is defined as the quantity of enzyme that releases oligoglucans so that the absorbance is increased by 0.9 units under the conditions of the assay. ¹⁸

The endo‐1,4‐β‐xylanase has a temperature optimum around 50°C (pH 4.8) and a pH optimum around 3.5 (30°C). ¹⁹

The endo‐1,3(4)‐β‐glucanase has a temperature optimum around 55°C (pH 4.6) and a pH optimum around pH 4.5 (30°C). ²⁰

Thermostability was tested by pre‐incubation of the food enzyme for 10 min at different temperatures (pH 4.35). Endo‐1,4‐β‐xylanase activity decreased above 45°C showing no residual activity at 60°C ²¹ and endo‐1,3(4)‐β‐glucanase activity decreased above 50°C showing no residual activity at 65°C. ²²

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation, one of which (batch 3) was also used for the toxicological tests (Table 1). ²³ The mean total organic solids (TOS) of the three batches was 8.5% and the mean enzyme activity/TOS ratio was 218.9 AXAU/mg TOS for the endo‐1,4‐β‐xylanase and 58.9 AGLU/mg TOS for the endo‐1,3(4)‐β‐glucanase.

TABLE 1.

Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3 a
Endo‐1,4‐β‐xylanase activity	AXAU/g b	19,144	16,716	20,022
Endo‐1,3(4)‐β‐glucanase activity	AGLU/g c	5109	4543	5385
Protein	%	6.3	5.7	6.0
Ash	%	0.6	0.8	0.7
Water	%	90.7	91.0	90.7
Total organic solids (TOS) d	%	8.7	8.2	8.6
Endo‐1,4‐β‐xylanase activity/TOS ratio	AXAU/mg TOS	220.0	203.9	232.8
Endo‐1,3(4)‐β‐glucanase activity/TOS ratio	AGLU/mg TOS	58.7	55.4	62.6

^a Batch used for the toxicological studies.

^b AXAU: acid xylanase unit (see Section 3.3.1).

^c AGLU: β‐glucanase unit (see Section 3.3.1).

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

3.3.3. Purity

The lead content in the three batches was below 0.3 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, arsenic, cadmium and mercury concentrations were below the limits of quantification (LOQ) of the employed methods. ^{25 , 26}

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). 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 presence of aflatoxins (B1, B2, G1 and G2), zearalenone, ochratoxin A, deoxynivalenol, fumonisins (B1 and B2), T2‐toxin and HT2‐toxin was examined in all food enzyme batches and all were below the LoQ of the applied analytical methods, except for zearalenone which was detected in all batches in concentrations up to 7 μg/kg. ^{29 , 30}

Using the highest estimated dietary exposure of 2.635 mg TOS/kg bw per day as the reference (see Section 3.5.2), European consumers could be exposed up to 0.217 ng/kg bw per day to zearalenone. As this estimate is below the tolerable daily intake (TDI) for zearalenone (0.25 μg/kg bw per day, EFSA CONTAM Panel, 2011), the Panel considered these concentrations as of no concern. 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 was 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 three independent batches analysed in triplicate. Ten millilitres of product was diluted in 90 mL of aqueous solution, and 10 mL of the solution was filtered through a 0.45‐μm pore diameter membrane filter. The filters were then placed on non‐selective agar plates and incubated at 30°C for 4 days. No colonies were produced. A positive control was included. ³¹

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 one of the commercial 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). ³²

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

The first experiment was carried out using seven concentrations of the food enzyme (5, 16, 50, 160, 500, 1600 and 5000 μg TOS/plate); whereas in the second, six concentrations of the food enzyme (160, 300, 625, 1250, 2500 and 5000 μg TOS/plate) were tested.

Toxic effects, evident as a reduced background bacterial lawn and a reduction in the number of revertant colonies, occurred in TA1535 strain without S9‐mix at 2500 μg TOS/plate and above in the second experiment. 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 containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase 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. ³³ 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).

Based on the results of a range finding test 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 2000, 4000 and 5000 μg TOS/mL in a short‐term treatment (3‐h exposure and 21‐h recovery period) either with or without S9‐mix and in a long‐term treatment (24‐h exposure without recovery period) without S9‐mix.

A maximum cytotoxicity (21%) was seen at the highest concentration tested in the long‐term treatment. 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 containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase 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). ³⁴

Groups of 10 male and 10 female Hsd:Sprague–Dawley SD rats received the food enzyme by gavage in doses of 308, 615 or 1230 mg TOS/kg bw per day. Controls received the vehicle (reverse osmosis water).

One high‐dose male and one high‐dose female were found dead on day 72 and 86 of administration, respectively. The Panel considered the death of the high‐dose male as incidental in the absence of clinical symptoms preceding the death or gross post‐mortem and histopathological findings, and the death of the high‐dose female as due to miss‐dosing based on the clinical symptoms prior to the death and the necropsy findings.

The body weight gain was statistically significantly decreased on day 22 of administration in mid‐ and high‐dose females (−11% both). The Panel considered the change as not toxicologically relevant, as it was only recorded at a single time interval, in one sex, there was no dose–response relationship, and the change was without a statistically significant effect on the final body weight and/or the final body weight gain.

The feed consumption was statistically significantly decreased on day 71 of administration in low‐ and high‐dose males (−8% both); on day 85 in low‐, mid‐ and high‐dose males (−8%, −8% and −11%); on day 15 in mid‐ and high‐dose females (−7% both); on day 29 in high‐dose females (−8%); on day 50 in low‐, mid‐ and high‐dose females (−7%, −10% and −10%); and on day 85 in mid‐dose females (−13%). The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically, there was no dose–response relationship (all, except day 29), there was no statistically significant change in the final feed consumption, the body weight and/or the body weight gain.

In the functional observations, statistically significant decreases in the number of rearing events in mid‐ and high‐dose males on day 33 of administration (−45% and −40%) and in mid‐ and high‐dose females on day 69 (−22% and −19%) were observed. Statistically significant increases in the number of rearing events were observed in high‐dose males on day 12 (+107%), in mid‐dose males on day 40 (+32%), in low‐ and high‐dose females on day 48 (+18% both), in high‐dose females on day 76 (+26%) and in low‐dose females on day 90 (+16%). A statistically significant decrease in the mean landing foot splay was observed in low‐dose males on day 82 (LAM, −13%). A decrease in a forelimb grip second reading (−27%) was observed in high‐dose males on day 82. The Panel considered the changes as not toxicologically relevant, as they were only recorded sporadically, and there was no consistency between the changes at different time intervals (rearing events), it was recorded only in one sex (LAM, forelimb grip) and there was no dose–response relationship (LAM).

Haematological investigations revealed in mid‐dose females a statistically significant increase in absolute and relative count of neutrophils (+60% and +34%) and in absolute and relative count of monocytes (+75% and +53%) and a decrease in relative count of lymphocytes (−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 (all parameters), there were no changes in other relevant parameters (other white blood cell parameters) and there were no histopathological changes in the haematopoietic organs.

Clinical chemistry investigations revealed a statistically significant increase in alkaline phosphatase activity (AP) in high‐dose females (+26%), a decrease in alanine aminotransferase activity (ALT) in mid‐dose females (−28%) and in aspartate aminotransferase activity (AST) in low‐, mid‐ and high‐dose females (−26%, −17% and −25%), a decrease in bile acids concentration (BA) in low‐dose males (−24%), an increase in total bilirubin concentration (TB) in high‐dose females (+28%); moreover, the following changes in serum electrolyte concentrations were observed: decrease in chloride (Cl) in high‐dose males (−2%) and increases in low‐, mid‐ and high‐dose females (+1%, +2% and +2%), increases in calcium (Ca) in high‐dose females (+2%), in sodium (Na) in mid‐ and high‐dose females (+1% both) and in potassium (K) in mid‐ and high‐dose females (+12% and +10%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (AP, ALT, AST, BA, TB, Ca, Na, K), there was no consistency between the change in males and females (Cl), there was no dose–response relationship (ALT, AST, BA, Na, K) and there were no histopathological changes in liver and kidneys.

Statistically significant changes detected in organ weights were a decrease in absolute thymus weight in mid‐ and high‐dose males (−20% and −24%) and in mid‐ and high‐dose females (−16% and −22%), a decrease in relative thymus weight in mid‐ and high‐dose males (−21% and −24%) and in high‐dose females (−19%), a decrease in absolute adrenal glands weight in low‐ and mid‐dose females (−10% and −17%), a decrease in absolute heart weight in mid‐ and high‐dose females (−11% and −12%) and a decrease in absolute kidneys weight in high‐dose females (−8%). The Panel considered the changes as not toxicologically relevant, as they were only observed in one sex (absolute weights of adrenal glands, heart and kidneys), there was no dose–response relationship (absolute adrenal glands) and there were no correlated histopathological changes in thymus, adrenal glands, heart and kidneys.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1230 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‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase activities produced with Aspergillus tubingensis strain CBS 138353 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, no match was found. ³⁵

No reports on oral and respiratory sensitisation or elicitation reactions of the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase activities under assessment have been published.

Respiratory allergy, e.g. baker's asthma, following occupational exposure to xylanase has been described (Baur et al., 1998; Elms et al., 2003; Lipińska‐Ojrzanowska et al., 2016; Martel et al., 2010; Merget et al., 2001). Several studies have shown that individuals respiratorily sensitised to a food enzyme are usually able to ingest the corresponding enzyme without acquiring clinical symptoms of food allergy (Armentia et al., 2009; Cullinan et al., 1997; Poulsen, 2004). Adverse reactions upon dietary exposure to xylanases in individuals sensitised through the respiratory route have not been reported.

Allergenic 1,3‐β‐glucanases have been identified as candidates in latex–pollen–vegetable food cross‐reactivity (Callero et al., 2012; Palomares et al., 2005). In addition, 1,3‐β‐glucanases in olives (Palomares et al., 2003), banana (Choudhury et al., 2009) and bell pepper (Callero et al., 2012) have been indicated to be allergenic after oral exposure. However, the Panel noted that none of these allergens have a homology match with the endo‐1,3(4)‐β‐glucanase under assessment.

The Panel considered that the results of the sequence homology search and the available literature do not indicate a risk of allergic reactions upon dietary exposure to the 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.

■■■■■, a product from ■■■■■ that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/2011 ³⁶ ), is used as raw material. In addition, ■■■■■, a known source of allergens, are present in the culture medium. During the fermentation process, this product 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), 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 alcohol, 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.

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in 11 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. ³⁷

Food manufacturing process	Raw material (RM)	Maximal recommended use level (mg TOS/kg RM) a
Processing of cereals and other grains
Production of flour	Cereals	50
Production of starch and gluten fractions	Cereals	70
Production of baked products	Flour	15
Production of cereal‐based products other than baked	Flour, Cereals	70
Production of brewed products	Cereal	70
Production of distilled alcohol	Cereals	20
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	50
Production of fruit and vegetable products other than juices 38	Fruit and vegetables	50
Production of wine and wine vinegar	Grapes	50
Processing of plant‐ and fungal‐derived products
Production of plant extracts (as flavouring preparations)	Plant materials	160
Processing of yeast and yeast products	Yeast	1250

^a The numbers in bold represent the maximum recommended use levels, which were used for calculation.

In the production of flour and in the production of starch and gluten fractions, the food enzyme is added to grain together with water during mixing ³⁹ to reduce viscosity and increase yield. The food enzyme–TOS are removed from the gluten and starch fractions by repeated washing (EFSA CEP Panel, 2023) but remain in the flour.

In the production of baked and other cereal‐based products, the food enzyme is added to flour during the preparation of the dough ⁴⁰ to catalyse the hydrolysis of glucans and (arabino)xylans and reduce dough stiffness. The food enzyme–TOS remains in the final foods.

In the production of brewed products, the food enzyme is added to cereals during mashing ⁴¹ or to the wort during fermentation ⁴² to reduce viscosity and turbidity. The food enzyme–TOS remains in the brewed products.

In the production of distilled alcohol, the food enzyme is added during slurry mixing, pre‐saccharification, liquefaction or fermentation ⁴³ to reduce viscosity and to increase yield. The food enzyme–TOS is not carried over into the distilled alcohol (EFSA CEP Panel, 2023).

In the production of juices and in the production of fruit and vegetable products other than juices, the food enzyme is added to mashed fruit and vegetables ^{44 , 45} to facilitate the removal of peels and to reduce the viscosity. The food enzyme–TOS remains in the final foods.

During the production of juice concentrates from fruits and vegetables, flavouring preparations recovered by distillation are also obtained, ⁴⁶ which may be used to restore the flavour of fruit juices made from concentrates, or purees and jams. The distillation is expected to remove the food enzyme–TOS from the 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 endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase) and no nitrogen was detected. ⁴⁷ The Panel considered the technical information and experimental data sufficient to demonstrate the removal (> 99%) of the food enzyme–TOS from the flavouring preparations.

In the production of wine and wine vinegar, the food enzyme is added to grapes during pressing, maceration, fermentation and clarification. The food enzyme can also be added directly to the wine during filtration. ⁴⁸ The enzymatic reaction facilitates the release of colouring and flavouring substances. The subsequent downstream processing that may include ultrafiltration 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. ^{49 , 50} However, the LoD of the method corresponds to a concentration of the food enzyme 200 times higher than that used in wine making. ⁵¹ 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 are transferred into the wines and wine vinegars.

In the production of plant extracts, the food enzyme is added to the plant materials (e.g. garlic, leek, onion) during maceration. ⁵² The subsequent downstream processing that includes distillation to obtain flavouring preparations is 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 endo‐1,3(4)‐β‐glucanase and endo‐1,4‐β‐xylanase) and no nitrogen was detected. ⁵³ 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.

In processing of yeast and yeast products, the food enzyme is added to yeast before cell lysis ⁵⁴ to degrade cell wall glucans, improving the extraction process of cellular components from yeast. ⁵⁵ The food enzyme–TOS remains in the final yeast products.

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 in most of the food manufacturing processes listed in Table 2 in which the food enzyme–TOS remain. However, it may remain in its active form in brewed products, wines, wine vinegars and in flour, depending on the heat treatment conditions.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the eight 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 webtool ⁵⁶ 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 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 2.635 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	4–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.182–0.941 (14)	0.506–1.763 (17)	0.381–1.051 (21)	0.199–0.715 (23)	0.172–0.456 (23)	0.130–0.361 (25)
Min–max 95th percentile (number of surveys)	0.441–2.258 (13)	1.131–2.635 (16)	0.705–2.606 (21)	0.411–1.582 (22)	0.409–1.220 (23)	0.315–0.896 (24)

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
Although only yeast extracts and yeast cell walls are produced by the enzymatic treatment, 57 food categories chosen for calculation included also foods relevant to yeast autolysates.	+
Assumption that the food enzyme–TOS are fully transferred into wines	+
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	+/−
Exclusion of three processes from the exposure estimation: – Production of starch and gluten fractions – Production of distilled alcohol – Production of plant extracts (as flavouring preparations)	–

Note: +: 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.

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

3.6. Margin of exposure

A comparison of the NOAEL (1230 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.130–1.763 mg TOS/kg bw per day at the mean and from 0.315 to 2.635 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 467.

4. CONCLUSIONS

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase produced with the non‐genetically modified Aspergillus tubingensis strain CBS 138353 does not give rise to safety concerns under the intended conditions of use.

5. REMARK

The use of this food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase from the non‐genetically modified Aspergillus tubingensis strain CBS 138353 is not considered to raise a safety concern when used in the production of fruit and vegetable juices. However, the Panel noted that, according to Directive 2012/12/EU, the use of endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase is not permitted in the treatment of fruits for juice production.

6. DOCUMENTATION AS PROVIDED TO EFSA

Technical dossier xylanase and technical dossier beta‐glucanase. March 2023. Submitted by Solyve.

Additional information. September 2023 and June 2024. Submitted by Solyve.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

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

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMM

genetically modified microorganism

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

MNBN

bi‐nucleated cells with micronuclei

MOE

margin of exposure

OECD

Organisation for Economic Cooperation and Development

TOS

total organic solids

WHO

World Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00230, EFSA‐Q‐2023‐00235

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

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 16 weeks on up to and including 11 months of age	Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Poland, Portugal, Slovenia, Spain
Toddlers	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Montenegro,* Netherlands, Poland, Portugal, Republic of North Macedonia,* Serbia,* Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* Netherlands, Poland, 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,* Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro,* Netherlands, Poland, 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, Poland, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Croatia, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro,* Netherlands, Poland, Portugal, Romania, Serbia,* Slovenia, Spain, Sweden

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

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

EFSA FEZ Panel (EFSA Panel on Food Enzymes) , Zorn, H. , Barat Baviera, J. M. , Bolognesi, C. , Catania, F. , Gadermaier, G. , Greiner, R. , Mayo, B. , Mortensen, A. , Roos, Y. H. , Solano, M. L. M. , Van Loveren, H. , Vernis, L. , Lunardi, S. , Andryszkiewicz, M. , Cavanna, D. , Kovalkovicova, N. , & Liu, Y. (2026). Safety evaluation of the food enzyme containing endo‐1,4‐β‐xylanase and endo‐1,3(4)‐β‐glucanase from the non‐genetically modified Aspergillus tubingensis strain CBS 138353. EFSA Journal, 24(2), e9958. 10.2903/j.efsa.2026.9958