Safety evaluation of the food enzyme mannan endo‐1,4‐β‐mannosidase from the non‐genetically modified Aspergillus niger strain ACH 12‐525

TL;DR

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

Contribution

The study provides a comprehensive safety evaluation of mannan endo-1,4-β-mannosidase from a non-GMO Aspergillus niger strain.

Findings

Genotoxicity tests showed no safety concerns for the food enzyme.

The no observed adverse effect level was 1331 mg TOS/kg bw per day in rats.

The enzyme's amino acid sequence showed no homology to known allergens.

Abstract

The food enzyme mannan endo‐1,4‐β‐mannosidase (1,4‐β‐D‐mannan mannanohydrolase; EC 3.2.1.78) is produced with the non‐genetically modified Aspergillus niger strain ACH 12‐525 by Shin Nihon Chemical Co., Ltd. The food enzyme was considered free from viable cells of the production organism. The food enzyme is intended to be used in three food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 0.579 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 1331 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 2299. A search for the homology of the amino acid sequence of the mannan endo‐1,4‐β‐mannosidase 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.

Tables

TABLE 1. Composition of the food enzyme.

Parameters	Unit	Batches
		1	2	3 a	4 b
Mannan endo‐1,4‐β‐mannosidase activity	U/g c	35,900	35,100	35,200	37,500
Protein	%	9.8	9.9	9.6	10.3
Ash	%	0.2	0.2	0.2	0.2
Water	%	87.7	87.8	87.7	87.0
Total organic solids (TOS) d	%	12.1	12.0	12.1	12.8
Activity/TOS ratio	U/mg TOS	297	293	291	293

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 35

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM) b
Processing of fruits and vegetables
Production of wine and wine vinegar	Grapes	42
Processing of plant‐ and fungal‐derived products
Production of coffee extracts	Coffee beans	169
Processing of sugars
Production of oligosaccharides (manno‐oligosaccharides)	Coffee beans, copra meal, palm kernel meal, guar gum	339

TABLE 3. Summary of the estimated dietary exposure to food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.077–0.253 (12)	0.011–0.112 (15)	0.005–0.018 (19)	0.003–0.014 (21)	0.028–0.157 (22)	0.025–0.210 (23)
Min–max 95th percentile (number of surveys)	0.258–0.579 (11)	0.056–0.255 (14)	0.012–0.052 (19)	0.008–0.066 (20)	0.134–0.408 (22)	0.063–0.463 (22)

TABLE 4. Qualitative evaluation of the influence of uncertainties on the dietary exposure estimate.

Sources of uncertainties	Direction of impact
Model input data
Consumption data: different methodologies/representativeness/underreporting/misreporting/no portion size standard	+/−
Use of data from food consumption surveys of a few days to estimate long‐term (chronic) exposure for high percentiles (95th percentile)	+
Possible national differences in categorisation and classification of food	+/−
Model assumptions and factors
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
Assumption that the food enzyme‐TOS are fully transferred in wines and in MOS	+
MOS are mainly found in supplements; such consumption data are not yet well captured in the EU	+/−
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	+/−

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 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.

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 Union list may be placed on the market as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/20081^1^ 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 1331/2008^2^, 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.

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 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 1332/2008^1^ 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 food enzyme Cellulase, Glucanase and Hemicellulase covering Xylanase and Mannanase from Aspergillus niger submitted by AMFEP.

The application was submitted initially as a joint dossier3 and identified as the EFSA‐Q‐2015‐00340, EFSA‐Q‐2018‐01034 and EFSA‐Q‐2018‐01035. During a meeting between EFSA, the European Commission and the AMFEP,4 it was agreed that joint dossiers will be split into individual data packages.

The current opinion addresses one data package originating from the former joint dossier EFSA‐Q‐2015‐00340/EFSA‐Q‐2018‐01034/EFSA‐Q‐2018‐01035. This data package is identified as EFSA‐Q‐2023‐00242 and concerns the food enzyme mannan endo‐1,4‐β‐mannosidase produced from the Aspergillus niger strain ACH 12‐525 and submitted by Shin Nihon Chemical Co., Ltd.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in March 2023 in support of the application for authorisation of the food enzyme cellulase from a non‐genetically modified Aspergillus niger (strain ACH 12‐525). The dossier was updated in July 2023 as an ‘application for the authorisation of endo‐1,4‐β‐mannosidase from aspergillus niger strain ACH 12‐525’.

Additional information was requested from the applicant during the assessment phase on 13 July 2023 and 13 March 2024 and was received on 20 July 2023 and 12 June 2024, respectively (see Section 5).

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, 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).

ASSESSMENT

3

IUBMB nomenclatureMannan endo‐1,4‐β‐mannosidaseSystematic name1,4‐β‐D‐mannan mannanohydrolaseSynonymsEndo‐1,4‐β‐mannanase; β‐mannanase; endo‐β‐mannanase; β‐D‐mannanaseIUBMB No3.2.1.78CAS No37288‐54‐3EINECS No253‐446‐5

Mannan endo‐1,4‐β‐mannosidases catalyse the random hydrolysis of 1,4‐β‐mannosidic linkages of mannans, galactomannans and related polysaccharides.

The food enzyme under assessment is intended to be used in three food manufacturing processes as defined in the EFSA guidance (EFSA CEP Panel, 2023): (1) processing of fruits and vegetables for the production of wine and wine vinegar; (2) processing of plant‐ and fungal‐derived products for the production of coffee extracts and (3) processing of sugars for the production of oligosaccharides.

Source of the food enzyme

3.1

The mannan endo‐1,4‐β‐mannosidase is produced with the non‐genetically modified filamentous fungus Aspergillus niger strain ACH 12‐525, which is deposited at the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan), with the deposition number ■■■■■.5 The production strain was identified as A. niger by ■■■■■.6 ^,^ 7

■■■■■.8

Production of the food enzyme

3.2

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 water, and the biomass is removed from the fermentation broth by centrifugation, followed by microfiltration. The filtrate containing the enzyme is 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.

Characteristics of the food enzyme

3.3

Properties of the food enzyme

3.3.1

The mannan endo‐1,4‐β‐mannosidase is a single polypeptide chain of ■■■■■ amino acids.13 The molecular mass of the mature protein, calculated from the amino acid sequence, is ■■■■■ kDa.14 The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis.15 A consistent protein pattern was observed across all batches. The gel showed a major protein band migrating between the protein markers of ■■■■■ and ■■■■■ kDa in all batches, consistent with the molecular mass of the enzyme.

No other enzyme activities were reported.16

The applicant's in‐house determination of mannan endo‐1,4‐β‐mannosidase activity is based on the hydrolysis of galactomannan ■■■■■. The enzyme activity is expressed in β‐mannanase activity units (U)/g. One unit is defined as the amount of enzyme that reduces the viscosity of 100 mL of test solution by half in 60 s under the conditions of this assay.17

The food enzyme has a temperature optimum around 70°C (■■■■■) and a pH optimum around pH 4.0 (■■■■■). Thermostability was tested by pre‐incubation of the food enzyme for 30 min at different temperatures (■■■■■). The enzyme activity decreased above 45°C, showing no residual activity at 80°C.18

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for four batches used for commercialisation, among which batches 3 and 4 were also used for the toxicological tests (Table 1).19 The mean total organic solids (TOS) was 12.3% and the mean enzyme activity/TOS ratio was 294 U/mg TOS.

Purity

3.3.3

The lead content in all batches was below 5 mg/kg,20 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 arsenic content was below the limit of quantification (LoQ) of the employed method.21 ^,^ 22

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

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, G2), fumonisins (B1, B2), ochratoxin A, sterigmatocystin, T‐2 toxin and zearalenone was examined in three food enzyme batches and was below the LoQ of the applied methods.25 ^,^ 26 Adverse effects caused by the possible presence of other secondary metabolites are addressed by the toxicological examination of the food enzyme.

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

Viable cells and DNA of the production strain

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 were detected. A positive control was included.27

Toxicological data

3.4

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

The batches 3 and 4 (Table 1) used in these studies were used for commercialisation and have a similar enzyme activity/TOS ratio as the other two batches, and thus are considered suitable as test items.

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, 1997a), the Japanese Guidelines (Notification No. 29, MHW 1996) and following good laboratory practice (GLP).28 A range‐finding test and three main experiments were carried out in triplicate.

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA were used with or without metabolic activation (S9‐mix), applying the pre‐incubation or ‘treat and wash’ method.

Based on the results of a dose finding study, the main study applying the pre‐incubation method was carried out using S. Typhimurium TA98, TA100, TA1535, TA1537 and E. coli WP2uvrA without S9‐mix and S. Typhimurium TA100, TA1537 and E. coli WP2uvrA with S9‐mix and six concentrations of the food enzyme of 420, 830, 1670, 3330, 6660 and 13,311 μg TOS/plate. The growth of the bacterial lawn was stimulated in the absence of S9‐mix: at ≥ 6655 μg TOS/plate in TA100 and TA1535 strains, at ≥ 3328 μg TOS/plate in TA98 and TA1537 strains and at ≥ 416 μg TOS/plate in E. coli WP2uvrA and in the presence of S9‐mix: at ≥ 416 μg TOS/plate in E. coli WP2uvrA, at ≥ 1666 μg TOS/plate in TA100 strain and at ≥ 6655 μg TOS/plate in TA1537 strain. An increase in the number of revertant colonies above the control values was observed at 13,311 μg TOS/plate in TA100 strain and at 6655 and 13,311 μg TOS/plate in TA98 strain without S9‐mix and at 13,311 μg TOS/plate in TA100 strain with S9‐mix.

The stimulation of the growth of the bacterial lawn, observed in the dose finding and the main study applying the pre‐incubation method, was attributed to the presence of free amino acids in the food enzyme by the study authors.

The main study applying the treat and wash method was carried out using S. Typhimurium TA100 and TA98 without S9‐mix and S. Typhimurium TA100, TA98, TA1535 with S9‐mix and seven concentrations of the food enzyme of 20, 50, 160, 490, 1480, 4440 and 13,311 μg TOS/plate. The growth of the bacterial lawn was stimulated at ≥ 1478 μg TOS/plate in TA100 and TA98 strains in the absence of S9‐mix. 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.

A confirmative study applying the treat and wash method was carried out using TA100 and TA98 strains and six concentrations of the food enzyme of 420, 830, 1670, 3330, 6660 and 13,311 μg TOS/plate without S9‐mix and using TA100, TA98 and TA1535 strains and eight concentrations of the food enzyme of 100, 210, 420, 830, 1670, 3330, 6660 and 13,311 μg TOS/plate with S9‐mix. The growth of the bacterial lawn was stimulated at ≥ 833 μg TOS/plate in TA100 and TA98 strains in the absence of S9‐mix. 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 mannanase did not induce gene mutations under the test conditions applied in this study.

In vitro mammalian chromosomal aberration test

3.4.1.2

The in vitro mammalian chromosomal aberration test was carried out according to Japanese Guidelines (Notification No. 29, MHW 1996), OECD Test Guideline 473 (OECD, 1997b) and following GLP.29 An experiment was performed with duplicate cultures of a Chinese hamster lung fibroblast cell line (CHL/IU). The cell cultures were treated with the food enzyme either with or without metabolic activation (S9‐mix).

In the range finding test, no cytotoxicity above 50% was seen at any concentration tested up to 13,311 μg TOS/mL.

Based on these results, in the main experiment, cells were exposed to the food enzyme and scored for chromosomal aberrations at concentrations of 3328, 6655 and 13,311 μg TOS/mL in a short‐term treatment (6‐h exposure and 18‐h recovery period) either with or without 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 (with or without S9‐mix) or in long‐term treatment. The frequency of structural and numerical aberrations 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 mannanase did not induce an increase in the frequency of structural and numerical aberrations under the test conditions applied in this study.

In vitro mammalian cell micronucleus test

3.4.1.3

The in vitro mammalian cell micronucleus test was carried out according to OECD Test Guideline 487 (OECD, 2016) and following GLP.30 Two separate experiments were 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 above 50% was seen at any concentration tested up to 5000 μg TOS/mL with and without metabolic activation (S9‐mix).

In the first experiment, cells were exposed to the food enzyme and scored for the frequency of binucleated cells with micronuclei (MNBN) at concentrations of 1000, 2000 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 and 24‐h recovery period) without S9‐mix. No cytotoxicity was seen either in the short‐term with and/or without S9‐mix or in the long‐term treatment. In the long‐term treatment, the frequency of MNBN was statistically significantly different from the negative controls at all concentrations tested, with concentration response, however, within the 95% of the historical control range.

In the second confirmatory experiment, cells were exposed to the food enzyme and scored for MNBN at concentrations of 1000, 2000 and 5000 μg TOS/mL in a long‐term treatment (24‐h exposure and 24‐h recovery period) without S9‐mix. No cytotoxicity was seen at any concentration tested. 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 mannanase did not induce an increase in the frequency of MNBNs 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 followed the Japanese Guidelines (Notification No. 29, MHW 1996), OECD Test Guideline 408 (OECD, 1998) and GLP31 with the following deviation: functional observation tests were not performed. The Panel considered that this deviation is minor and does not impact on the evaluation of the study.

Groups of 10 male and 10 female Sprague–Dawley Crj:CD(SD)IGS [SPF] rats received the food enzyme by gavage in doses of 13, 133 and 1331 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

Clinical chemistry investigation revealed a statistically significant increase in the blood urea nitrogen concentrations (BUN) in low‐ and high‐dose males (+14%, +19%, respectively) and a decrease in γ‐glutamyl transpeptidase (γ‐GTP) activities in high‐dose males (−25%). 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 (BUN), the change was small (γ‐GTP), there were no changes in other relevant parameters (creatinine, other enzymes) and there were no histopathological changes in liver and kidneys.

The urinalysis revealed a statistically significant increase in osmotic pressure in high‐dose males (+18%), a decrease in sodium (Na) concentrations in low‐dose females (−30%) and an increase in Na excretion in mid‐dose males (+34%). 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 (Na concentrations and excretion), there were no changes in other relevant parameters (other electrolytes) and there were no histopathological changes in kidneys.

Statistically significant changes detected in organ weights were an increase in the absolute lung weight in high‐dose females (+7%), a decrease in the absolute weight of the adrenal glands in low‐dose males (−16%), an increase in the relative kidney weight in high‐dose males (+11%) and a decrease in the relative weight of the adrenal glands in low‐dose males (−17%). 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 (the absolute and relative weights of the adrenal glands), there were no histopathological changes in these organs and the changes were within the historical control values.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1331 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 mannan endo‐1,4‐β‐mannosidase produced with the Aspergillus niger strain ACH 12‐525 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.32

No reports on oral or respiratory sensitisation or elicitation reactions of the endo‐1,4‐β‐mannosidase under assessment have been published.33

A mannan endo‐1,4‐β‐mannosidase was identified as a putative tomato allergen using mass spectrometry (Welter et al., 2013). No further information on this IgE‐binding protein is available, and it is not listed in allergen databases.

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 mannan endo‐1,4‐β‐mannosidase 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 wheat that may cause allergies or intolerances (listed in the Regulation (EU) No 1169/201134), is used as raw material. In addition, ■■■■■ and ■■■■■, known sources of allergens, are present in the culture medium. During the fermentation process, these products will mostly be degraded and utilised by the production strain.

Taken together, concerning the potential allergic reactions due to the production strain and the raw material in the culture medium, the Panel considered that residual amounts of allergenic proteins could be present in the food enzyme. Taking into account the level of dietary exposure (see Section 3.5.2), this would result in minute amounts in the final foods, from which allergic reactions are usually not expected.

In conclusion, the Panel considered that, under the intended conditions of use, a risk of allergic reactions upon dietary exposure to this food enzyme cannot be excluded, but that the likelihood is low.

Dietary exposure

3.5

Intended use of the food enzyme

3.5.1

The food enzyme is intended to be used in three food manufacturing processes at the recommended use levels summarised in Table 2.

TABLE 2: Intended uses and recommended use levels of the food enzyme as provided by the applicant. 35

In the production of wine and wine vinegar, the food enzyme may be added to the grape must during maceration, fermentation or pressing steps.36 The endo‐1,4‐β‐mannanase degrades the structural polysaccharides, lowering the viscosity and facilitating the release of phenolic compounds. The subsequent processing steps during wine making, such as ultrafiltration, could theoretically remove the food enzyme–TOS from the wines. However, in the absence of analytical data demonstrating the extent of such removal,37 the Panel opted for a conservative scenario, assuming that 100% of the food enzyme–TOS are transferred into the wines.

In the production of coffee extracts, the food enzyme is added to the aqueous extract of coffee.38 The endo‐1,4‐β‐mannanase hydrolyses coffee mannans, reducing the viscosity of coffee extracts and gel formation. The food enzyme–TOS remain in the coffee products.

In the production of manno‐oligosaccharides (MOS), the food enzyme is added to mannan‐rich plant materials (coffee beans, copra meal, guar gum39 and palm kernel meal)40 to hydrolyse mannans, galactomannans and related polysaccharides. The resulting polysaccharide hydrolysate contains mannans of lower molecular mass and manno‐oligosaccharides. The subsequent purification processing includes treatment with activated charcoal and fractionation by ion exchange chromatography, which could theoretically remove the food enzyme–TOS from the MOS. However, in the absence of analytical data demonstrating the extent of such removal,41 the Panel opted for a conservative scenario and assumed that 100% of the food enzyme–TOS are transferred into the MOS, which are used as ingredients in a variety of foods and beverages including infant formulae.42

Based on the 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 will be inactivated in coffee extracts and MOS, but may remain in its active form in wines and wine vinegar, depending on the heat treatment conditions.

Dietary exposure estimation

3.5.2

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool43 by combining the maximum recommended use level with individual consumption data (EFSA CEP Panel, 2021). The estimation involved selection of relevant food categories and application of technical conversion factors (EFSA CEP Panel, 2023).

Table 3 provides an overview of the derived exposure estimates across all surveys. Detailed mean and 95th percentile exposure to the food enzyme–TOS per age class, country and survey, as well as contribution from each FoodEx category to the total dietary exposure, are reported in Appendix A – Tables 1 and 2. For the present assessment, food consumption data were available from 48 dietary surveys (covering infants, toddlers, children, adolescents, adults and the elderly), carried out in 26 European countries (Appendix B). The highest dietary exposure was estimated to be 0.579 mg TOS/kg bw per day in infants at the 95th percentile.

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 4.

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.

Margin of exposure

3.6

A comparison of the NOAEL (1331 mg TOS/kg bw per day) identified from the 90‐day rat study with the derived exposure estimates of 0.003–0.253 mg TOS/kg bw per day at the mean and from 0.008 to 0.579 mg TOS/kg bw per day at the 95th percentile resulted in a margin of exposure of at least 2299.

CONCLUSIONS

4

Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme mannan endo‐1,4‐β‐mannosidase produced with the non‐genetically modified Aspergillus niger strain ACH 12‐525 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for the authorisation of mannan endo‐1,4‐β‐mannosidase from Aspergillus niger strain ACH 12‐525 as a food enzyme in the European Union. July 2023. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information July 2023. Submitted by Shin Nihon Chemical Co., Ltd.

Additional information June 2024. Submitted by Shin Nihon Chemical Co., Ltd.

ABBREVIATIONSbwbody weightBUNblood urea nitrogen concentrationsCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFAOFood and Agricultural Organization of the United NationsFEZEFSA Panel on Food enzymesGLPGood Laboratory PracticeGMOgenetically modified organismγ‐GTPγ‐glutamyl transpeptidaseIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditiveskDakiloDaltonLOQlimit of quantificationMHWMinistry of Health and WelfareMNBNbi‐nucleated cells with micronucleiMOSmanno‐oligosaccharidesNOAELno observed adverse effect levelOECDOrganisation for Economic Cooperation and DevelopmentRMraw materialTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00242

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PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Francesco Catania, Gabriele Gadermaier, Ralf Greiner, Baltasar Mayo, Alicja Mortensen, Yrjö Henrik Roos, Marize de Lourdes Marzo Solano, Henk Van Loveren, Laurence Vernis and Holger Zorn.

NOTE

The full opinion will be published in accordance with Article 12 of Regulation (EC) No 1331/2008 once the decision on confidentiality is received from the European Commission.

Supporting information

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