Safety evaluation of an extension of use of the food enzyme mannan endo‐1,4‐β‐mannosidase from the non‐genetically modified Aspergillus niger strain AE‐HCM

TL;DR

This study evaluates the safety of expanding the use of a food enzyme produced by a non-genetically modified fungus in 13 food manufacturing processes.

Contribution

The study provides a revised safety assessment for extended use of mannan endo-1,4-β-mannosidase in additional food processes.

Findings

Dietary exposure to the enzyme was estimated at up to 0.586 mg TOS/kg body weight per day.

The margin of exposure was calculated to be at least 1242, indicating no safety concerns.

The enzyme is considered safe under the revised intended conditions of use.

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 microorganism Aspergillus niger strain AE‐HCM by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in three food manufacturing processes. Subsequently, the applicant requested to extend its use to include ten additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme when used in a total of 13 food manufacturing processes. As the food enzyme–total organic solids (TOS) are not carried into the final foods in one food manufacturing process, the dietary exposure to the food enzyme–TOS was estimated for the remaining 12 processes. The dietary exposure was calculated to be up to 0.586 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (728 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 1242. Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Tables

TABLE 1. Updated intended uses and use levels of the food enzyme. 7

Food manufacturing process a	Raw material (RM)	Maximum recommended use level (mg TOS/kg RM)
		Current evaluation b	Previous evaluation b , c
Processing of cereals and other grains
Production of baked products	Flour	5.4
Production of cereal‐based products other than baked	Cereals	5.4
Production of brewed products	Cereals	1.1
Production of distilled alcohol	Cereals	453.6
Processing of fruits and vegetables
Production of juices	Fruit and vegetables	5.4
Production of fruit and vegetable products other than juices	Fruit and vegetables	5.4
Production of wine and wine vinegar	Grapes	5.4
Production of alcoholic beverages other than grape wine	Fruit and vegetables	5.4
Processing of plant‐ and fungal‐derived products
Production of coffee extracts	Coffee beans	324	11₋324
Production of tea and other herbal and fruit infusions	Tea leaves	10.8
Production of plant‐based analogues of milk and milk products	Cereals, nuts, seeds	54	1₋54
Production of partially hydrolysed polysaccharides from galacto‐mannan gums d	Gums containing 1,4‐β‐d‐mannosidic linkages	1080	11₋1080
Processing of yeast and yeast products	Yeast	10.8

TABLE 2. Updated dietary exposure to the food enzyme–TOS in six population groups.

Population group	Estimated exposure (mg TOS/kg body weight per day)
	Infants	Toddlers	Children	Adolescents	Adults	The elderly
Age range	3–11 months	12–35 months	3–9 years	10–17 years	18–64 years	≥ 65 years
Min–max mean (number of surveys)	0.014–0.120 (12)	0.062–0.289 (15)	0.057–0.149 (19)	0.030–0.097 (21)	0.056–0.203 (22)	0.034–0.240 (23)
Min–max 95th percentile (number of surveys)	0.035–0.234 (11)	0.159–0.315 (14)	0.113–0.315 (19)	0.070–0.234 (20)	0.137–0.469 (22)	0.078–0.586 (22)

TABLE 3. 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 are produced by the enzymatic treatment, 24 food categories chosen for calculation included foods relevant not only to yeast extract, but also to yeast autolysates and yeast cell wall.	+
Exposure to food enzyme–TOS was always calculated based on the recommended maximum use level	+
Selection of broad FoodEx categories for the exposure assessment	+
Use of recipe fractions to disaggregate FoodEx categories	+/–
Use of technical factors in the exposure model	+/–
The estimation of the dietary exposure to the food enzyme–TOS via the partially hydrolysed polysaccharides from galacto‐mannan gums was based on the consumption data of two authorised food additives: guar gum (E412) and xanthan gum (E415).	+
The use of a conservative scenario to consider that the food enzyme–TOS fully remain in the partially hydrolysed polysaccharides from galacto‐mannan gums.	+
The applicant claimed that the partially hydrolysed polysaccharides are not consumed by infants. However, the calculation considered the exposure to all the population groups	+
Exclusion of one process from the exposure assessment: – production of distilled alcohol	–

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 European Union market and intended to remain on that market, as well as all new food enzymes, shall be subjected to a safety evaluation by the European Food Safety Authority (EFSA) and approval via an EU Community list.

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 1332/2008^1^ on food enzymes.

Mannan endo‐1,4‐β‐mannosidase from a non‐genetically modified strain of Aspergillus niger (strain AE‐HCM) is a food enzyme included in the Register of food enzymes3 to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 22 December 2022, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the conditions of use for the above food enzyme in several food processes.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessment and the assessment of possible confidentiality requests of an extension of the condition of use for the following food enzyme: Mannan endo‐1,4‐β‐mannosidase from a non‐genetically modified strain of Aspergillus niger (strain AE‐HCM), in accordance with Regulation (EC) No 1331/2008 establishing a common authorization procedure for food additives, food enzymes and food flavourings.4

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of the food enzyme mannan endo‐1,4‐β‐mannosidase from non‐genetically modified Aspergillus niger (AE‐HCM).

Additional information, requested from the applicant during the assessment process on 25 February 2025, was received on 17 March 2025 (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 existing guidance documents of EFSA Scientific Committee.

The ‘Scientific Guidance for the submission of dossiers on food enzymes’ (EFSA CEP Panel, 2021) and the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023) have been followed for the evaluation.

Public consultation

2.3

According to Article 32c(2) of Regulation (EC) No 178/20025 and to the Decision of EFSA's Executive Director laying down the practical arrangements on pre‐submission phase and public consultations, EFSA carried out a public consultation on the non‐confidential version of the technical dossier from 22 January to 12 February 2024.6 No comments were received.

ASSESSMENT

3

IUBMB nomenclatureMannan endo‐1,4‐β‐mannosidaseSystematic name1,4‐β‐d‐mannan mannanohydrolaseSynonymsβ‐mannanase; endo‐β‐1,4‐mannase; endo‐β‐mannanase; β‐d‐mannanaseIUBMB No.3.2.1.78CAS No.37288‐54‐3EINECS No.253‐446‐5

Mannan endo‐1,4‐β‐mannosidases catalyse the random hydrolysis of 1,4‐β‐d‐glycosidic linkages in mannans, galactomannans and glucomannans, resulting in the generation of β‐1,4‐manno oligosaccharides.

All aspects concerning the safety of this food enzyme, when used in three food manufacturing processes, were evaluated in May 2025 (EFSA FEZ Panel, 2025).

Following a request to update the intended uses (adding 10 additional uses), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme, when used in 13 food manufacturing processes.

Dietary exposure

3.1

The current dietary exposure supersedes Section 3.5 of the previous evaluation (EFSA FEZ Panel, 2025).

Revised intended use of the food enzyme

3.1.1

The food enzyme is intended to be used in 13 food manufacturing processes at the recommended use levels summarised in Table 1.

TABLE 1: Updated intended uses and use levels of the food enzyme. 7

The additional 10 uses of the food enzyme are described below.

In the additional food manufacturing processes, the food enzyme acts on the mannans, galactomannans and glucomannans present in the different raw materials, contributing to improved sensory properties or yields of the final products.8

In the production of baked products, the food enzyme is added to the flour during the preparation of dough.9 The food enzyme–TOS remain in the final baked foods.

In the production of cereal‐based products other than baked, the food enzyme is added to cereals after milling.10 The food enzyme–TOS remain in the final cereal products.

In the production of brewed products, the food enzyme is added during the mashing step for beer production,11 while it can be added to cereals during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps for the production of fermented beverages like sake or rice wine.12 The food enzyme–TOS remain in the final brewed products.

In the production of distilled alcohol, the food enzyme can be added to cereals during the slurry mixing, the liquefaction, the pre‐saccharification or the fermentation steps.13 The food enzyme–TOS are not carried over with the distilled alcohols (EFSA CEP Panel, 2023).

In the production of juices, the food enzyme is added to the depectinised fruit juice14 to aid clarification, while in the production of fruit and vegetable products other than juices, it is added to mashed fruit and vegetables after the straining step15 to improve the yield.16 The food enzyme–TOS remain in the final foods.

In the production of wine and wine vinegars, the food enzyme can be added to grapes during the maceration or during the fermentation steps.17 The food enzyme–TOS remain in wine and wine vinegars.

In the production of alcoholic beverages other than grape wine, the food enzyme can be added to the mashed fruits or to the juice before fermentation.18 The food enzyme–TOS remain in the alcoholic beverages.

In the production of tea and other herbal and fruit infusions, the food enzyme is added to tea leaves at the beginning of the food manufacturing process.19 The food enzyme–TOS remain in the final foods.

In the processing of yeast and yeast products, the food enzyme can be added to yeast during lysis or during the incubation step20 to obtain yeast extracts,21 which are used as ingredients for various savoury foods, ready‐to‐eat vegetable meals, soups, bouillons and sauces. The food enzyme–TOS remain in the yeast extracts.

The food enzyme has a temperature optimum around 80°C (pH 5.0) and a pH optimum around pH 5.0 (40°C). After a pre‐incubation of the food enzyme at different temperatures for 60 min (pH 5.0), the food enzyme activity decreased above 70°C showing no residual activity above 80°C (EFSA FEZ Panel, 2025). Based on these data, the Panel concluded that the food enzyme is inactivated in beers. It may remain in its active form in other foods, in which the food enzyme–TOS remain, depending on the heat treatment conditions during the respective food manufacturing process.

Dietary exposure estimation

3.1.2

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated for the 12 food manufacturing processes where the food enzyme–TOS remain in the final foods.

Chronic exposure to the food enzyme–TOS was calculated using the FEIM webtool22 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) together with the information provided in Appendix C.

Partially hydrolysed guar gum (E412) and xanthan gum (E415) are authorised food additives in the EU according to Annex II and Annex III of Regulation (EC) No 1333/2008 on food additives.23 The EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS Panel) re‐evaluated guar gum (EFSA ANS Panel, 2017a) and xanthan gum (EFSA ANS Panel, 2017b) when used as food additives. To enable a single calculation of the food enzyme–TOS from all dietary sources, in this assessment, the FoodEx categories considered by the ANS Panel were disaggregated to the more detailed hierarchy level 7 to estimate the dietary exposure to the food enzyme–TOS via partially hydrolysed polysaccharides from galacto‐mannan gums (see Appendix C).

Table 2 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 0.586 mg TOS/kg bw per day in the elderly at the 95th percentile.

Uncertainty analysis

3.1.3

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 3.

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 one food manufacturing process from the exposure assessment was based on > 99% of TOS removal. This is not expected to impact on the overall estimate derived.

Margin of exposure

3.2

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 728 mg TOS/kg body weight (bw) per day, the highest dose tested, resulting in a margin of exposure (MoE) of at least 1282 (EFSA FEZ Panel, 2025).

A comparison of the NOAEL with the newly derived exposure estimates of 0.014–0.289 mg TOS/kg bw per day at the mean and from 0.035 to 0.586 mg TOS/kg bw per day at the 95th percentile resulted in a MoE of at least 1242.

Despite a higher number of food manufacturing processes was considered in the current assessment, the newly derived dietary exposure is only slightly increased, leading to a slightly lower MoE when compared to the previous evaluation (EFSA FEZ Panel, 2025). This is because the major contributing foods came from the food manufacturing processes previously assessed, but not from the extended uses.

CONCLUSION

4

Based on the new data, the revised margin of exposure and the previous evaluation, the Panel concluded that the food enzyme mannan endo‐1,4‐β‐mannosidase produced with the non‐genetically modified A. niger strain AE‐HCM does not give rise to safety concerns under the revised intended conditions of use.

REMARKS

5

The production of the food additives guar gum E412 and xanthan gum E415 as defined in Regulation (EU) No 231/2012 laying down specification for food additives, is not covered by this assessment.

This food enzyme is intended to be used in the manufacturing of partially hydrolysed polysaccharides from galacto‐mannan gums. The safety evaluation of partially hydrolysed polysaccharides from galacto‐mannan gums as such is outside the remit of the EFSA FEZ Panel.

The use of this mannan endo‐1,4‐β‐mannosidase from the non‐genetically modified A. niger strain AE‐HCM 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 the Directive 2012/12/EU, the use of mannan endo‐1,4‐β‐mannosidase is not permitted in the treatment of fruits for juice production.

DOCUMENTATION AS PROVIDED TO EFSA

6

Application for authorisation of mannan endo‐1,4‐β‐mannosidase from Aspergillus niger AE‐HCM in accordance with the Regulation (EC) No 1331/2008. December 2022. Submitted by Amano Enzymes Inc.

Additional information. March 2025. Submitted by Amano Enzymes Inc.ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsECEuropean CommissionEINECSEuropean Inventory of Existing Commercial Chemical SubstancesEUEuropean UnionFEZEFSA Panel on Food EnzymesIUBMBInternational Union of Biochemistry and Molecular BiologyMOEmargin of exposureNOAELno observed adverse effect levelRMRaw MaterialTOStotal organic solids

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00414

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

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

Supporting information

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