Safety evaluation of an extension of use of the food enzyme glucan 1,4‐α‐glucosidase from the non‐genetically modified Rhizopus arrhizus strain AE‐G

Abstract

The food enzyme glucan 1,4‐α‐glucosidase (4‐α‐d‐glucan glucohydrolase; EC 3.2.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐G 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 one food manufacturing process. Subsequently, the applicant requested to extend its use to nine additional processes and revised the use levels. In this assessment, EFSA updated the safety evaluation of this food enzyme for uses in a total of 10 food manufacturing processes. As the food enzyme–total organic solids (TOS) is removed from the final foods in two food manufacturing processes, the dietary exposure to the food enzyme–TOS was estimated only for the remaining eight processes. Dietary exposure was up to 0.424 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level previously reported (1868 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 4406. Based on the data provided for the previous evaluation and the margin of exposure revised in the present evaluation, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

1. INTRODUCTION

Article 3 of the Regulation (EC) No 1332/2008 ¹ provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or 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 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.

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 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 on food enzymes.

Glucoamylase from a non‐genetically modified strain of Rhizopus oryzae (AE‐G) is a food enzyme included in the Register of food enzymes ³ to be considered for inclusion in the Union list and thus subject to a risk assessment by the European Food Safety Authority (EFSA).

On 11 November 2022, a new application has been introduced by the applicant “Amano Enzyme Inc.” for an extension of the conditions of the use of the food enzyme glucoamylase from a non‐genetically modified strain of Rhizopus oryzae (AE‐G).

1.1.2. Terms of Reference

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: glucoamylase from a non‐genetically modified strain of Rhizopus oryzae (AE‐G), in accordance with Regulation (EC) No 1331/2008 establishing a common authorisation procedure for food additives, food enzymes and food flavourings. ⁴

1.1.3. Interpretation of the Terms of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of an extension of the conditions of use for the glucoamylase from the non‐genetically modified Rhizopus oryzae strain AE‐G.

The production microorganism was reclassified as Rhizopus arrhizus at the species level in the previously published scientific opinion (EFSA CEP Panel, 2023a). Therefore, the same species name Rhizopus arrhizus is used also in the present opinion.

2. DATA AND METHODOLOGIES

2.1. Data

The applicant has submitted a dossier in support of the application for the authorisation of the extension of use of the food enzyme glucoamylase from a non‐genetically modified Rhizopus oryzae strain AE‐G.

Additional information, requested from the applicant during the assessment process on 8 November 2023, were received on 24 November 2023 (see ‘Documentation provided to EFSA’).

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, 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, 2023b) have been followed for the evaluation of the application.

2.3. Public consultation

According to Article 32c(2) of Regulation (EC) No 178/2002 ⁵ 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.

3. ASSESSMENT

IUBMB nomenclature	Glucan 1,4‐α‐glucosidase
Systematic name	4‐α‐d‐glucan glucohydrolase
Synonyms	Glucoamylase, amyloglucosidase
IUBMB no	EC 3.2.1.3
CAS no	9032‐08‐0
EINECS no	232‐877‐2

Glucan 1,4‐α‐glucosidases catalyse the hydrolysis of terminal (1–4)‐linked α‐d‐glucose residues successively from the non‐reducing ends of amylopectin and amylose with the release of glucose.

All aspects concerning the safety of the food enzyme under assessment were evaluated in December 2022, when used in one food manufacturing process (EFSA CEP Panel, 2023a). Following a request to update the intended uses (adding nine food manufacturing processes and revising the use levels), EFSA revises the exposure assessment and updates the safety evaluation of this food enzyme when used in 10 food manufacturing processes.

3.1. Dietary exposure

The current dietary exposure supersedes section 3.5 in the previous evaluation (EFSA CEP Panel, 2023a).

3.1.1. Revised intended use of the food enzyme

The food enzyme is intended to be used in 10 food manufacturing processes, as described in the EFSA guidance (EFSA CEP Panel, 2023b), at the revised use levels summarised in Table 1.

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	8.8	79
Production of brewed products	Cereals	1.8
Production of cereal‐based products other than baked	Flour, grains, cereals	8.8
Production of glucose syrups and other starch hydrolysates	Starch	1055.1
Production of distilled alcohol	Cereals	703.4
Processing of fruits and vegetables
Production of non‐wine vinegar	Cereals	17.6
Production of juices	Fruit and vegetables	8.8
Processing of plant‐ and fungal‐derived products
Production of tea and other herbal and fruit infusions	Leaf extract rich in glucosylated steviol glycoside	17.6
Production of coffee extracts	Coffee beans, coffee extracts	87.9	4800 d
Production of plant‐based analogues of milk and milk products	Cereals	8.8

^a The name has been harmonised by EFSA according to the ‘Food manufacturing processes and technical data used in the exposure assessment of food enzymes’ (EFSA CEP Panel, 2023b).

^b The numbers in bold were used for calculation.

^c The previous evaluation is made for the food enzyme application EFSA‐Q‐2015‐00272.

^d This use level was proposed in EFSA‐Q‐2015‐00272 but not considered in the exposure assessment of the previous evaluation.

The Panel noted a substantial decrease in the use level recommended in the current assessment for the production of baked products when compared to the previous one. The applicant ascribes this change to a modification of the current food manufacturing process. ⁸

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

In the production of beer, the food enzyme is added to cereals during the mashing step. ⁹ For the production of fermented beverages like sake or rice wine, it may be added during the slurry mixing, the liquefaction, the fermentation or the pre‐saccharification steps. ¹⁰ The glucan 1,4‐α‐glucosidase hydrolyses starch in the mash to release glucose for fermentation. The food enzyme–TOS remains in the beer and other fermented beverages.

In the production of cereal‐based products other than baked and in the production of plant‐based analogues of milk and milk products, the food enzyme is added to the cereal slurry ¹¹ to release glucose from starch. The food enzyme–TOS remains in the final foods.

In the production of glucose syrups, the food enzyme is added during the saccharification step, ¹² where it hydrolyses starch and starch degradation products to release glucose. ¹³ The food enzyme–TOS is removed from the final glucose syrups by treatment with activated charcoal or similar and with ion‐exchange resins. This conclusion is extended to other starch hydrolysates (EFSA CEP Panel, 2023b).

In the production of distilled alcohol, the food enzyme is added during the pre‐saccharification and the fermentation steps. ¹⁴ It converts starch into a glucose‐rich solution, increasing the amounts of fermentable sugars to produce alcohol. ¹⁵ The food enzyme–TOS is not carried over with the distilled alcohols (EFSA CEP Panel, 2023b).

In the production of non‐wine vinegar, the food enzyme is added to milled grains or cereals during slurry mixing. ¹⁶ The glucan 1,4‐α‐glucosidase hydrolyses the starch content of the slurry to release glucose for fermentation. The food enzyme–TOS remains in the final foods.

In the production of juices, the food enzyme is added to the crushed fruits or vegetables during the depectinisation step. ¹⁷ The glucan 1,4‐α‐glucosidase catalyses the hydrolysis of starch, leading to higher yields, improved processability and reduced juice haziness. ¹⁸ The food enzyme–TOS remains in the juices.

In the processing of tea, herbal and fruit infusions, the food enzyme is added to the leaf extracts from Stevia. ¹⁹ The food enzyme acts on steviol glycosides in the Stevia leaves, which alters their glycosylation pattern and thus their sensory properties. ²⁰ The food enzyme–TOS remains in the final processed foods.

In the production of coffee extracts, the food enzyme is added to the ground coffee beans. ²¹ The glucan 1,4‐α‐glucosidase acts in combination with galactomannan degrading enzymes. The latter degrade the polysaccharides to oligosaccharides, on which the glucan 1,4‐α‐glucosidase can exert its activity. The hydrolysis catalysed by this food enzyme contributes to the reduction of turbidity and improves the filtration rate. ^{22 , 23} The food enzyme–TOS remains in the coffee extracts.

Based on the thermostability evaluated previously (EFSA CEP Panel, 2023a) and the downstream processing steps applied in the food processes, it is expected that the food enzyme is inactivated during the food manufacturing processes in which the food enzyme–TOS is not removed, with the exception of juice and non‐wine vinegar production. In these cases, the enzyme may remain active, depending on the processing conditions.

3.1.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), dietary exposure was calculated only 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 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, 2023b). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight (bw). This was done for all individuals across all surveys, resulting in distributions of individual average exposure. Based on these distributions, the mean and 95th percentile exposures were calculated per survey for the total population and per age class. Surveys with only 1 day per subject were excluded and high‐level exposure/intake was calculated for only those population groups in which the sample size was sufficiently large to allow calculation of the 95th percentile (EFSA, 2011).

Table 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 0.424 mg TOS/kg bw per day in toddlers at the 95th percentile.

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)	0.009–0.081 (12)	0.043–0.275 (15)	0.038–0.146 (19)	0.030–0.100 (21)	0.030–0.086 (22)	0.018–0.088 (23)
Min–max 95th percentile (number of surveys)	0.034–0.245 (11)	0.125–0.424 (14)	0.071–0.423 (19)	0.058–0.253 (20)	0.063–0.215 (22)	0.038–0.171 (22)

3.1.3. 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 3.

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
The consumption data covered all types of tea and herbal infusion products, not only the stevia infusion.	+
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 two processes from the exposure estimation: – production of glucose syrups and other starch hydrolysates – production of distilled alcohol	−

Abbreviations: +, uncertainty with potential to cause overestimation of exposure; –, uncertainty with potential to cause underestimation of exposure.

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

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

3.2. Margin of exposure

In the previous evaluation, the Panel identified a no observed adverse effect level (NOAEL) of 1868 mg TOS/kg bw per day, the highest dose tested, resulting in a margin of exposure (MOE) of at least 1987 (EFSA CEP Panel, 2023a).

A comparison of the NOAEL with the newly derived exposure estimates of 0.009–0.275 mg TOS/kg bw per day at the mean and from 0.034 to 0.424 mg TOS/kg bw per day at the 95th percentile resulted in a MOE of at least 4406.

Despite the fact that more uses were considered in the current assessment, the newly derived MOE is higher than that previously calculated. This is due to the revision of food groups and technical factors used for each food manufacturing process (EFSA CEP Panel, 2023b). In addition, dietary surveys have been updated in the EFSA food consumption database and the use levels have been updated by the applicant.

4. CONCLUSION

Based on the data provided for the previous evaluation and the revised MOE estimation, the Panel concluded that the food enzyme glucoamylase produced with the non‐genetically modified Rhizopus arrhizus strain AE‐G does not give rise to safety concerns under the revised intended conditions of use.

5. DOCUMENTATION AS PROVIDED TO EFSA

Application for authorisation of Glucoamylase from Rhizopus oryzae AE‐G in accordance with Regulation (EC) No 1331/2008. November 2022. Submitted by Amano Enzyme Inc.

Additional information. November 2023. Submitted by Amano Enzyme Inc.

ABBREVIATIONS

bw

body weight

CAS

Chemical Abstracts Service

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EC

European Commission

EINECS

European Inventory of Existing Commercial Chemical Substances

EU

European Union

IUBMB

International Union of Biochemistry and Molecular Biology

MOE

margin of exposure

NOAEL

no observed adverse effect level

RM

raw material

TOS

total organic solids

CONFLICT OF INTEREST

If you wish to access the declaration of interests of any expert contributing to an EFSA scientific assessment, please contact interestmanagement@efsa.europa.eu.

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2023‐00381

COPYRIGHT FOR NON‐EFSA CONTENT

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PANEL MEMBERS

José Manuel Barat Baviera, Claudia Bolognesi, Andrew Chesson, Pier Sandro Cocconcelli, Riccardo Crebelli, David Michael Gott, Konrad Grob, Claude Lambré, Evgenia Lampi, Marcel Mengelers, Alicja Mortensen, Gilles Rivière, Inger‐Lise Steffensen, Christina Tlustos, Henk Van Loveren, Laurence Vernis and Holger Zorn.

Supporting information

Dietary exposure estimates to the food enzyme–TOS in details

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

A.1.

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

B.1.

Population	Age range	Countries with food consumption surveys covering more than 1 day
Infants	From 12 weeks on up to and including 11 months of age	Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Italy, Latvia, Portugal, Slovenia, Spain
	From 12 months up to and including 35 months of age	Belgium, Bulgaria, Cyprus, Denmark, Estonia, Finland, France, Germany, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia*, Serbia*, Slovenia, Spain
Children	From 36 months up to and including 9 years of age	Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Netherlands, Portugal, Republic of North Macedonia*, Serbia*, Spain, Sweden
Adolescents	From 10 years up to and including 17 years of age	Austria, Belgium, Bosnia and Herzegovina*, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
	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, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden
The elderly a	From 65 years of age and older	Austria, Belgium, Cyprus, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Ireland, Italy, Latvia, Montenegro*, Netherlands, Portugal, Romania, Serbia*, Slovenia, Spain, Sweden

^*Consumption data from these pre‐accession countries are not reported in Table 2 of this opinion, however, they are included in Appendix A 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 CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré, C. , Barat Baviera, J. M. , Bolognesi, C. , Cocconcelli, P. S. , Crebelli, R. , Gott, D. M. , Grob, K. , Lampi, E. , Mengelers, M. , Mortensen, A. , Rivière, G. , Steffensen, I.‐L. , Tlustos, C. , Van Loveren, H. , Vernis, L. , Zorn, H. , Cavanna, D. , Liu, Y. , di Piazza, G. , & Chesson, A. (2024). Safety evaluation of an extension of use of the food enzyme glucan 1,4‐α‐glucosidase from the non‐genetically modified Rhizopus arrhizus strain AE‐G . EFSA Journal, 22(5), e8773. 10.2903/j.efsa.2024.8773