Safety evaluation of the food enzyme β‐galactosidase from the non‐genetically modified Kluyveromyces lactis strain LAC‐01

TL;DR

This paper evaluates the safety of a food enzyme produced by a non-genetically modified yeast strain and concludes it is safe for use in food manufacturing.

Contribution

The study confirms the safety of β-galactosidase from Kluyveromyces lactis LAC-01 under intended food use conditions.

Findings

The production strain meets the qualified presumption of safety (QPS) criteria.

No homology to known allergens was found in the enzyme's amino acid sequence.

Safety concerns are not expected under the intended use conditions.

Abstract

The food enzyme β‐galactosidase (β‐d‐galactoside galactohydrolase; EC 3.2.1.23) is produced with the non‐genetically modified Kluyveromyces lactis strain LAC‐01 by Nagase (Europa) GmbH. The production strain meets the requirements for the qualified presumption of safety (QPS) approach. The food enzyme is intended to be used in two food manufacturing processes. Dietary exposure to the food enzyme–total organic solids (TOS) was estimated to be up to 2.133 mg TOS/kg body weight per day in European populations. Given the QPS status of the production strain and the absence of concern arising from the food enzyme manufacturing process, toxicity tests were considered unnecessary by the Panel. A search for the homology of the amino acid sequence of the β‐galactosidase 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 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
β‐Galactosidase activity	U/g a	170,000	230,000	211,000
Protein	%	70.0	82.5	73.5
Ash	%	1.6	2.4	1.7
Water	%	4.3	1.5	0.9
Total organic solids (TOS) b	%	94.1	96.1	97.4
Activity/TOS ratio	U/mg TOS	181	239	217

TABLE 2. Intended uses and recommended use levels of the food enzyme as provided by the applicant. 23

Food manufacturing process a	Raw material (RM)	Recommended use level (mg TOS/kg RM) b
Processing of dairy products
Production of lactose‐reduced dairy products	Milk, whey	19.5–30	Before thermal treatment. For drinking milk, dairy powder, ice‐cream, dairy desserts, whey syrups
		1.6–2.5	After thermal treatment, only for drinking milk
Production of fermented dairy products	Milk	2.0–3.0

TABLE 3. Summary of 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)	0.027–0.456 (12)	0.023–0.851 (15)	0.167–0.790 (19)	0.009–0.288 (21)	0.011–0.107 (22)	0.006–0.097 (23)
Min–max 95th (number of surveys)	0.073–1.406 (11)	0.581–2.133 (14)	0.466–1.386 (19)	0.023–0.618 (20)	0.028–0.351 (22)	0.098–0.248 (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
Selection of broad FoodEx categories for the exposure assessment	+
Exposure to food enzyme–TOS always calculated based on the recommended maximum use level	+
For the production of lactose‐reduced dairy products, the higher use level (before thermal treatment) was used for the calculation	+
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 1332/2008^1^ on food enzymes

Five applications have been introduced by the companies “Danisco US Inc.” for the authorisation of the food enzyme Hexose oxidase from a genetically modified strain of Hansenula polymorpha (strain DP‐Jza21); “Novozymes A/S.” for the authorisation of the food enzyme Pectin lyase from a genetically modified strain of Aspergillus niger (strain NZYM‐PN); “Puratos NV” for the authorisation of the food enzyme Xylanase from a genetically modified strain of Bacillus subtilis (strain LMG‐S‐27588); the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) for the authorisation of the food enzyme Beta‐galactosidase from Kluyveromyces lactis and “AB Enzymes GmbH” for the authorisation of the food enzyme Lysophospholipase from a genetically modified strain of Trichoderma reesei (strain RF7206).

Following the requirements of Article 12.1 of Regulation (EC) No 234/2011 implementing Regulation (EC)No 1331/2008, the Commission has verified that the five applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

Terms of Reference

1.1.2

The European Commission requests the European Food Safety Authority to carry out the safety assessments on the food enzymes Hexose oxidase from a genetically modified strain of Hansenula polymorpha (strain DP‐Jza21), Pectin lyase from a genetically modified strain of Aspergillus niger (strain NZYM‐PN), Xylanase from a genetically modified strain of Bacillus subrilis (strain LMG‐S‐27588), Beta‐galactosidase from Kluyveromyces lactis and Lysophospholipase from a genetically modified strain of Trichoderma reesei (strain RF7206)in accordance with Article 17.3 of Regulation (EC) No 1332/2008 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 Beta galactosidase from Kluyveromyces lactis submitted by Association of Manufacturers and Formulators of Enzyme Products (AMFEP).

The application was submitted initially as a joint dossier3 and identified as the EFSA‐Q‐2015‐00409. Agreement to split the joint dossiers into individual data packages was made between EFSA, the European Commission and the Association of Manufacturers and Formulators of Enzyme Products (AMFEP).4

The current opinion addresses one data package originating from the former joint dossier. This data package, identified as EFSA‐Q‐2022‐00598, concerns the food enzyme Beta galactosidase produced with K. lactis strain LAC‐01 and submitted by Nagase (Europa) GmbH.

DATA AND METHODOLOGIES

2

Data

2.1

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme Beta galactosidase from the non‐genetically modified K. lactis strain LAC‐01.

Additional information was requested from the applicant during the assessment process on 18 September 2023 and received on 13 December 2023 (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, 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 nomenclatureβ‐GalactosidaseSystematic nameβ‐d‐galactoside galactohydrolaseSynonymsLactase; β‐lactosidase; β‐d‐lactosidase; β‐d‐galactanaseIUBMB NoEC 3.2.1.23CAS No9031‐11‐2EINECS No232‐864‐1

β‐Galactosidases catalyse the hydrolysis of the β‐(1,4)‐glycosidic linkage of lactose (β‐d‐galactosyl‐1,4‐d‐glucoside) resulting in the release of d‐galactose and d‐glucose.

The food enzyme under assessment is intended to be used in two food manufacturing processes as described in the EFSA guidance (EFSA CEP Panel, 2023): processing of dairy products for the production of (1) lactose‐reduced dairy products and (2) fermented products.

Source of the food enzyme

3.1

The β‐galactosidase is produced with the non‐genetically modified yeast K. lactis strain LAC‐01, 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 K. lactis by phylogenetic analysis ■■■■■6

The species K. lactis is included in the list of organisms for which the qualified presumption of safety (QPS) may be applied (EFSA, 2007; EFSA BIOHAZ Panel, 2022). The production strain has been unequivocally identified as K. lactis. Therefore, the production strain is considered to qualify for the QPS approach to safety assessment.

Production of the food enzyme

3.2

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/2004,7 with food safety procedures based on Hazard Analysis and Critical Control Points and in accordance with current Good Manufacturing Practice.8

The production strain is grown as a pure culture using a typical industrial medium in a submerged, batch fermentation system with conventional process controls in place. After completion of the fermentation, the solid biomass is removed from the fermentation broth by centrifugation and ■■■■■ is added to kill the cells and extract the released intracellular enzyme. The extract containing the enzyme is centrifuged or filtered to remove the cell debris and then concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass material passes the filtration membrane and is discarded.9 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.10

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 β‐galactosidase is a single polypeptide chain of 1025 amino acids.11 The molecular mass of the mature protein, calculated from the amino acid sequence, is 117.6 kDa. The food enzyme was analysed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE). A consistent protein pattern was observed across all batches. The gel showed a major protein band corresponding to an apparent molecular mass of about 120 kDa, consistent with the expected mass of the enzyme.12 The food enzyme contained protease activities.13 No other enzyme activities were reported.

The applicant's in‐house determination of β‐galactosidase activity is based on the hydrolysis of ■■■■■ (reaction conditions: ■■■■■). The enzyme activity is determined by measuring the release of ■■■■■ spectrophotometrically at 420 nm. The enzyme activity is expressed in Units/g (U/g). One Unit is defined as the amount of enzyme which produces 1 μmol of ■■■■■ per minute under the conditions of the assay.14

The food enzyme has a temperature optimum around 40°C (pH 6.5) and a pH optimum around pH 6.5 (30°C). Thermostability was tested after a pre‐incubation of the food enzyme for 10 min at different temperatures (pH 6.5). β‐Galactosidase activity decreased above 50°C showing no residual activity above 55°C.15

Chemical parameters

3.3.2

Data on the chemical parameters of the food enzyme were provided for three batches intended for commercialisation (Table 1).16 The mean total organic solids (TOS) of the three food enzyme batches intended for commercialisation was 95.9% and the mean enzyme activity/TOS ratio was 212 U/mg TOS.

Purity

3.3.3

The lead content in the three commercial batches was below 0.02 mg/kg17 ^,^ 18 which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006).

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).19 No antimicrobial activity was detected in any of the tested batches.20

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

Toxicological data

3.4

As the production strain qualifies for the QPS approach of safety assessment and no issues of concern arising from the production process of the food enzyme were identified (see Sections 3.1, 3.3), the Panel considered that no toxicological studies other than the assessment of allergenicity were necessary (EFSA CEP Panel, 2021).

Allergenicity

3.4.1

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 β‐galactosidase produced with the non‐genetically modified K. lactis strain LAC‐01 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.21

No reports on oral or respiratory sensitisation or elicitation reactions of the β‐galactosidase under assessment have been published.22

Cases of occupational allergy following exposure by inhalation of β‐galactosidase have been reported (Stöcker et al., 2016). 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). Two case reports describing allergic reactions upon ingestion of β‐galactosidase pills, which were confirmed by antigen challenge, have been reported (Binkley, 1996; Voisin & Borici‐Mazi, 2016).

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 β‐galactosidase under assessment.

■■■■■, a known source of allergens, is present in the culture medium. During the fermentation process, this product will mostly be degraded and utilised by the production strain.

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 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 two 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. 23

In all food manufacturing processes, the β‐galactosidase is used to hydrolyse lactose to galactose and glucose. Lactose‐reduced dairy products are more suitable than untreated dairy products for lactose‐intolerant individuals and sweeter.24

In the production of lactose‐reduced dairy products, the food enzyme can be added to milk before25 or after the thermal treatment.26 The enzymatically treated milk can be consumed directly, but it can also be used as an ingredient in a variety of foods.27 The food enzyme–TOS remain in the final foods.

When used for the production of whey syrups, the food enzyme is added to demineralised whey before the pasteurisation step28 to increase the sweetness.29 The food enzyme–TOS remain in the whey syrups.

In the production of fermented dairy products, the food enzyme is added to milk at the beginning of the fermentation process.30 The food enzyme–TOS remain in the final foods (e.g. lactose‐reduced yoghurt and similar products).

Based on the data provided on thermostability (see Section 3.3.1), the Panel considered that this β‐galactosidase is inactivated in most of the final foods. When added after final thermal treatment, such as in the production of drinking milk and yoghurt, the enzyme may remain in its active form, depending on the processing conditions.

Dietary exposure estimation

3.5.2

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, 2023). Exposure from all FoodEx categories was subsequently summed up, averaged over the total survey period (days) and normalised for body weight. 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 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 2.133 mg TOS/kg bw per day in toddlers 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 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

Since no toxicological assessment was considered necessary by the Panel, a margin of exposure was not calculated.

CONCLUSIONS

4

Based on the data provided, the QPS status of the production strain and the absence of issues of concern arising from the production process, the Panel concluded that the food enzyme β‐galactosidase produced with the non‐genetically modified K. lactis strain LAC‐01 does not give rise to safety concerns under the intended conditions of use.

DOCUMENTATION AS PROVIDED TO EFSA

5

Application for authorisation of β‐galactosidase from Kluyveromyces lactis LAC‐01 in accordance with Regulation (EC) No 1331/2008. 23 September 2022. Submitted by Nagase (Europa) GmbH.

Additional information. December 2023. Submitted by Nagase (Europa) GmbH.

ABBREVIATIONSbwbody weightCASChemical Abstracts ServiceCEPEFSA Panel on Food Contact Materials, Enzymes and Processing AidsEINECSEuropean Inventory of Existing Commercial Chemical SubstancesFAOFood and Agricultural Organization of the United NationsGMOgenetically modified organismIUBMBInternational Union of Biochemistry and Molecular BiologyJECFAJoint FAO/WHO Expert Committee on Food AdditivesLODlimit of detectionMOEmargin of exposureQPSqualified presumption of safetySDS‐PAGEsodium dodecyl sulfate‐polyacrylamide gel electrophoresisTOStotal organic solidsWHOWorld Health Organization

REQUESTOR

European Commission

QUESTION NUMBER

EFSA‐Q‐2022‐00598

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 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

Dietary exposure estimates to the food enzyme–TOS in details