Safety evaluation of the food enzyme triacylglycerol lipase from the non‐genetically modified Rhizopus arrhizus strain AE‐N

Abstract

The food enzyme triacylglycerol lipase (EC 3.1.1.3) is produced with the non‐genetically modified Rhizopus arrhizus strain AE‐N by Amano Enzyme Inc. It is considered free from viable cells of the production organism. The food enzyme is intended to be used in an immobilised form in the modification of fats and oils by interesterification. Since residual amounts of total organic solids (TOS) are removed during refinement of the fats and oils, dietary exposure was not calculated. 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 1,806 mg TOS/kg body weight (bw) per day, the highest dose tested. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that, under the intended conditions of use, the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

1. Introduction

Article 3 of the Regulation (EC) No. 1332/20081 provides definition for ‘food enzyme’ and ‘food enzyme preparation’.

‘Food enzyme’ means a product obtained from plants, animals or micro‐organisms or products thereof including a product obtained by a fermentation process using micro‐organisms: (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.

The ‘Guidance on submission of a dossier on food enzymes for safety evaluation’ (EFSA, 2009a) lays down the administrative, technical and toxicological data required.

1.1. Background and Terms Of Reference as provided by the requestor

1.1.1. Background as provided by the European Commission

Only food enzymes included in the European Union (EU) Community list may be placed on the marked as such and used in foods, in accordance with the specifications and conditions of use provided for in Article 7 (2) of Regulation (EC) No 1332/2008 on food enzymes.

Four applications have been introduced by the companies “Advanced Enzyme Technologies Ltd”, “DuPont Nutrition Biosciences ApS”, “Amano Enzyme Inc.” and “Puratos NV sa” for the authorization of the food enzymes Amylase from Bacillus amyloliquefaciens (strain BANSC), Beta‐amylase from Barley (Hordeum vulgare), Triacylglycerol lipase from Rhizopus niveus (strain AE‐N) and Xylanase from a genetically modified strain of Bacillus subtilis TD160(229).

Following the requirements of Article 12.1 of Regulation (EC) No 234/20113 implementing Regulation (EC) No 1331/2008, the Commission has verified that the four applications fall within the scope of the food enzyme Regulation and contain all the elements required under Chapter II of that Regulation.

1.1.2. Terms of Reference

The European Commission requests the European Food Safety Authority to carry out the safety assessments of the food enzymes Amylase from Bacillus amyloliquefaciens (strain BANSC), Beta‐amylase from Barley (Hordeum vulgare), Triacylglycerol lipase from Rhizopus niveus (strain AE‐N) and Xylanase from a genetically modified strain of Bacillus subtilis TD160(229) in accordance with Article 17.3 of Regulation (EC) No 1332/2008 on food enzymes.

1.2. Interpretation of the Terms Of Reference

The present scientific opinion addresses the European Commission's request to carry out the safety assessment of a food enzyme triacylglycerol lipase from R. niveus strain AE‐N.

Recent data identified the production microorganism as Rhizopus arrhizus (see Section 3.1). Therefore, this name will be used in this opinion instead of R. niveus.

2. Data and Methodologies

2.1. Data

The applicant has submitted a dossier in support of the application for authorisation of the food enzyme triacylglycerol lipase from R. niveus strain AE‐N.

Additional information was requested from the applicant during the assessment process on 22 March 2021, 4 November 2021 and 22 March 2022 and has subsequently been provided (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, 2009b) and following the relevant guidance documents of the EFSA Scientific Committee.

The current ‘Scientific Guidance for the submission of dossiers on Food Enzymes’ (EFSA CEP Panel, 2021) has been followed for the evaluation of the application.

3. Assessment

IUBMB nomenclature	Triacylglycerol lipase
Systematic name	Triacylglycerol acylhydrolase
Synonyms	Lipase; triglyceride lipase; glycerol ester hydrolase
IUBMB No	EC 3.1.1.3
CAS No	9001‐62‐1
EINECS No	232‐619‐9

Triacylglycerol lipases catalyse, in the absence or at very low concentrations of water, the interesterification of fatty acids in glycerides. The triacylglycerol lipase is intended to be used in modification of fats and oils by interesterification.

3.1. Source of the food enzyme

The triacylglycerol lipase is produced with the non‐genetically modified filamentous fungus R. arrhizus strain AE‐N ■■■■■, which is deposited at the National Institute of Technology and Evaluation (NITE) Biological Resource Center (Japan) with the deposit number NITE SD 00490.4

The production strain was identified as R. arrhizus by ■■■■■.5 It was derived from a natural isolate by ■■■■■ and selection.

3.2. Production of the food enzyme

The food enzyme is manufactured according to the Food Hygiene Regulation (EC) No 852/20046, with food safety procedures based on Hazard Analysis and Critical Control Points, and in accordance with current Good Manufacturing Practice.7

The production strain is grown as a pure culture using a typical industrial medium in a solid state fermentation system with conventional process controls in place. After completion of the fermentation, water is added and then the solid biomass is removed from the resulting fermentation broth by filtration, leaving a filtrate containing the food enzyme. The filtrate is then further purified and concentrated, including an ultrafiltration step in which enzyme protein is retained, while most of the low molecular mass compounds pass the filtration membrane and are discarded. 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.8

The food enzyme is used in immobilised form. Immobilisation is achieved by adsorption of the food enzyme onto a solid‐state carrier (■■■■■). It is then dried and packed into a column.9

The Panel considered that sufficient information has been provided on the manufacturing process and the quality assurance system implemented by the applicant to exclude issues of concern.

3.3. Characteristics of the food enzyme

3.3.1. Properties of the food enzyme

The triacylglycerol lipase is a single polypeptide chain of ■■■■■ amino acids. The molecular mass of the mature protein derived from the amino acid sequence was ■■■■■ kDa.10 The food enzyme was analysed by size exclusion chromatography and the chromatograms of all food enzyme batches showed a consistent protein pattern.11 The food enzyme also shows protease activity.12 No other enzymatic activities were reported.

The determination of triacylglycerol lipase hydrolytic activity is based on the hydrolysis of olive oil. The activity is determined by titration of the free fatty acids released from an olive oil emulsion (reaction conditions: pH 7.0, 37°C, 30 min). The activity is measured in fat digestive activity units (U). One U is defined as the quantity of enzyme that releases 1 μmol of fatty acids per minute under the conditions of the assay.13

The interesterification activity is quantified by the formation of methyl caprate from tricaprylin and methyl stearate (reaction conditions: pH 7.0, 60°C, 1–24 h), which is measured by gas chromatography. The activity is measured in transesterification Units (TEU). One TEU is defined as the quantity of enzyme required to form 1 nmol of methyl caprate per minute.14

The optimum hydrolytic activity was observed at a temperature of around 40°C (pH 3.0) and the pH optimum was determined around pH 7.0 (37°C). Thermostability was tested after a pre‐incubation of the food enzyme for 60 min at different temperatures (pH 3.0). Enzyme activity decreased above 40°C, showing only minor residual activity above 50°C.15

3.3.2. Chemical parameters

Data on the chemical parameters of the food enzyme were provided for three batches used for commercialisation and for one batch produced for the toxicological tests (Table 1).16 The mean total organic solids (TOS) of the three food enzyme batches for commercialisation was 92.1% and the mean enzyme hydrolysis activity/TOS ratio was 48.9 U/mg TOS.

Table 1.

Composition of the food enzyme

Parameters	Unit	Batches
		1	2	3	4 (a)
Triacylglycerol activity (hydrolysis)	U/g (b)	39,700	43,300	52,100	41,400
Protein	%	48.9	45.6	45.2	45.8
Ash	%	4.0	3.8	4.2	4.8
Water	%	3.6	3.8	4.3	4.9
Total organic solids (TOS) (c)	%	92.4	92.4	91.5	90.3
Hydrolysis activity/TOS	U/mg TOS	43.0	46.9	56.9	45.8

^(a)Batch used for the toxicological studies.

^(b)U: Fat digestive activity units (see Section 3.3.1).

^(c)TOS calculated as 100% ‐ % water ‐ % ash.

3.3.3. Purity

The lead content in the three commercial batches and in the batch used for toxicological studies was on average 0.08 mg/kg which complies with the specification for lead as laid down in the general specifications for enzymes used in food processing (FAO/WHO, 2006). For arsenic, cadmium and mercury, the average concentration determined in the commercial batches was 0.096, 0.082 and 0.001 mg/kg, respectively.17 ^, 18 The Panel considered these concentrations as of no concern.

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). No antimicrobial activity was detected in any of these batches.19

Strains of Rhizopus, in common with most filamentous fungi, have the capacity to produce a range of secondary metabolites. The presence of aflatoxin (B1, B2, G1, G2), fumonisins (B1, B2), HT‐2 toxin, T‐2 toxin, zearalenone, ochratoxin A, sterigmatocystin and rhizoxin was examined in the four food enzyme batches and all were below the limit of detection (LoD) of the respective methods.20 ^, 21 Adverse effects caused by the possible presence of toxic secondary metabolites derived from the production strain is addressed by the toxicological examination of the food enzyme TOS.

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

3.3.4. Viable cells of the production strain

The absence of viable cells of the production strain in the food enzyme was demonstrated in three independent batches analysed in triplicate. Three × 10 g of product were diluted in 90 mL of sterile water, 10 mL of each solution was then filtered with a membrane filter. The filters were placed on agar plates and incubated at 27°C for 7 days. Colonies morphologically different from the production strain were produced. A positive control was included.22

3.4. Toxicological data

A battery of toxicological tests, including a bacterial gene mutation assay (Ames test), an in vitro mammalian chromosomal aberration test and a repeated dose 90‐day oral toxicity study in rats, was provided. The batch 4 (Table 1) used in these studies has a chemical composition and activity/mg TOS value which falls within the range of the commercial batches and is considered suitable as a test item.

3.4.1. Genotoxicity

3.4.1.1. Bacterial reverse mutation test

A bacterial reverse mutation assay (Ames test) was performed according to the test Guidelines and GLP of the Japanese Ministry of Health and Labor Welfare.23

Four strains of Salmonella Typhimurium (TA98, TA100, TA1535 and TA1537) and Escherichia coli WP2uvrA were used in the presence or absence of metabolic activation (S9‐mix), applying the preincubation method. In a concentration‐range experiment, growth inhibition was observed at 5,000 μg food enzyme/plate corresponding to 4,515 μg TOS/plate for TA98, TA100, TA1535 and TA1537 with S9‐mix and for strain TA100 and TA 1535 without S9‐mix. Growth inhibition was noted starting from 1,129 μg TOS/plate for TA98 and TA1537 without S9‐mix. Based on these results, the main experiment was carried out in duplicate at a range of concentrations from 156 to 5,000 μg food enzyme/plate corresponding to 141 to 4,515 μg TOS/plate for TA100 and TA1535 with and without S9‐mix and for TA98 and TA1537 with S9‐mix. A range of concentrations from 39.1 to 1,250 μg food enzyme/plate corresponding to 35.3 to 1,129 μg TOS/plate was also tested in TA98 and TA1537 without S9‐mix. For E. coli WP2urvA, five concentrations were tested with and without S9‐mix in the range 313 to 5,000 μg food enzyme/plate corresponding to 282.6 to 4,515 μg TOS/plate.

Upon treatment with the food enzyme, there was no significant increase in revertant colony numbers above the control values in any strain tested, with or without S9‐mix.

The Panel concluded that the food enzyme did not induce gene mutations under the test conditions employed in this study.

3.4.1.2. In vitro mammalian chromosomal aberration test

The in vitro mammalian chromosomal aberration test was carried out in cultured Chinese hamster lung fibroblasts (CHL/IU) according to the test Guidelines and GLP of the Japanese Ministry of Health and Labor Welfare.24

A dose‐finding study was performed at eight concentrations ranging from 39.1 to 5,000 μg/mL of food enzyme, corresponding to 35.3 and 4,515 μg TOS/mL, and no inhibition of cell growth by 50% or more was observed. Based on these results, the cells were exposed to the food enzyme at 1,129, 2,258 and 4,515 μg TOS/mL in a short‐term treatment (6 h followed by 18 h recovery period) with and without metabolic activation (S9‐mix) and in a continuous treatment (24 and 48 h) in the absence of S9‐mix. Cytotoxic effects were observed only in the short‐term treatment in the presence of S9‐mix at the highest concentration tested (relative growth rate = 68%). The frequency of structural and numerical chromosomal aberrations in treated cultures was comparable to the values detected in negative controls.

The Panel concluded that food enzyme did not induce chromosome aberrations under the test conditions employed for this study.

3.4.2. Repeated dose 90‐day oral toxicity study in rodents

The repeated dose 90‐day oral toxicity study was performed in accordance with guidelines of the Japanese Ministry of Health and Welfare (1996 and 1999) and following GLP. The study is in accordance with OECD Test Guideline 408 (OECD, 1998) with the following deviations: detailed clinical observations and functional observations were not performed, urea was not determined, epididymides were not weighed, and only two areas of the brain and one level of the spinal cord were examined in the microscopy. The Panel considered that these deviations are not major and do not impact on the evaluation of the study.25

Groups of 12 male and 12 female Sprague–Dawley SPF (Crl:CD(SD)) rats received by gavage 500, 1,000 and 2,000 mg food enzyme/kg body weight (bw) per day corresponding to 451.5, 903 and 1,806 mg TOS/kg bw per day. Controls received the vehicle (water for injection).

No mortality was observed.

The feed consumption was statistically significantly decreased on days 14 to 28 and on day 35 for high‐dose males (−11% on all occasions). The Panel considered the changes as not toxicologically relevant as they were only recorded sporadically and they were only observed in one sex.

The haematological investigation revealed a statistically significant decrease in monocyte percentage in low‐dose males (−33%), an increase in concentration of fibrinogen (FIB) in high‐dose males (+14%) and a decrease in activated partial thromboplastin time (APTT) in mid‐dose females (−15%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), the changes were small (monocyte percentage), there was no dose–response relationship (monocyte percentage, APTT) and there were no changes in other relevant parameters (for monocyte percentage in white blood cell count, for fibrinogen and APTT in other coagulation parameters).

The clinical chemistry investigation revealed a statistically significant increase in the sodium concentration in mid‐dose males (+1%), a decrease in levels of total protein in mid‐ and high‐dose males (−3% at both doses) and decreased levels of triglycerides in low‐dose females (−39%). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), the changes were small (sodium, total protein) and there was no dose–response relationship (all parameters).

The urinalysis revealed a statistically significant increase in urine volume (+65%), a decrease in osmolarity (−23%) and a slight presence of crystal phosphate salts (8/12 vs. 2/12 in the control) in high‐dose males, a slight presence of protein in the urine of high‐dose females (5/12: 10–25 mg/dL and 2/12: 26–250 mg/dL vs. <10 mg/dL in all controls). The Panel considered the changes as not toxicologically relevant as they were only observed in one sex (all parameters), the changes were related to a slightly higher water intake in comparison to controls (urine volume and osmolality), the changes were small (phosphate salt crystals, protein) and there were no histopathological changes in the kidneys.

Statistically significant changes in organ weights included an increase in the relative weight of lungs (+11%) and adrenals (+17%) in mid‐ and low‐dose females, respectively. The Panel considered these changes as not toxicologically relevant because they were only observed in one sex, there was no dose–response relationship and there were no histopathological changes in these organs.

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

The Panel identified a no observed adverse effect level (NOAEL) of 1,806 mg TOS/kg bw per day, the highest dose tested.

3.4.3. Allergenicity

The allergenicity assessment considered only the food enzyme and not any carrier or other excipient, which may be used in the final formulation.

The potential allergenicity of the triacylglycerol lipase produced with the R. arrhizus strain AE‐N was assessed by comparing its amino acid sequence with those of known allergens according to the ‘Scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derived food and feed of the Scientific Panel on Genetically Modified Organisms’ (EFSA GMO Panel, 2010). Using higher than 35% identity in a sliding window of 80 amino acids as the criterion, no match was found.26

No information is available on oral and respiratory sensitisation or elicitation reactions of this triacylglycerol lipase.

Respiratory allergy following occupational inhalation of triacylglycerol lipase has been reported (Elms et al., 2003; Brant et al., 2004; Martel et al., 2010). Several studies have shown that adults with occupational asthma to a food enzyme may be able to ingest the corresponding allergen without acquiring clinical symptoms of food allergy (Cullinan et al., 1997; Poulsen, 2004; Armentia et al., 2009).

Soybean flour, wheat bran and flour, products that may cause allergies (listed in the Regulation (EU) No 1169/201127), are used as raw materials. However, during the fermentation process, these products will be degraded and utilised by the microorganisms for cell growth, cell maintenance and production of enzyme protein. In addition, the fungal biomass and fermentation solids are removed. Taking into account the fermentation process and downstream processing, the Panel considered that no potentially allergenic residues are present in the food enzyme.

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

3.5. Dietary exposure

3.5.1. Intended use of the food enzyme

The food enzyme is intended to be used in an immobilised form to modify fats and oils by interesterification at the recommended use levels of 128–1,280 mg TOS/kg plant and animal fats/oils.28

In the fat and oil modification process, fats and oils of plant and animal origin are passed through a column, packed with the immobilised food enzyme.29 The triacylglycerol lipase catalyses the exchange of fatty acids at the 1‐ and 3‐position of the triglycerides, modifying the properties of the resulting triglycerides. The modified fats and oils are subsequently refined. The applicant analysed inter‐esterified oils obtained from two different immobilisation carriers for traces of protein and of the immobilisation carrier, and the respective values were below the limits of detection.30

The Panel accepted the data as evidence for the removal of the food enzyme–TOS from the final inter‐esterified fats and oils. Thus, no activity of the enzyme is expected in the final product.

3.5.2. Dietary exposure estimation

In accordance with the guidance document (EFSA CEP Panel, 2021), since the food enzyme TOS is removed in the final foods, a dietary exposure was not calculated.

3.6. Margin of exposure

Since the calculation of dietary exposure was not considered necessary, a margin of exposure was not calculated.

4. Conclusions

Based on the data provided and the removal of TOS from the final foods, the Panel concluded that the food enzyme triacylglycerol lipase produced with R. arrhizus strain AE‐N does not give rise to safety concerns under the intended conditions of use.

5. Documentation as provided to EFSA

Technical dossier ‘Application for authorisation of Triacylglycerol lipase from Rhizopus niveus AE‐N in accordance with Regulation (EC) No 1331/2008’ September 2014. Submitted by Amano Enzyme Inc.

Additional information. July 2022. Submitted by Amano Enzyme Inc.

Abbreviations

bw

body weight

CAS

Chemical Abstracts Service

CEF

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

CEP

EFSA Panel on Food Contact Materials, Enzymes and Processing Aids

EINECS

European Inventory of Existing Commercial Chemical Substances

FAO

Food and Agricultural Organization of the United Nations

GLP

Good Laboratory Practice

GMO

genetically modified organism

IUBMB

International Union of Biochemistry and Molecular Biology

JECFA

Joint FAO/WHO Expert Committee on Food Additives

kDa

kilo Dalton

LoD

limit of detection

LoQ

limit of quantification

NOAEL

no observed adverse effect level

OECD

Organisation for Economic Cooperation and Development

TOS

total organic solids

WHO

World Health Organization

Suggested citation: EFSA CEP Panel (EFSA Panel on Food Contact Materials, Enzymes and Processing Aids) , Lambré C, Barat Baviera JM, Bolognesi C, Cocconcelli PS, Crebelli R, Gott DM, Grob K, Lampi E, Mengelers M, Mortensen A, Rivière G, Steffensen I‐L, Tlustos C, Van Loveren H, Vernis L, Zorn H, Andryszkiewicz M, Liu Y, Lunardi S, Nielsen E, Nørby K and Chesson A, 2023. Scientific Opinion on the safety evaluation of the food enzyme triacylglycerol lipase from the non‐genetically modified Rhizopus arrhizus strain AE‐N . EFSA Journal 2023;21(1):7756, 11 pp. 10.2903/j.efsa.2023.7756