Information and Consultation Document on Health Canada's Proposal to Enable the use of the Food Additive 'Hexose Oxidase' in mozzarella cheese, ultra-heat treated milk, and dough for bread products

Notice to the reader:

The online consultation is now closed.

As a result of this process, a modification was made to the List of Permitted Food Enzymes to enable the use of hexose oxidase from hansenula polymorpha (B13-HOX4-Mut45) as a food enzyme in certain cheese, bakery and milk products on June 4, 2013. The proposed use of this food additive in Canada as described is now enabled.

For more information on this initiative, please contact the Bureau of Chemical Safety's Chemical Health Hazard Assessment Division at bcs-bipc@hc-sc.gc.ca.

November 2012

Table of Contents

Purpose

This document outlines Health Canada's evaluation of the use of hexose oxidase as a food additive in pizza mozzarella cheese, part-skim pizza mozzarella cheese, ultra-heat treated (UHT) milk, and dough for bread, baking and pasta. Based on the conclusions of this evaluation, Health Canada proposes that hexose oxidase be legally enabled for use in the foods listed above. Health Canada is seeking comments on this proposal.

Background

In Canada, all food additives are regulated under the Food and Drug Regulations (Regulations) and are subject to rigorous controls under the Food and Drugs Act.  Before a food additive is permitted for use, a submission must be filed with Health Canada's Food Directorate so the Department can conduct a thorough safety evaluation of the proposed use(s) of the additive.  Food manufacturers are not permitted to use the additive in foods to be sold in Canada until it has been approved by Health Canada and steps have been taken to legally enable its use.

Health Canada received a submission requesting legal approval of the use of a new enzyme, hexose oxidase,in pizza mozzarella cheese, part-skim pizza mozzarella cheese, ultra-heat treated (UHT) milk, and dough for bread, baking and pasta in accordance with Good Manufacturing Practices. Hexose oxidase currently has no approved food additive uses in Canada.

The hexose oxidase enzyme is obtained from a genetically modified strain of yeast called Hansenula polymorpha (B13-HOX4-Mut45) which expresses a gene, from the alga Chondrus crispus (Irish moss or carrageenan seaweed), that encodes a hexose oxidase enzyme. During fermentation of the yeast, lauryl trimethyl ammonium bromide (LTAB) is added to cause permeability of the yeast cells and allow extraction of the enzyme. The hexose oxidase enzyme is then extracted, and undergoes centrifugation, filtration, and ultrafiltration which result in a cell- and debris-free concentrated liquid product that is spray-dried onto wheat starch or other food grade carriers.  Distributing the enzyme in a carrier facilitates its handling and use in food applications.

Both Hansenula polymorpha and Chondrus crispus have a history of safe food use. Hansenula polymorpha is yeast and is considered to be a safe production organism that poses no risk to humans or the environment. Chondrus crispus is red algae from which the hexose oxidase coding sequence is derived.  It too has a long history of safe use in foods and has not been associated with the production of any known toxins or allergens.

The hexose oxidase enzyme has the International Union of Biochemistry and Molecular Biology (IUBMB) Number EC 1.1.3.5 and the Chemical Abstract Services (CAS) Registry Number 9028-75-5.

Health Canada's Assessment of Hexose Oxidase

Food Directorate scientists at Health Canada conducted a detailed evaluation of the submission requesting approval of hexose oxidase, focusing on safety and efficacy.  Their evaluation considered the chemical, microbiological, and toxicological aspects of the proposed use of hexose oxidase as a food additive, and is further described in the following sections.

Technical Efficacy

The common name of hexose oxidase is D-hexose:oxygen 1-oxidoreductase.  The enzyme, which catalyses the oxidation of various hexoses, is proposed to be used in pizza mozzarella cheese, part-skim pizza mozzarella cheese, ultra-heat treated (UHT) milk, and dough for bread, baking and pasta.

The purpose of hexose oxidase in pizza mozzarella cheese and part-skim pizza mozzarella cheese is to reduce browning (Maillard reaction) of the cheese during baking of the pizza. Mono- and disaccharides in cheese (e.g., galactose) are responsible for the browning of cheese when baked.  Oxidizing these mono- and disaccharides in the cheese leads to reduced browning during baking. The need for hexose oxidase is limited to pizza mozzarella and part-skim mozzarella due to the higher amount of galactose available for the Maillard reaction relative to other types of cheese.

UHT milk is milk that is heated for a few seconds to a temperature exceeding 135 degrees Celsius to inactivate thermoresistant bacterial spores. Milk normally contains dissolved oxygen in small amounts. During UHT treatment of milk, the dissolved oxygen could initiate oxidative reactions that could adversely affect the flavour. In UHT milk, hexose oxidase functions as an oxygen scavenger, eliminating the oxygen and thereby protecting the milk's flavour.

The hexose oxidase enzyme strengthens dough used in bread, baking and pasta  by strengthening the gluten network.

Chemical Assessment

Food Directorate scientists have not identified any safety concerns, from a chemical perspective, in relation to the use of hexose oxidase in pizza mozzarella cheese and part-skim mozzarella cheese, UHT milk, and in dough used in break, baking and pasta, and under the conditions of use proposed in the submission. The product meets the specifications for enzyme preparations of both the Food Chemicals Codex (FCC), 7th Edition, and of the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

Dietary Exposure

As for other enzymes, the use level is proposed to be consistent with Good Manufacturing Practice (GMP).  For example, GMP was identified in the petition as being between 800 and 2400 mg Total Organic Solids (TOS) per 100 kg flour.  Based on the directions for use of commercial preparations of this enzyme, the typical and maximum exposure levels of the hexose oxidase enzyme were estimated by the petitioner to be approximately 1 and 8.6 mg TOS/kg bw/day, respectively. These estimations of exposure were considered acceptable for the Food Directorate safety assessment.  For the purposes of the safety assessment, it was conservatively assumed that exposure is to the active enzyme. However, the enzyme is not stable at temperatures above 30 degrees Celsius and will typically be denatured during the heat treatment (baking, pasteurization) of food.

Residues of LTAB, used to facilitate extraction of the enzyme from the production cells, remain in the final enzyme preparation at a concentration of approximately 50 mg/kg of commercial enzyme product. Typical and maximum exposure levels of LTAB were calculated to be 0.62 and 5.4 μg/kg bw/day, respectively. A worst-case theoretical intake scenario was also estimated where a 2-year-old (10 kg) child consuming 500 g of bread per day containing 0.27 mg/kg LTAB would be exposed to 13.5 µg/kg bw/day LTAB.

Microbiological Assessment

Data on hexose oxidase was generated using validated, internationally accepted methodology to determine microbial safety. Apart from the production of the hexose oxidase and the loss of the ability to sporulate, the biological activity, growth and physiological characteristics of the production strain were not observed to be any different from that of unmodified Hansenula polymorpha.

A description of the manufacturing process and the control measures that are applied to prevent microbial contamination and to ensure quality have also been assessed and are considered acceptable.

As previously mentioned, both Hansenula polymorpha and Chondrus crispus have a history of safe use and pose no risk to humans or the environment.  Neither is associated with any pathogenicity or allergenicity concerns. H. polymorpha is considered to be a safe production organism and C. crispus has a long history of safe use in foods for human consumption.

The assessment found that the genetic material transferred to the microorganism is well-characterized and limited in size, that the genetic stability of the production strain was shown to be maintained across all inoculated cultures assayed in the course of approximately 35 generations, that the genetically modified production organism is not present in the final enzyme product, and that the hexose oxidase preparation complies with internationally accepted microbial specifications for enzyme preparations.

Based on this information, Food Directorate scientists have no microbiological concerns with the use of hexose oxidase produced by the genetically modified Hansenula polymorpha in those foods and under the conditions of use proposed in the submission.

Toxicological Assessment

A toxicological review of the submission also found the host organism Hansenula polymorpha to be non-toxicogenic. A no observed effect level (NOEL) equivalent to approximately 900 mg TOS/kg bw/day was determined based on a 2-week range-finding study and a 13-week oral toxicity study in rats. There is a margin of exposure of greater than 100 between the NOEL and the maximum anticipated consumption, and of approximately 1000 for typical consumption levels.

With respect to the presence of residues of LTAB in the enzyme preparation, the petitioner submitted a 13-week oral toxicity study in which rats were exposed to the enzyme as well as 11.3 mg LTAB/kg bw/day, or 2000 times more than the maximum exposure level of a 90th percentile consumer. No effects were observed. The Danish Food Agency has established an acceptable daily intake (ADI) of 0.25 mg/kg bw for all quaternary ammonium compounds (which would include LTAB) from all sources and recommends an ADI from food sources of no more than 10% or 0.025 mg/kg bw. The worst-case intake calculated by the petitioner (0.0135 mg/kg bw/day) is about 5% of the ADI from all sources (or half of the ADI from food sources).

The low exposure levels, the low rate of absorption of a related compound (cetyl trimethyl ammonium bromide) through the digestive system, and the low toxicity of compounds structurally related to LTAB, support the safety of LTAB as used in the production of hexose oxidase.

Based on the assessment, Food Directorate scientists have no toxicological concerns with the use of hexose oxidase produced by the genetically modified Hansenula polymorpha in those foods and under the conditions of use proposed in the submission.

Environmental Assessment

Hexose oxidase is not on the Domestic Substances List (DSL) and it is obtained from a genetically modified organism. Therefore, it may be subject to the New Substances Notifications Regulations of the Canadian Environmental Protection Act, 1999.  The petitioner has been informed of its responsibilities in this regard.

International Status

This hexose oxidase enzyme has been approved by a GRAS (Generally Recognized as Safe) panel in the United States. Hexose oxidase has also been evaluated by the Danish Food Agency in accordance with the guidelines for the Scientific Committee for Food of the Commission of the European Communities, which considered it acceptable for use in food. Hexose oxidase is also listed as a processing aid in Australia and New Zealand, as well as in the Inventory of Processing Aids of the Codex Committee on Food Additives.

The JECFA has specifications for hexose oxidase from Chondrus crispus expressed in Hansenula polymorpha.  These specifications were prepared at the 63rd JECFA (2004) and published in Food and Nutrition Paper 52 Add 12 (2004).  An allowable daily intake (ADI) "not specified" was established at the 63rd JECFA (2004).

Previous Consultation

Since this proposal includes uses of a food additive in foods that are standardized under the Regulations, the Baking Association of Canada, the Canadian Food Inspection Agency (Dairy Program), and the Dairy Processors Association of Canada were consulted during the development of this proposal. The Baking Association of Canada and the Dairy Processors Association of Canada have expressed their support of the proposal to legally permit the use of hexose oxidase.

The Dairy Program of the Canadian Food Inspection Agency had no objections to the use of hexose oxidase in pizza mozzarella cheese, part-skim pizza mozzarella cheese, UHT milk, and dough for bread, baking and pasta. It was also noted that the Dairy Products Regulations (Canada Agricultural Products Act)  list "part skim pizza mozzarella" and "pizza mozzarella" as varieties of cheese in section 28.1, the table to which specifies maximum percentage of moisture and minimum percentage of milk fat for a large variety of different cheeses.

Recommendations

Health Canada completed a detailed safety assessment of a submission requesting approval of the use of hexose oxidase. No health or safety concerns with the proposed uses of the enzyme have been identified. Given the acceptability of the food additive use of hexose oxidase on chemical, microbiological, and toxicological grounds, and the fact that the submission meets the requirements for a food additive submission outlined in Section B.16.002 of the Regulations, it is proposed that hexose oxidase be approved as a food additive for use in pizza mozzarella cheese, part-skim pizza mozzarella cheese, ultra-heat treated (UHT) milk, and dough for bread, baking and pasta.

Hexose oxidase enzyme from Hansenula polymorpha (B13-HOX4-Mut45) would be enabled for use according to the conditions shown below:

Proposed Amendment to the List of Permitted Food Additives
Additive Permitted Source Permitted in or Upon Maximum Level of Use
Hexose oxidase Hansenula polymorph (B13-HOX4-Mut45) 1) Bread; Flour; Whole wheat flour 1) Good Manufacturing Practice
2) Milk, partly skimmed milk and skim milk heat-treated above 100 °C; sterilized milk 2) Good Manufacturing Practice
3) Part skim pizza mozzarella cheese; Pizza mozzarella cheese 3) Good Manufacturing Practice
4) Unstandardized bakery products 4) Good Manufacturing Practice

The foods in which hexose oxidase would be permitted for use include those which are standardized under Division 8 (Dairy Products) and Division 13 (Grain and Bakery Products) of the Regulations.  Amendment of these standards may also be required to reflect the possible use of this additive.

For example, the amendments might appear as follows:

(1) Section B.08.003 be amended by adding a new paragraph (c) to read as follow:

(c) in the case of milk that has been heat-treated above 100 °C, may contain hexose oxidase at a maximum level of use consistent with good manufacturing practice.

(2)  Section B.08.004 be amended adding a new paragraph (d) to read as follow:

(d) in the case of skim milk that has been heat-treated above 100 °C, may contain hexose oxidase at a maximum level of use consistent with good manufacturing practice.

(3)  Section B.08.005 be amended adding a new paragraph (d) to read as follow:

(d)  in the case of partly skimmed milk that has been heat-treated above 100 °C, may contain hexose oxidase at a maximum level of use consistent with good manufacturing practice.

(4) Section B.08.007 amended adding a new paragraph (e) to read as follow:

(e) may contain hexose oxidase at a maximum level of use consistent with good manufacturing practice.

(5) Subsection B.08.033(3) be amended by adding a new paragraph (g) to read as follow:

(g) hexose oxidase derived from Hansenula polymorpha (B13-HOX4-Mut45) in the manufacture of pizza mozzarella cheese and part skim pizza mozzarella cheese.

Subparagraph B.13.001 (e) (iii) be amended by adding the words "hexose oxidase" after the words "glucose oxidase".

Subparagraph B.13.005 (d) (iii) be amended by adding the words "hexose oxidase" after the words "glucose oxidase".

Subparagraph B.13.021 (h) be amended by adding the words "hexose oxidase" after the words "glucose oxidase".

Comments

Comments on this proposal may be submitted in writing, either electronically or by regular mail.  If you are submitting your comments electronically, please use the words "hexose oxidase" in the subject box of your e-mail.  Comments must be received by 11:59 p.m. EST, January 3, 2013.

Mailing address:
Bureau of Chemical Safety
251 Sir Frederick Banting Driveway
Tunney's Pasture, PL: 2203B
Ottawa, Ontario K1A 0L2

Additional Information

For more information on this initiative, please contact the Bureau of Chemical Safety at bcs-bipc@hc-sc.gc.ca, using the words "hexose oxidase" in the subject box of your e-mail.

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