ANNEX 1

GENERAL SPECIFICATIONS FOR ENZYME PREPARATIONS USED IN FOOD PROCESSING

Prepared at the 35th JECFA (1989) and published in FNP 49 (1990) and in FNP 52 (1992), superseding general specifications prepared at the 25th JECFA (1981) and published in FNP 19 (1981) and in FNP 31/2 (1984). Amended at the 55^th JECFA (1999) and partially published in FNP 52 Add 7 (1999).

Enzyme Nomenclature

Recommendations (1984) of the Nomenclature Committee of the International Union of Biochemistry, Academic Press (1984).

DEFINITION

Enzyme preparations used in food processing are usually named according to the substrate to which they are applied, such as protease or amylase. Some traditional names are also in use, such as malt, pepsin and rennet. Active components consist of biologically active proteins, at times combined with metals, carbohydrates and/or lipids. Known molecular weights range from approximately twelve thousand to several hundred thousand. They are obtained from animal, vegetable or microbial sources and may consist of whole cells, parts of cells, or cell-free extracts of the source used. They may contain one or more active components as well as carriers, solvents, preservatives, antioxidants and other substances consistent with good manufacturing practice. They may be liquid, semi-liquid, dry or in an immobilized form (immobilized enzyme preparations are preparations which have been made insoluble in their intended food matrix by physical and/or chemical means). Their colour may vary from virtually colourless to dark brown.

ACTIVE COMPONENTS

The principle activities are characterized by their systematic names and Enzyme Commission Numbers.

SOURCE MATERIALS

Animal tissues used for the preparation of enzymes must comply with meat-inspection requirements and be handled in accordance with good hygienic practice.

Plant material used in the production of enzyme preparations must consist of components which leave no residues harmful to health in the processed finished food under normal conditions of use.

Microbial sources used in the production of enzyme preparations may be native strains or variants of micro-organisms, or be derived from native strains or variants by the processes of selective serial culture or genetic manipulation. They must be discrete and stable strains or variants which are sufficiently well characterised to enable them to be assigned unique identities as the sources of the enzyme preparations which are the subject of individual specifications. They must be maintained under conditions which ensure the absence of strain drift and when used in the production of enzyme preparations must be subjected to methods and culture conditions which are applied consistently and reproducibly from batch to batch. Such conditions must ensure the absence of toxin production by the source organism and prevent the introduction of micro-organisms which could be the source of toxic materials and other undesirable substances. Culture media used for the growth of microbial sources must consist of components which leave no residues harmful to health in the processed finished food under normal conditions of use.

When a non-pathogenic, non-toxicogenic strain belongs to a species that includes pathogenic and toxicogenic strains, the source section of the monograph for the enzyme should include a requirement that the strain be non-pathogenic and non-toxicogenic. Citation of a suitable strain number may be included by way of example.

CARRIERS AND OTHER ADDITIVES AND INGREDIENTS

The carriers, diluents, excipients, supports and other additives and ingredients (including processing aids) used in the production, distribution and application of enzyme preparations must be substances that are acceptable for the relevant food uses of the enzyme preparations concerned, or substances which are insoluble in food and removed from the food material after processing.

In the case of immobilized enzyme preparations, leakage of carriers, immobilization agents and active enzymes must be kept within acceptable limits as specified in the individual specifications.

In order to distinguish the proportion of the enzyme preparation derived from the source material from that contributed by diluents and other additives and ingredients, individual specifications may require a statement of percentage Total Organic Solids (T.O.S.) which is defined as follows:

% T.O.S. = 100 - (A + W + D)

where

A = % ash, W = % water and D = % diluents and/or other additives and ingredients.

HYGIENE

Enzyme preparations are produced in accordance with good food manufacturing practice. They cause no increase in the total microbial count in the treated food, over the level accepted for the respective food.

PURITY

Arsenic (FNP 5)

Not more than 3 mg/kg (Method II)

Lead (FNP 5)

Not more than 10 mg/kg

Heavy metals (FNP 5)

Not more than 40 mg/kg

Test 2g of the sample as directed in the Limit Test (Method II)

Microbiological criteria

Salmonella: Absence by suitable method using a 25 g sample (e.g. Chapter 7: Isolation and identification of Salmonella; FDA Bacteriological Analytical Manual, Sixth Edition (1984)).

Coliforms: Not more than 30 per gram by suitable method (eg ISO 4832: 1978: Microbiology - General guidance for enumeration of Coliforms. Colony count technique at 30^o)

Escherichia coli: Absence by suitable method using 25 g of sample (eg Chapter 6: Enteropathogenic Escherichia coli; FDA Bacteriological Analytical Manual, Sixth Edition (1984))

Total viable count: Not more than 5 x 10⁴ per gram by a suitable method (eg ISO 4833: 1978: Microbiology - General guidance for enumeration of micro-organisms. Colony count technique at 30^o)

Antibiotic activity

Absent in preparations from microbial sources when determined in accordance with Appendix A.

Aflatoxin B1, ochratoxin A, sterigmatocystin, T-2 toxin or zearalenone

Preparations from fungal sources shall not contain detectable amounts, determined according to the method of Patterson and Roberts (Patterson, D.S. and Roberts, B.A., J. Assoc. of Analyt. Chemists, 62, 1265 (1979))

DETERMINATION OF ANTIBIOTIC ACTIVITY

SCOPE

This procedure is designed for the determination of antibiotic activity in enzyme preparations derived from microbial sources.

PRINCIPLE

The assay is based on the measurement of inhibition of bacterial growth under specific circumstances.

CULTURE PLATES

Six organisms are tested: Staphylococcus aureus ATCC 6538; Escherichia coli ATCC 11229; Bacillus cereus ATCC 2; Bacillus circulans ATCC 4516; Streptococcus pyrogenes ATCC 12344; and Serratia marcescens ATCC 14041. Make a test plate of each organism by preparing a 1:10 dilution of a 24 hour Trypticase Soya Broth culture in Trypticase Agar (TSA) (for Streptococcus pyrogenes ATCC 12344 a 1: 20 dilution).

Pour 15 ml of plain TSA into a Petri dish and allow the medium to harden. Overlay with 10 ml of seeded TSA and allow to solidify. Place a paper disk (prepared according to disk preparation below) of the tested enzyme on each of the 6 inoculated plates.

DISK PREPARATION

Make a 10% solution of the enzyme by adding 1 g of enzyme to 9 ml of sterile, distilled water. Mix thoroughly with a Vortex mixer to obtain a homogeneous suspension. Autoclave suitable paper disks (for instance, S&S Analytical Filter Papers No. 740-E, 12.7 mm in diameter), then saturate them with the enzyme by application of 0.1 ml (about 3 drops) of a 10% solution of the enzyme to the disk surface. Prepare 6 disks (1 for each of the 6 organisms) for each enzyme: place one disk on the surface of the 6 inoculated agar plates.

INCUBATION

Keep the 6 plates in the refrigerator overnight to obtain proper diffusion. Incubate the plates at 37^o for 24 h. Examine the plates for any inhibition zones that may have been caused by the enzyme preparation.

INTERPRETATION

A visually clear zone around a disk (total diameter: 16 mm) indicates the presence of antibacterial components in the enzyme preparation. If an enzyme preparation shows obvious antibacterial activity against 3 (or more) organisms it is concluded that antibiotic agents are present.

GENERAL CONSIDERATIONS AND SPECIFICATIONS FOR ENZYME PREPARATIONS FROM GENETICALLY MODIFIED MICROORGANISMS

Prepared at the 53^rd JECFA (1998) and published in FNP 52 Add 6 (1998), superseding Appendix prepared at the 37 th JECFA (1990) and published FNP 52 (1992). Amended at the 55^th JECFA (1999) and partially published in FNP 52 Add 7 (1999).

GENERAL CONSIDERATIONS

For the proper evaluation of enzyme preparations derived from genetically modified microorganisms, information should be provided on the host microorganism, the genetic material introduced into the host microorganism, and the recombinant production organism. Annex 1 of FNP 52 addresses factors relevant to all microbial sources (conventional and recombinant) used in the production of enzyme preparations and to fermentation and recovery procedures. The following points need emphasis when considering the production of enzyme preparations from genetically modified microorganisms:

1. The host microorganism should be taxonomically and genetically characterized.

2. Documentation that the host microorganism is non-pathogenic and non-toxicogenic should be provided.

3. The genetic material (i.e., the expression vector or expression plasmid) intended for introduction into the host microorganism should be characterized and a description of its construction provided. As appropriate, it should be demonstrated that the genetic material does not contain genes coding for virulence factors, protein toxins, or enzymes that may be involved in the synthesis of mycotoxins or any other toxic or undesirable substances. The source of the DNA encoding the enzyme of interest should be identified where possible.

4. The production microorganism should be characterized with respect to the introduced DNA, its genetic stability, and its growth properties.

5. If the production microorganism is capable of producing proteins that inactivate clinically useful antibiotics, documentation should be provided that the finished enzyme preparation contains neither antibiotic-inactivating proteins at concentrations that would interfere with antibiotic treatment nor DNA that is capable of transforming microorganisms, which potentially could lead to the spread of antibiotic resistance.

6. All enzyme preparations should be evaluated for their potential to elicit allergenic reactions. As a general rule, if the food is known to cause an allergic reaction in humans, its use as a source of DNA encoding the enzyme of interest should be avoided. In exceptional cases, where there is a demonstrated need to use an allergenic source of DNA, documentation should be provided indicating that the enzyme is not associated with the allergic reaction. The most common allergenic foods on a world-wide basis are fish, crustacea, peanuts, tree nuts, soybeans, milk, eggs, and wheat.

Points 1-6, above, cover the major issues pertinent to the development of enzyme preparations derived from genetically modified microorganisms. These issues emphasize and supplement those that must be considered in the safety evaluation of enzyme preparations containing non-recombinant enzymes, which relate to the avoidance of undesirable impurities, in general.

The following properties might be useful in characterizing the recombinant enzyme: molecular weight; isoelectric point; substrate specificity; reaction kinetics; activity as a function of pH and temperature; amino acid composition; amino acid sequence; a peptide map; and DNA base sequence coding for the enzyme.

SPECIFICATIONS

An enzyme preparation obtained from genetically modified microorganisms should meet the same general specification limits for arsenic, lead, heavy metals.(as lead) and microbial contaminants as a conventional preparation. The format for the specifications monograph should also parallel the monograph far a conventional enzyme preparation. In elaborating the SOURCE section of the monograph, however, the following should be noted:

Microbial strain numbers

Any microbial strain that meets the considerations described above should be a safe and suitable host for the introduced DNA. Citation in the monograph of the genus and species of the host organism is usually adequate for those that have been determined to be safe and suitable. Identification at the strain level may impose unnecessary constraints on the development of production micro-organisms used to produce food-grade enzymes. In the case of a non-pathogenic, non-toxicogenic strain that belongs to a species that includes pathogenic and toxicogenic strains (e.g. Escherichia coli), there should be a requirement in the monograph that the strain be non-pathogenic and non-toxicogenic. Citation of a suitable strain number may be included by way of example.

Vectors

Any well-characterized plasmid vector may be selected to construct the expression plasmid by appropriate modification and insertion of regulatory and coding DNA sequences. The documentation on the production microorganism, including the introduced DNA, can be used to verify the appropriateness of the selected expression plasmid. As with the citation of strains, the citation of specific expression plasmids is generally unnecessary and may impose unnecessary restrictions on the development of enzyme preparations.