The following general specifications were prepared by the Committee at its sixty-seventh meeting (2006) for publication in FAO JECFA Monographs 3 (2006), superseding the general specifications prepared at the fifty-seventh meeting (1) and published in FAO JECFA Monographs 1 (2). These specifications were originally prepared by the Committee at its twenty-fifth meeting (3) and published in FAO Food and Nutrition Papers No. 19 and No. 31/2 (4,5). Subsequent revisions were made by the Committee at its thirty-fifth meeting and published in FAO Food and Nutrition Paper No. 52 (6). Additional amendments were made at the fifty-first meeting and published in FAO Food and Nutrition Paper No. 52 Add. 6 (7), and at the fifty-third meeting (8) and partially published in FAO Food and Nutrition Paper No. 52 Add. 7 (9).
Classification and nomenclature of enzymes
Enzymes are proteins that catalyse chemical reactions. The Enzyme Commission of the International Union of Biochemistry and Molecular Biology (formerly the International Union of Biochemistry) classified enzymes into six main classes: oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases (10). Based on the type of reaction catalysed, enzymes are assigned to one of these classes and given an Enzyme Commission (EC) number, a systematic name, and a common name. Other names are also provided, if available. Enzymes used in food processing are often referred to by their common or traditional names such as protease, amylase, malt, or rennet. For enzymes derived from microorganisms, the name of the source microorganism is usually specified, for example, “α-amylase from Bacillus subtilis.” For enzymes derived from microorganisms modified by using recombinant DNA techniques (referred to as recombinant-DNA microorganisms or genetically modified microorganisms), the names of both the enzyme source (donor organism) and the production microorganism are provided, for example, “α-amylase from Bacillus licheniformis expressed in Bacillus subtilis.”
Enzyme preparations
Enzymes are used in food processing as enzyme preparations. An enzyme preparation contains an active enzyme (in some instances a blend of two or more enzymes) and intentionally added formulation ingredients such as diluents, stabilizing agents, and preserving agents. The formulation ingredients may include water, salt, sucrose, sorbitol, dextrin, cellulose, or other suitable compounds. Enzyme preparations may also contain constituents of the source organism (i.e. an animal, plant, or microbial material from which an enzyme was isolated) and compounds derived from the manufacturing process, for example, the residues of the fermentation broth. Depending on the application, an enzyme preparation may be formulated as a liquid, semi-liquid or dried product. The colour of an enzyme preparation may vary from colourless to dark brown. Some enzymes are immobilized on solid support materials.
Active components
Enzyme preparations usually contain one principal enzyme that catalyses one specific reaction during food processing. For example, α-amylase catalyses the hydrolysis of 1,4-α-D-glucosidic linkages in starch and related polysaccharides. However, some enzyme preparations contain a mixture of enzymes that catalyse two or more different reactions in food. Each principal enzyme present in an enzyme preparation is characterized by its systematic name, common name, and EC number. The activity of each enzyme is measured using an appropriate assay and expressed in defined activity units per weight (or volume) of the preparation.
Source materials
Enzymes used in food processing are derived from animal, plant, and microbial sources. Animal tissues used for the preparation of enzymes should comply with meat inspection requirements and be handled in accordance with good hygienic practice.
Plant material and microorganisms used in the production of enzyme preparations should not leave any residues harmful to health in the processed finished food under normal conditions of use.
Microbial strains used in the production of enzyme preparations may be native strains or mutant strains derived from native strains by the processes of serial culture and selection or mutagenesis and selection or by the application of recombinant DNA technology. Although nonpathogenic and nontoxigenic microorganisms are normally used in the production of enzymes used in food processing, several fungal species traditionally used as sources of enzymes are known to include strains capable of producing low levels of certain mycotoxins under fermentation conditions conducive to mycotoxin synthesis (11–15). Enzyme preparations derived from such fungal species should not contain toxicologically significant levels of mycotoxins that could be produced by these species.
Microbial production strains should be taxonomically and genetically characterized and identified by a strain number or other designation. The strain identity may be included in individual specifications, if appropriate. The strains should be maintained under conditions that ensure the absence of genetic drift and, when used in the production of enzyme preparations, should be subjected to methods and culture conditions that are applied consistently and reproducibly from batch to batch. Such conditions should prevent the introduction of microorganisms that could be the source of toxic and other undesirable substances. Culture media used for the growth of microbial sources should consist of components that leave no residues harmful to health in the processed finished food under normal conditions of use.
Enzyme preparations should be produced in accordance with good food manufacturing practice and cause no increase in the total microbial count in the treated food over the level considered to be acceptable for the respective food.
Substances used in processing and formulation
Substances used in processing and formulation of enzyme preparations should be suitable for their intended uses.
In the case of immobilized enzyme preparations, leakage of active enzymes, support materials, crosslinking agents and/or other substances used in immobilization should be kept within acceptable limits established in the individual specifications.
To distinguish the proportion of the enzyme preparation derived from the source material and manufacturing process from that contributed by intentionally added formulation ingredients, the content of total organic solids (TOS) is calculated as follows:
% TOS = 100 - (A + W + D)
where:
A = % ash, W = % water and D = % diluents and/or other formulation ingredients.
Purity
Lead:
Not more than 5 mg/kg
Determine using an atomic absorption spectroscopy/inductively coupled atomic-emission spectroscopy (AAS/ICP-AES) technique appropriate to the specified level. The selection of the sample size and the method of sample preparation may be based on the principles described in the Compendium of Food Additive Specifications, Volume 4.
Microbiological criteria:
Salmonella species: absent in 25 g of sample
Total coliforms: not more than 30 per gram
Escherichia coli: absent in 25 g of sample
Determine using procedures described in Volume 4.
Antimicrobial activity:
Absent in preparations from microbial sources.
Other considerations
Safety assessment of food enzyme preparations has been addressed in a number of publications and documents. Pariza & Foster (11) proposed a decision tree for determining the safety of microbial enzyme preparations. Pariza & Johnson (16) subsequently updated this decision tree and included information on enzyme preparations derived from recombinant-DNA microorganisms. The Scientific Committee on Food (17) issued guidelines for the presentation of data on food enzymes. The document includes a discussion on enzymes from genetically modified organisms including microorganisms, plants, and animals. Several international organizations, government agencies, and expert groups have also published discussion papers or guidelines that address safety assessment of food and food ingredients derived from recombinant-DNA plants and microorganisms (18–28). Certain information in these documents may be applicable to enzyme preparations derived from recombinant sources.
An overall safety assessment of each enzyme preparation intended for use in food processing should be performed. This assessment should include an evaluation of the safety of the production organism, the enzyme component, side activities, the manufacturing process, and the consideration of dietary exposure. Evaluation of the enzyme component should include considerations of its potential to cause an allergic reaction. For enzyme preparations from recombinant-DNA microorganisms, the following should also be considered:
1. The genetic material introduced into and remaining in the production microorganism should be characterized and evaluated for function and safety, including evidence that it does not contain genes encoding known virulence factors, protein toxins, and enzymes involved in the synthesis of mycotoxins or other toxic or undesirable substances.
2. Recombinant-DNA production microorganisms might contain genes encoding proteins that inactivate clinically useful antibiotics. Enzyme preparations derived from such microorganisms should contain neither antibiotic inactivating proteins at concentrations that would interfere with antibiotic treatment nor transformable DNA that could potentially contribute to the spread of antibiotic resistance.
References