Safety and regulatory aspects of enzyme use

Only very few enzymes present hazards, because of their catalytic activity, to those handling them in normal circumstances but there are several areas of potential hazard arising from their chemical nature and source. These are allergenicity, activity-related toxicity, residual microbiological activity, and chemical toxicity.

All enzymes, being proteins, are potential allergens and have especially potent effects if inhaled as a dust. Once an individual has developed an immune response as a result of inhalation or skin contact with the enzyme, re-exposure produces increasingly severe responses becoming dangerous or even fatal. Because of this, dry enzyme preparations have been replaced to a large extent by liquid preparations, sometimes deliberately made viscous to lower the likelihood of aerosol formation during handling. Where dry preparations must be used, as in the formulation of many enzyme detergents, allergenic responses by factory workers are a very significant problem particularly when fine-dusting powders are employed. Workers in such environments are usually screened for allergies and respiratory problems. The problem has been largely overcome by encapsulating and granulating dry enzyme preparations, a procedure that has been applied most successfully to the proteases and other enzymes used in detergents. Enzyme producers and users recognise that allergenicity will always be a potential problem and provide safety information concerning the handling of enzyme preparations. They stress that dust in the air should be avoided so weighing and manipulation of dry powders should be carried out in closed systems. Any spilt enzyme powder should be removed immediately, after first moistening it with water. Any waste enzyme powder should be dissolved in water before disposal into the sewage system. Enzyme on the skin or inhaled should be washed with plenty of water. Liquid preparations are inherently safer but it is important that any spilt enzyme is not allowed to dry as dust formation can then occur. The formation of aerosols (e.g., by poor operating procedures in centrifugation) must be avoided as these are at least as harmful as powders.

Activity-related toxicity is much rarer but it must be remembered that proteases are potentially dangerous, particularly in concentrated forms and especially if inhaled. No enzyme has been found to be toxic, mutagenic or carcinogenic by itself as might be expected from its proteinaceous structure. However, enzyme preparations cannot be regarded as completely safe as such dangerous materials may be present as contaminants, derived from the enzyme source or produced during its processing or storage.

The organisms used in the production of enzymes may themselves be sources of hazardous materials and have been the chief focus of attention by the regulatory authorities. In the USA, enzymes must be Generally Regarded As Safe (GRAS) by the FDA (Food and Drug Administration) in order to be used as a food ingredient. Such enzymes include a-amylase, b-amylase, bromelain, catalase, cellulase, ficin, a-galactosidase, glucoamylase, glucose isomerase, glucose oxidase, invertase, lactase, lipase, papain, pectinase, pepsin, rennet and trypsin. In the UK, the Food Additives and Contaminants Committee (FACC) of the Ministry of Agriculture, Fisheries and Food classified enzymes into five classes on the basis of their safety for presence in the foods and use in their manufacture.

Group A.  Substances that the available evidence suggests are acceptable for use in food.

Group B.  Substances that on the available evidence may be regarded as provisionally acceptable for use in food but about which further information must be made available within a specified time for review.

Group C.  Substances for which the available evidence suggests toxicity and which ought not to be permitted for use in food until adequate evidence of their safety has been provided to establish their acceptability.

Group D.  Substances for which the available information indicates definite or probable toxicity and which ought not to be permitted for use in food.

Group E.  Substances for which inadequate or no toxicological data are available and for which it is not possible to express an opinion as to their acceptability for use in food.

This classification takes into account the potential chemical toxicity from microbial secondary metabolites such as mycotoxins and aflotoxins. The growing body of knowledge on the long-term effects of exposure to these toxins is one of the major reasons for the tightening of legislative controls.

The enzymes that fall into group A are exclusively plant and animal enzymes such as papain, catalase, lipase, rennet and various other proteases. Group B contains a very wide range of enzymes from microbial sources, many of which have been used in food or food processing for many hundreds of years. The Association of Microbial Food Enzyme Producers (AMFEP) has suggested subdivisions of the FACC's group B into:

Class ain8 microorganisms that have traditionally been used in food or in food processing, including Bacillus subtilis, Aspergillus niger, Aspergillus oryzae, Rhizopus oryzae, Saccharomyces cerevisiae, Kluyveromyces fragilis, Kluyveromyces lactis and Mucor javanicus. 

Class bin8 microorganisms that are accepted as harmless contaminants present in food, including Bacillus stearothermophilus, Bacillus licheniformis, Bacillus coagulans, and Klebsiella in8aerogenes.

Class cin8 microorganisms that are not included in Classes b and c, including Mucor miehei, Streptomyces albus, Trichoderma reesei, Actinoplanes missouriensis, and Penicillium emersonii.

It was proposed that Class a should not be subjected to testing and that Classes b and c should be subjected to the following tests:

1. acute oral toxicity in mice and rats,
2. subacute oral toxicity for 4 weeks in rats,
3. oral toxicity for 3 months in rats, and 
4. in vitro mutagenicity. 

In addition Class c should be tested for microorganism pathogenicity and, under exceptional circumstances, in vivo mutagenicity, teratogenicity, and carcinogenicity.

The cost of the various tests needed to satisfy the legal requirements are very significant and must be considered during the determination of process costs. Plainly the introduction of an enzyme from a totally new source will be a very expensive matter. It may prove more satisfactory to clone such an enzyme into one of AMFEP's Class a organisms but this will first require new legislation to regulate the use of cloned microbes in foodstuffs. Some of the safety problems associated with the use of free enzymes may be overcome by using immobilised enzymes (see Chapter 3). This is an extremely safe technique, so long as the materials used are acceptable and neither they, nor the immobilised enzymes, leak into the product stream.

The production of enzymes is subject, in the UK, to the Health and Safety at Work Act 1974, to ensure the health and safety of employees. Good manufacturing practice is employed and controls ensure that enzyme production is performed by a pure culture of the producing microbes.