Enzymes are commonly used in food processing and in the production of food ingredients. Enzymes traditionally isolated from culturable microorganisms, plants, and mammalian tissues are often not well-adapted to the conditions used in modern food production methods. The use of recombinant DNA technology has made it possible to manufacture novel enzymes suitable for specific food-processing conditions. Such enzymes may be discovered by screening microorganisms sampled from diverse environments or developed by modification of known enzymes using modern methods of protein engineering or molecular evolution. As a result, several important food-processing enzymes such as amylases and lipases with properties tailored to particular food applications have become available. Another important achievement is improvement of microbial production strains. For example, several microbial strains recently developed for enzyme production have been engineered to increase enzyme yield by deleting native genes encoding extracellular proteases. Moreover, certain fungal production strains have been modified to reduce or eliminate their potential for production of toxic secondary metabolites. In this article, we discuss the safety of microorganisms used as hosts for enzyme-encoding genes, the construction of recombinant production strains, and methods of improving enzyme properties. We also briefly describe the manufacture and safety assessment of enzyme preparations and summarize options for submitting information on enzyme preparations to the US Food and Drug Administration.

Caulobacters are prosthecate (stalked) bacteria that elaborate an attachment organelle called a holdfast at the tip of the cellular stalk. We examined the binding of lectins to the holdfasts of 16 marine Caulobacter strains and 10 freshwater species or strains by using a panel of fluorescein-conjugated lectins and fluorescence microscopy. The holdfasts of all the marine isolates bound to only wheat germ agglutinin (WGA) and other lectins that bind N-acetylglucosamine (GlcNac) residues. The freshwater caulobacters showed more variability in holdfast composition. Some bound only to WGA and comparable lectins as the marine strains did. Others bound additional or other lectins, and some did not bind to the lectins tested. The binding of WGA appeared to involve the regions of the holdfast involved with adhesion; a holdfast bound to WGA was significantly less adhesive to glass. Competition experiments with WGA-binding holdfasts and oligomers of GlcNac demonstrated that trimers of GlcNac (the preferred substrate for WGA binding) were more effective than dimers or monomers in preventing WGA binding to holdfasts, suggesting that stretches of contiguous GlcNac residues occur in the WGA-binding holdfasts. In addition, differences between freshwater and marine holdfasts in the strength of WGA binding were noted. The eect of a number of proteolytic and glycolytic enzymes on holdfast integrity was examined; the proteases had no,effect for all caulobacters. None of the glycolytic enzymes had an effect on marine caulobacter holdfasts, but chitinase and lysozyme (both attack oligomers of GlcNac) disrupted the holdfasts of those freshwater caulobacters that bound WGA. Despite some similarity to chitin, holdfasts did not bind Calcofluor and no measurable effects on holdfast production were detectable after cell growth in the presence of diflubenzuron or polyoxin D, inhibitors of chitin synthesis in other systems. Finally, the holdfasts of all caulobacters bound to colloidal gold particles, without regard to the coating used to stabilize the gold particles. This binding was stronger or more specific than WGA binding; treatment with colloidal gold particles prevented WGA binding, but the reverse was not the case.

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Patulin is a mycotoxin produced primarily by Penicillium expansum, a mold responsible for rot in apples and other fruits. The growth of this fungus and the production of patulin are common in fruit that has been damaged. However, patulin can be detected in visibly sound fruit. The purpose of this project was to determine how apple quality, storage, and washing treatments affect patulin levels in apple cider. Patulin was not detected in cider pressed from fresh tree-picked apples (seven cultivars) but was found at levels of 40.2 to 374 microg/liter in cider pressed from four cultivars of fresh ground-harvested (dropped) apples. Patulin was not detected in cider pressed from culled tree-picked apples stored for 4 to 6 weeks at 0 to 2 degrees C but was found at levels of 0.97 to 64.0 microg/liter in cider pressed from unculled fruit stored under the same conditions. Cider from controlled-atmosphere-stored apples that were culled before pressing contained 0 to 15.1 microg of patulin per liter, while cider made from unculled fruit contained 59.9 to 120.5 microg of patulin per liter. The washing of ground-harvested apples before pressing reduced patulin levels in cider by 10 to 100%, depending on the initial patulin levels and the type of wash solution used. These results indicate that patulin is a good indicator of the quality of the apples used to manufacture cider. The avoidance of ground-harvested apples and the careful culling of apples before pressing are good methods for reducing patulin levels in cider.