Food-processing enzymes from recombinant microorganisms--a review.

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.

Influence of fruit variety, harvest technique, quality sorting, and storage on the native microflora of unpasteurized apple cider.

Apple variety, harvest, quality sorting, and storage practices were assessed to determine their impact on the microflora of unpasteurized cider. Seven apple varieties were harvested from the tree or the ground. The apples were used fresh or were stored at 0 to 4 degrees C for < or = 5 months and were pressed with or without quality selection. Cider yield, pH, Brix value, and titratable acidity were measured. Apples, postpressing apple pomace, and cider samples were analyzed for aerobic bacteria, yeasts, and molds. Aerobic bacterial plate counts (APCs) of ciders from fresh ground-picked apples (4.89 log CFU/ml) were higher than those of ciders made from fresh, tree-picked apples (3.45 log CFU/ml). Quality sorting further reduced the average APC to 2.88 log CFU/ml. Differences among all three treatment groups were significant (P < 0.0001). Apple and pomace microbial concentrations revealed harvest and postharvest treatment-dependent differences similar to those found in cider. There were significant differences in APC among apple varieties (P = 0.0001). Lower counts were associated with varieties exhibiting higher Brix values and higher titratable acidity. Differences in APC for stored and fresh apples used for cider production were not significant (P > 0.05). Yeast and mold counts revealed relationships similar to those for APCs. The relationship between initial microbial load found on incoming fruit and final cider microbial population was curvilinear, with the weakest correlations for the lowest apple microflora concentrations. The lack of linearity suggests that processing equipment contributed to cider contamination. Tree-picked quality fruit should be used for unpasteurized cider production, and careful manufacturing practices at cider plants can impact both safety and quality of the final product.

Potential for internalization, growth, and survival of Salmonella and Escherichia coli O157:H7 in oranges.

Internalization potential, survival, and growth of human pathogens within oranges were investigated in a series of laboratory experiments. Submerging oranges into dye solutions at various temperature differentials was used to assess internalization potential. Conditions in which dye internalization was observed were further studied by applying Escherichia coli O157:H7 or Salmonella onto the stem scar, subjecting the oranges to a temperature differential, juicing, and measuring numbers of pathogens in the resulting juice. Pathogens for growth and survival studies were applied to or injected into simulated peel punctures. Oranges with small peel holes of selected sizes were also placed into solutions containing these pathogens. Bacterial survival was also evaluated in orange juice at 4 and 24 degrees C. Oranges internalized pathogens at a frequency of 2.5 to 3.0%, which mirrored dye internalization frequency (3.3%). Pathogens were internalized at an uptake level of 0.1 to 0.01% of the challenge applied. Bacteria grew within oranges at 24 degrees C, but not at 4 degrees C. Thirty-one percent of oranges with 0.91-mm surface holes showed pathogen uptake, whereas 2% of oranges with 0.68-mm holes showed pathogen uptake. Pathogens added to fresh orange juice and incubated at 24 degrees C declined 1 log CFU/ml within 3 days. These results suggest that internalization, survival, and growth of human bacterial pathogens can occur within oranges intended for producing unpasteurized juice.

Apple quality, storage, and washing treatments affect patulin levels in apple cider.

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.

Efficacy of sanitation and cleaning methods in a small apple cider mill.

The efficacy of cleaning and sanitation in a small apple cider processing plant was evaluated by surface swab methods as well as microbiological examination of incoming raw ingredients and of the final product. Surface swabs revealed that hard-to-clean areas such as apple mills or tubing for pomace and juice transfer may continue to harbor contaminants even after cleaning and sanitation. Use of poor quality ingredients and poor sanitation led to an increase of approximately 2 logs in aerobic plate counts of the final product. Reuse of uncleaned press cloths contributed to increased microbiological counts in the finished juice. Finally, using apples inoculated with Escherichia coli K-12 in the plant resulted in an established population within the plant that was not removed during normal cleaning and sanitation. The data presented in this study suggest that current sanitary practices within a typical small cider facility are insufficient to remove potential pathogens.