Angela Boyce

Comparison of Selected Physicochemical Characteristics of Commercial Phytases Relevant to their Application in Phosphate Pollution Abatement

The enzyme phytase catalyses the dephosphorylation of phytic acid and its salts, phytates. Supplementation of monogastric animal feed with microbial-derived phytase increases the bioavailability of phytic acid bound phosphate. This facilitates a reduction in the addition of inorganic phosphate to the feed and reduces phosphorus excretion. To achieve maximum efficacy in terms of phosphate pollution abatement, supplemental phytases added to animal feed must survive thermal processing of the feed, resist inactivation by the proteolytic enzymes encountered in the animals digestive tract and display high activity at physiological temperature and pH. A series of in vitro experiments were carried out to determine the relative suitability of four major commercial phytase products for use in animal feed. The enzymes assessed lost between 14% and 72% of their original activities after heating to 80°C for 5 minutes. After exposure to simulated upper digestive tract conditions, the phytases assessed retained between 0 and 28% of their original activities. The commercial phytases displayed between 98% and 67% of their maximum activities at 39°C and all phytases assessed had an optimum pH between pH 4 and pH 5. None of the phytases assessed satisfied all of the criteria of an ideal phytase for use in animal feed.

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry.

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40°C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60°C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.

A phytase enzyme-based biochemistry practical particularly suited to students undertaking courses in biotechnology and environmental science

Courses in introductory biochemistry invariably encompass basic principles of enzymology, with reinforcement of lecture‐based material in appropriate laboratory practicals. Students undertaking practical classes are more enthusiastic, and generally display improved performance, when the specific experiments undertaken show direct relevance to their core degree area. Herein is described an enzyme‐based practical of particular relevance to students undertaking primary degrees in biotechnology, environmental science, nutrition, as well as animal and veterinary science. The practical entails assay of phytase, an enzyme that is now routinely included in monogastric animal feed in order to ameliorate the negative nutritional and environmental consequences of its substrate, dietary phytic acid. The practical is technically straightforward, requiring relatively basic laboratory equipment and reagents that are both easily obtained and relatively inexpensive. To be an effective digestive supplement, the enzyme must display a combination of physicochemical properties, which provide sufficient scope for an interesting and multifaceted post‐laboratory discussion.

Identification of fungal proteases potentially suitable for environmentally friendly cleaning-in-place in the dairy industry

Fourteen fungi were screened for ability to produce proteases with activity on milk protein. The proteases produced were assessed on a lab-scale in terms of their potential suitability for cleaning-in-place (CIP) in the dairy industry. Cleaning performance was assessed by determining the ability of the enzymes to remove an industrial-like milk fouling deposit from stainless steel. Based on the results observed, the extracellular protease activity produced by Schizophyllum commune was selected as most suitable for potential CIP application. A CIP procedure involving a sodium carbonate rinse followed by enzymatic cleaning with this fungal enzyme activity was developed. Satisfactory cleaning, judged by quantification of residual organic matter and protein on the stainless steel surface after cleaning, was achieved using the developed CIP procedure at 40°C. This CIP procedure, based on biodegradable enzymes working at low temperature is more environmentally favourable than conventional CIP methods using caustic based cleaning solutions at 70-80°C. Potential environmental benefits of the developed enzymatic CIP procedure include reduced energy consumption, decreased chemical usage and a reduced requirement for pH neutralisation of the resultant waste prior to release.

A Series of Enzymology-Based Experiments Designed to Mimic an Applied Research Project.

Four mini‐practicals are described in which the effects of temperature and pH on phytase activity are assessed, as well as the enzymes thermostability and the effect upon stability of simulated digestive tract conditions. Phytase is routinely incorporated into monogastric animal feed to ameliorate the negative nutritional and environmental consequences of its substrate, dietary phytic acid. In addition to illustrating selected basic concepts in enzymology, the combined experiments allow the students to determine the suitability of the test phytase for inclusion in animal feed. As such the practical mimics an applied research project and is particularly suited to biotechnology students undertaking courses in basic biochemistry. Students may be segregated into groups of 4, with each team member charged with undertaking one of the mini‐experiments. In this way students are given individual responsibility and learn to work as part of an integrated research grouping.