Advances and challenges in the production of extracellular thermoduric pullulanases by wild-type and recombinant microorganisms: a review.

Thermoduric pullulanases, acting as starch-debranching enzymes, are required in many industrial applications, mainly in the production of concentrated glucose, maltose, and fructose syrups. To date, however, a single pullulanase, from Bacillus acidopullulyticus, is available on the market for industrial purposes. This review is an investigation of the major advances as well as the major challenges being faced with regard to optimization of the production of extracellular thermoduric pullulanases either by their original hosts or by recombinant organisms. The critical aspects linked to industrial pullulanase production, which should always be considered, are emphasized, including those parameters influencing solubility, thermostability, and catalytic efficiency of the enzyme. This review provides new insights for improving the production of extracellular thermoduric pullulanases in the hope that such information may facilitate their commercial utilization and potentially be applied to the development of other industrially relevant enzymes.

Optimization of the production of an extracellular and thermostable amylolytic enzyme by Thermus thermophilus HB8 and basic characterization.

The objective of this study was to determine the potential of Thermus thermophilus HB8 for accumulating a high level of extracellular, thermostable amylolytic enzyme. Initial production tests indicated clearly that only very low levels of amylolytic activity could be detected, solely from cells after extraction using the mild, non-ionic detergent Triton X-100. A sequential optimization strategy, based on statistical designs, was used to enhance greatly the production of extracellular amylolytic activity to achieve industrially attractive enzyme titers. Focus was placed on the optimal level of initial biomass concentration, culture medium composition and temperature for maximizing extracellular amylolytic enzyme accumulation. Empirical models were then developed describing the effects of the experimental parameters and their interactions on extracellular amylolytic enzyme production. Following such efforts, extracellular amylolytic enzyme accumulation was increased more than 70-fold, with enzyme titers in the 76 U/mL range. The crude extracellular enzyme was thereafter partially characterized. The optimal temperature and pH values were found to be 80 °C and 9.0, respectively. 100% of the initial enzyme activity could be recovered after incubation for 24 h at 80 °C, therefore, proving the very high thermostability of the enzyme preparation.

Validation of a Modified Version of Actero™ Salmonella Enrichment Media for Rapid Detection of Salmonella spp. in Environmental and Food Samples.

BACKGROUND: Actero™ Salmonella Enrichment Media1 (Actero™ Salmonella) is a culture broth developed to recover Salmonella spp. from foods and environmental surfaces. Performance of Actero™ Salmonella broth has already been assessed and validated (AOAC Performance Tested MethodSM 041403) for the detection of Salmonella spp. in various foods, feeds and environmental samples. OBJECTIVE: This study aimed to validate the performance of a modified version of Actero™ Salmonella broth by incorporating one of the two liquid supplements into the powdered formula. METHODS: Inclusivity, exclusivity, stability, and lot-to-lot studies were carried out. Raw ground beef, chicken carcass rinse, dry pet food and stainless steel samples were enriched for 14-20 h at 35-39°C and analyzed using real-time PCR assay as well as by direct plating. RESULTS: The Probability of Detection assay confirmed the equivalent performance of the alternative methods as compared to the reference methods. All Salmonella strains, except Salmonella II : 57: z29:-, were able to grow in Actero™ Salmonella broth. One-half of the non-target strains did not grow in Actero™ Salmonella broth, whereas the atypical for Salmonella growth was observed for other non-target microorganisms subsequently plated onto selective and differential agars. Lot-to-lot consistency was demonstrated for three consecutively manufactured lots of the broth. The liquid broth was proven to be stable at 4°C for up to 9 weeks of storage. CONCLUSIONS AND HIGHLIGHTS: The incorporation of one of the two specific supplements into a powdered formula of Actero™ Salmonella broth made it more convenient to use without compromising the performance and accuracy.