Properties of biopolymers produced by transglutaminase treatment of whey protein isolate and gelatin.

Byproduct utilization is an important consideration in the development of sustainable processes. Whey protein isolate (WPI), a byproduct of the cheese industry, and gelatin, a byproduct of the leather industry, were reacted individually and in blends with microbial transglutaminase (mTGase) at pH 7.5 and 45 degrees C. When a WPI (10% w/w) solution was treated with mTGase (10 U/g) under reducing conditions, the viscosity increased four-fold and the storage modulus (G') from 0 to 300 Pa over 20 h. Similar treatment of dilute gelatin solutions (0.5-3%) had little effect. Addition of gelatin to 10% WPI caused a synergistic increase in both viscosity and G', with the formation of gels at concentrations greater than 1.5% added gelatin. These results suggest that new biopolymers, with improved functionality, could be developed by mTGase treatment of protein blends containing small amounts of gelatin with the less expensive whey protein.

Prion infected meat-and-bone meal is still infectious after biodiesel production.

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process.

Diffusion of protease into meat & bone meal for solubility improvement and potential inactivation of the BSE prion.

BACKGROUND: Government-imposed feed bans have created a need for new applications for meat & bone meal (MBM). Many potential new applications require MBM protein to be both soluble and free of infectious prion. Treatment with protease is generally effective in reducing insoluble, thermally-denatured proteins to soluble peptides. It has been reported in the literature that certain proteases, including Versazyme, are able to degrade infectious prions in a system where the prions are readily accessible to proteolytic attack. Prions distributed within MBM, however, may conceivably be protected from proteases. METHODOLOGY/PRINCIPAL FINDINGS: The overall rate of proteolytic MBM digestion depends greatly on whether the protease can penetrate deep within individual particles, or if the protease can only act near the surface of the particle. This research examined the barriers to the diffusion of Versazyme into particles of MBM. Confocal microscopy demonstrated differences in the density distributions between the bone and the soft tissue particles of MBM. By tracking the diffusion of fluorescently labeled Versazyme through individual particles, it was found that bone particles show full Versazyme penetration within 30 minutes, while penetration of soft tissue particles can take up to four hours, depending on the particle's diameter. From the variety of normal proteins comprising MBM, a specific protein was chosen to serve as a prion surrogate based on characteristics including size, solubility, distribution and abundance. This surrogate was used to measure the effect of several factors on Versazyme diffusion. CONCLUSIONS/SIGNIFICANCE: Results showed that surrogate distributed in bone particles was more susceptible to degradation than that in soft tissue particles. Three factors controllable by unit operations in an industrial-scale process were also tested. It was found that removing the lipid content and hydrating MBM prior to incubation both significantly increased the rate of surrogate degradation. In a test of particle size, the smallest collected diameter range demonstrated the largest degradation of the prion surrogate, suggesting milling would be beneficial.

Isolation, characterization and optimization of culture parameters for production of an alkaline protease isolated from Aspergillus tamarii.

Aspergillus tamarii expresses an extracellular alkaline protease that we show to be effective in removing hair from cattle hide. Large quantities of the enzyme will be required for the optimization of the enzymatic dehairing process so the growth conditions for maximum protease expression by A. tamarii were optimized for both solid-state culture on wheat bran and for broth culture. Optimal protease expression occurred, for both cultural media, at initial pH 9; the culture was incubated at 30 degrees C for 96 h using a 5% inoculum. The crude enzyme was isolated, purified and characterized using MALDI TOF TOF. The alkaline protease was homologous to the alkaline protease expressed by Aspergillus viridinutans.

Conversion of agricultural feedstock and coproducts into poly(hydroxyalkanoates).

Aside from their importance to the survival and general welfare of mankind, agriculture and its related industries produce large quantities of feedstocks and coproducts that can be used as inexpensive substrates for fermentative processes. Successful adoption of these materials into commercial processes could further the realization of a biorefinery industry based on agriculturally derived feedstocks. One potential concept is the production of poly(hydroxyalkanoate) (PHA) polymers, a family of microbial biopolyesters with a myriad of possible monomeric compositions and performance properties. The economics for the fermentative production of PHA could benefit from the use of low-cost agricultural feedstocks and coproducts. This mini-review provides a brief survey of research performed in this area, with specific emphasis on studies describing the utilization of intact triacylglycerols (vegetable oils and animal fats), dairy whey, molasses, and meat-and-bone meal as substrates in the microbial synthesis of PHA polymers.

Transesterification activity of lipases immobilized in a phyllosilicate sol-gel matrix.

Lipases from Pseudomonas cepacia (P.c.) and Thermomyces lanuginosa (T.l.) were immobilized in a phyllosilicate sol-gel matrix and studied for their ability to catalyze the alcoholysis of fats and oils to simple alkyl esters. At 50 degrees C and 48 h reaction immobilized T.l. lipase gave higher alkyl ester yields (70 to 100%) from fats and oils regardless of chain length or degree of unsaturation of the acyl groups in the triacylglycerols than did immobilized P.c. lipase (20-90%), which preferred unsaturated oils. Both immobilized lipases catalyzed ester formation (80-90%) from greases containing a range of free fatty acids (2.6 to 36%). Molecular sieves had no effect on ester yields in the immobilized T.l. lipase-catalyzed alcoholysis of greases but did improve yields (5-10%) in the immobilized P.c. lipase-catalyzed reactions.

Optimization of alkyl ester production from grease using a phyllosilicate sol-gel immobilized lipase.

Simple alkyl ester derivatives of restaurant grease were prepared using a lipase from Pseudomonoas cepacia immobilized within a phyllosilicate sol-gel matrix as biocatalyst. Alcoholysis reactions of grease were carried out in solvent-free media using a one-step addition of alcohol to the reaction mixture. The immobilized lipase was active from 40 to 70 degrees C. Ester yields (60-97%) were highest when using a ratio of reactants of 2 mmol grease to 8 mmol alcohol and the biocatalyst was 10% (w/w) of grease in the presence of molecular sieves.

Immobilized lipase-catalysed production of alkyl esters of restaurant grease as biodiesel.

Simple alkyl ester derivatives of restaurant grease were prepared using immobilized lipases as biocatalysts. The lipases studied included those of Thermomyces lanuginosa and Candida antarctica supported on granulated silica (gran- T.l. and gran- C.a., respectively), C. antarctica supported on a macroporous acrylic resin (SP435) and Pseudomonas cepacia immobilized within a phyllosilicate sol-gel matrix (IM PS-30). All alcoholysis reactions were carried out in solvent-free media employing a one-step addition of the alcohol to the reaction system. Of the lipases studied, IM PS-30 was found to be the most effective in catalysing the methanolysis and ethanolysis of grease. The processes catalysed by gran- T.l. and gran- C.a. lipases gave poor conversions to esters, and the SP435-catalysed reactions gave intermediate yields of ethyl and methyl esters. Water activity (a(w)) was an important factor in the methanolysis reactions; reaction media with a(w)<0.5 resulted in the highest conversions to methyl esters. Molecular sieves also improved methyl ester yields by as much as 20% in transesterification reactions catalysed by IM PS-30. The immobilized lipases also were evaluated for their ability to produce alkyl esters of grease with several additional normal and branched-chain alcohols.