Use of dairy industry side-stream lactose for tablet manufacturing - proof of concept study.

During recent years there have been shortages of certain drugs due to problems in raw material supply. These are often related to active ingredients but could also affect excipients. Lactose is one of the most used excipients in tableting and comes in two anomeric and several solid-state forms. The aim of this study was to utilize lactose from a dairy side-stream and compare it against a commercial reference in direct compression. This would be a sustainable option and would secure domestic availability during crises. Two types of lactose, spray-dried and freeze-dried, were evaluated. Lactose was mixed with microcrystalline cellulose in different ratios together with lubricant and glidant, and flowability and tabletability of the formulations was characterized. The fully amorphous and small particle-sized spray-dried lactose flowed inadequately but exhibited good tabletability. The larger particle-sized, freeze-dried lactose exhibited sufficient flow and better tabletability than the commercial reference. However, disintegration and drug release were slower when using the investigational lactose formulations. This was most likely due to remaining milk proteins, especially caseins, in the lactose. Overall, the investigational lactose provides promise for the use of such a side-stream product during crisis situations but enhancing their properties and/or purity would be needed.

Cases of trisomy 21 and trisomy 18 among historic and prehistoric individuals discovered from ancient DNA.

Aneuploidies, and in particular, trisomies represent the most common genetic aberrations observed in human genetics today. To explore the presence of trisomies in historic and prehistoric populations we screen nearly 10,000 ancient human individuals for the presence of three copies of any of the target autosomes. We find clear genetic evidence for six cases of trisomy 21 (Down syndrome) and one case of trisomy 18 (Edwards syndrome), and all cases are present in infant or perinatal burials. We perform comparative osteological examinations of the skeletal remains and find overlapping skeletal markers, many of which are consistent with these syndromes. Interestingly, three cases of trisomy 21, and the case of trisomy 18 were detected in two contemporaneous sites in early Iron Age Spain (800-400 BCE), potentially suggesting a higher frequency of burials of trisomy carriers in those societies. Notably, the care with which the burials were conducted, and the items found with these individuals indicate that ancient societies likely acknowledged these individuals with trisomy 18 and 21 as members of their communities, from the perspective of burial practice.

Production of manganese peroxidase and laccase in a solid-state bioreactor and modeling of enzyme production kinetics.

Lignin-modifying enzymes have various promising applications such as biobleaching, biopulping, the functionalization of lignocellulosic materials, the modification of wood fibers, the remediation of contaminated soil and effluents, as well as improvement of the enzymatic hydrolysis of lignocellulosic substrates. In this study, the production of laccase and manganese peroxidase (MnP) in solid-state cultivation was examined. Oat husks were used as an inexpensive substrate for the white-rot fungus Cerrena unicolor PM170798 (FBCC 387). The addition of a fines fraction (consisting of oat flour and finely ground husks) enhanced MnP production fivefold and laccase production almost threefold. The enzyme production was studied first on a 100 g scale, and the cultivation experiments were then repeated at a larger laboratory-scale (4 kg) in a solid-state bioreactor. High enzyme activity levels were obtained (MnP: 340 nkat g(-1) DM, laccase: 470 nkat g(-1) DM). In addition, the correlation between the CO2 evolution rate and enzyme production was mathematically modeled from the bioreactor experimental data. The model parameters could be used to predict enzyme production.

Mechanisms of laccase-mediator treatments improving the enzymatic hydrolysis of pre-treated spruce.

BACKGROUND: The recalcitrance of softwood to enzymatic hydrolysis is one of the major bottlenecks hindering its profitable use as a raw material for platform sugars. In softwood, the guaiacyl-type lignin is especially problematic, since it is known to bind hydrolytic enzymes non-specifically, rendering them inactive towards cellulose. One approach to improve hydrolysis yields is the modification of lignin and of cellulose structures by laccase-mediator treatments (LMTs). RESULTS: LMTs were studied to improve the hydrolysis of steam pre-treated spruce (SPS). Three mediators with three distinct reaction mechanisms (ABTS, HBT, and TEMPO) and one natural mediator (AS, that is, acetosyringone) were tested. Of the studied LMTs, laccase-ABTS treatment improved the degree of hydrolysis by 54%, while acetosyringone and TEMPO increased the hydrolysis yield by 49% and 36%, respectively. On the other hand, laccase-HBT treatment improved the degree of hydrolysis only by 22%, which was in the same order of magnitude as the increase induced by laccase treatment without added mediators (19%). The improvements were due to lignin modification that led to reduced adsorption of endoglucanase Cel5A and cellobiohydrolase Cel7A on lignin. TEMPO was the only mediator that modified cellulose structure by oxidizing hydroxyls at the C6 position to carbonyls and partially further to carboxyls. Oxidation of the reducing end C1 carbonyls was also observed. In contrast to lignin modification, oxidation of cellulose impaired enzymatic hydrolysis. CONCLUSIONS: LMTs, in general, improved the enzymatic hydrolysis of SPS. The mechanism of the improvement was shown to be based on reduced adsorption of the main cellulases on SPS lignin rather than cellulose oxidation. In fact, at higher mediator concentrations the advantage of lignin modification in enzymatic saccharification was overcome by the negative effect of cellulose oxidation. For future applications, it would be beneficial to be able to understand and modify the binding properties of lignin in order to decrease unspecific enzyme binding and thus to increase the mobility, action, and recyclability of the hydrolytic enzymes.

Effect of temperature on lignin-derived inhibition studied with three structurally different cellobiohydrolases.

Non-productive enzyme adsorption onto lignin inhibits enzymatic hydrolysis of lignocellulosic biomass. Three cellobiohydrolases, Trichoderma reesei Cel7A (TrCel7A) and two engineered fusion enzymes, with distinctive modular structures and temperature stabilities were employed to study the effect of temperature on inhibition arising from non-productive cellulase adsorption. The fusion enzymes, TeCel7A-CBM1 and TeCel7A-CBM3, were composed of a thermostable Talaromyces emersonii Cel7A (TeCel7A) catalytic domain fused to a carbohydrate-binding module (CBM) either from family 1 or from family 3. With all studied enzymes, increase in temperature was found to increase the inhibitory effect of supplemented lignin in the enzymatic hydrolysis of microcrystalline cellulose. However, for the different enzymes, lignin-derived inhibition emerged at different temperatures. Low binding onto lignin and thermostable structure were characteristic for the most lignin-tolerant enzyme, TeCel7A-CBM1, whereas TrCel7A was most susceptible to lignin especially at elevated temperature (55 °C).

The carbohydrate-binding module of xylanase from Nonomuraea flexuosa decreases its non-productive adsorption on lignin.

BACKGROUND: The enzymatic hydrolysis step converting lignocellulosic materials into fermentable sugars is recognized as one of the major limiting steps in biomass-to-ethanol process due to the low efficiency of enzymes and their cost. Xylanases have been found to be important in the improvement of the hydrolysis of cellulose due to the close interaction of cellulose and xylan. In this work, the effects of carbohydrate-binding module (CBM family II) of the xylanase 11 from Nonomuraea flexuosa (Nf Xyn11) on the adsorption and hydrolytic efficiency toward isolated xylan and lignocellulosic materials were investigated. RESULTS: The intact family 11 xylanase of N. flexuosa clearly adsorbed on wheat straw and lignin, following the Langmuir-type isotherm. The presence of the CBM in the xylanase increased the adsorption and hydrolytic efficiency on insoluble oat spelt xylan. But the presence of the CBM did not increase adsorption on pretreated wheat straw or isolated lignin. On the contrary, the CBM decreased the adsorption of the core protein to lignin containing substrates, indicating that the CBM of N. flexuosa xylanase did not contribute to the non-productive adsorption. CONCLUSION: The CBM of the N. flexuosa xylanase was shown to be a xylan-binding module, which had low affinity on cellulose. The CBM of the N. flexuosa xylanase reduced the non-specific adsorption of the core protein to lignin and showed potential for improving the hydrolysis of lignocellulosic materials to platform sugars.

The laccase-catalyzed modification of lignin for enzymatic hydrolysis.

The efficient use of cellulases in the hydrolysis of pretreated lignocellulosic biomass is limited due to the presence of lignin. Lignin is known to bind hydrolytic enzymes nonspecifically, thereby reducing their action on carbohydrate substrates. The composition and location of residual lignin therefore seem to be important for optimizing the enzymatic hydrolysis of lignocellulosic substrates. The use of lignin-modifying enzymes such as laccase may have potential in the modification or partial removal of lignin from the biomass. In this study, the effect of lignin modification by laccase on the hydrolysis of pretreated spruce (Picea abies) and giant reed (Arundo donax) was evaluated. The substrates were first treated with laccase and then hydrolyzed with commercial cellulases. Laccase modification improved the hydrolysis yield of spruce by 12%, but surprisingly had an adverse effect on giant reed, reducing the hydrolysis yield by 17%. The binding properties of cellulases on the untreated and laccase-treated lignins were further studied using isolated lignins. The laccase treatment reduced the binding of enzymes on modified spruce lignin, whereas with giant reed, the amount of bound proteins increased after laccase treatment. Further understanding of the reactions of laccase on lignin will help to control the unspecific-binding of cellulases on lignocellulosic substrates.

Morphology and laccase production of white-rot fungi grown on wheat bran flakes under semi-solid-state fermentation conditions.

In this paper, we studied the laccase production and the growth morphology of different white-rot fungi, i.e. Pleurotus ostreatus, Trametes pubescens, Cerrena unicolor and Trametes versicolor, cultured under semi-solid-state fermentation conditions using wheat bran flakes as a natural low-cost support substrate. Trametes versicolor exhibited the highest laccase activity per gram of total dry matter, followed by P. ostreatus (63.5 and 58.2Ug(-1) , respectively). In addition, they showed a time profile of laccase production that was quite similar. Growth morphology was studied using environmental microscopic images and analyzed by discrete Fourier transformation-based software to determine the mean diameter of the hyphae, the number of hypha layers and the global micromorphology. The four strains exhibited different micromorphologies of growth. Pleurotus ostreatus presented narrow hyphae, which formed many thick clumps, T. pubescens and T. versicolor showed clumps of different sizes and C. unicolor showed thick hyphae that formed larger clumps, but in less amounts.

Biomass measurement online: the performance of in situ measurements and software sensors.

Biomass measurement is one of the most critical measurements in biotechnological processes. The technologies developed for the measurement of biomass in situ have developed over the years. Because it has been over 10 years since the last review concentrating on practical issues concerning biomass measurements, it is time to evaluate recent developments in the field. This review concentrates on the applications of dielectric spectroscopy, optical density, infrared spectroscopy, and fluorescence for in situ measurement of biomass. The advantages offered by these methods and an economic way of estimating biomass concentration, the software sensors, are considered.

A chemostat study of Streptomyces peucetius var. caesius N47.

The behavior of Streptomyces peucetius var. caesius N47 was studied in a glucose limited chemostat with a complex cultivation medium. The steady-state study yielded the characteristic constants mu (max) over 0.10 h(-1), Y (XS) 0.536 g g(-1), and m(S) 0.54 mg g(-1) h(-1). The product of secondary metabolism, epsilon-rhodomycinone, was produced with characteristics Y (PX) 12.99 mg g(-1) and m (P) 1.20 mg g(-1) h(-1). Significant correlations were found for phosphate and glucose consumption with biomass and epsilon-rhodomycinone production. Metabolic flux analysis was conducted to estimate intracellular fluxes at different dilution rates. TCA, PPP, and shikimate pathway fluxes exhibited bigger values with production than with growth. Environmental perturbation experiments with temperature, airflow, and pH changes on a steady-state chemostat implied that an elevation of pH could be the most effective way to shift the cells from growing to producing, as the pH change induced the biggest transient increase to the calculated epsilon-rhodomycinone flux.