The impact of emulsion droplet size on in vitro lipolysis rate and in vivo plasma uptake kinetics of triglycerides and vitamin D3 in rats.

Emulsions play an important role in the process of triglyceride (TG) digestion (lipolysis). Through emulsification, the oil-water interface is increased by orders of magnitude. This often leads to faster and more efficient lipolysis, which is potentially beneficial for the intestinal uptake of oils and lipophilic compounds. In this paper, we first examined the effect of emulsion droplet size on the in vitro lipolysis rate. Then an in vivo experiment was performed, to examine the plasma uptake kinetics of TGs and vitamin D3 (vitD3) over a 24 hours period after oral administration of the emulsions in rats. Basic corn oil emulsions loaded with vitD3 were prepared using polysorbate 80 as the emulsifier, with three different droplet sizes (D[3,2]): ∼3 µm (large), ∼1 µm (medium) and ∼0.3 µm (small). In vitro lipolysis experiments showed, as expected, that smaller droplets were lipolyzed more rapidly. However, the medium emulsion had by far the highest rate of lipolysis per surface area. This was attributed to bile salt limitation, polysorbate 80 lipolysis inhibition and TG digestion product accumulation. In vivo, the two smallest emulsions showed the highest uptake (Cmax and AUC) of vitD3 and TG, while the largest emulsion and bulk oil control showed lower values. However, only the (incremental) TG plasma values and kinetics displayed some statistically significant differences. These findings may have relevance for the formulation of functional foods/beverages or delivery units containing oils or lipophilic bioactives.

Production of Value-Added Chemicals by Bacillus methanolicus Strains Cultivated on Mannitol and Extracts of Seaweed Saccharina latissima at 50°C.

The facultative methylotroph Bacillus methanolicus MGA3 has previously been genetically engineered to overproduce the amino acids L-lysine and L-glutamate and their derivatives cadaverine and γ-aminobutyric acid (GABA) from methanol at 50°C. We here explored the potential of utilizing the sugar alcohol mannitol and seaweed extract (SWE) containing mannitol, as alternative feedstocks for production of chemicals by fermentation using B. methanolicus. Extracts of the brown algae Saccharina latissima harvested in the Trondheim Fjord in Norway were prepared and found to contain 12-13 g/l of mannitol, with conductivities corresponding to a salt content of ∼2% NaCl. Initially, 12 B. methanolicus wild type strains were tested for tolerance to various SWE concentrations, and some strains including MGA3 could grow on 50% SWE medium. Non-methylotrophic and methylotrophic growth of B. methanolicus rely on differences in regulation of metabolic pathways, and we compared production titers of GABA and cadaverine under such growth conditions. Shake flask experiments showed that recombinant MGA3 strains could produce similar and higher titers of cadaverine during growth on 50% SWE and mannitol, compared to on methanol. GABA production levels under these conditions were however low compared to growth on methanol. We present the first fed-batch mannitol fermentation of B. methanolicus and production of 6.3 g/l cadaverine. Finally, we constructed a recombinant MGA3 strain synthesizing the C30 terpenoids 4,4'-diaponeurosporene and 4,4'-diapolycopene, experimentally confirming that B. methanolicus has a functional methylerythritol phosphate (MEP) pathway. Together, our results contribute to extending the range of both the feedstocks for growth and products that can be synthesized by B. methanolicus.

Macromolecular acidic coating increases shelf life by inhibition of bacterial growth.

The sensitivity of microorganisms to low pH can be utilized in food protection by preparing coatings based on macromolecular acids. Due to limited diffusivity of macromolecules low pH occurs primarily at the surface, while the interior parts of the food remain unaffected. This principle is demonstrated using food approved alginic acid in various types of coatings (aqueous, emulsions, dispersions, dry coating) on a wide range of foods including meat, fish, chicken, shrimp and boiled rice. Significant delay or inhibition of the natural flora is generally demonstrated, particularly when exposed to 'temperature abuse'. Specifically, we show that the coatings reduce or inhibit regrowth of pathogens (Bacillus cereus, B. weihenstephanensis, Listeria monocytogenes serotype 1 and Staphylococcus aureus). In special cases like boiled rice, alginic acid may largely replace acetic acid for acidification and preservation, as demonstrated studying regrowth of added spores of B. cereus. Most formulations allow easy removal prior to further processing (cooking, frying). Temporary side effects such as 'acid cooking' obtained for high acid concentrations on sensitive surfaces (e.g. salmon) disappear during processing, recovering the normal taste and texture. The coating is hence suitable for a large variety of foods.

Activation of chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712 by ethanol shock: insights from the promoter fusion studies.

BACKGROUND: Streptomyces venezuelae ATCC 10712 produces antibiotics chloramphenicol (Cml) and jadomycin (Jad) in response to nutrient limitation and ethanol shock (ES), respectively. Biosynthesis of Cml and Jad was shown to be reciprocally regulated via the action of regulatory proteins JadR1 and JadR2 encoded by the jad cluster, and mechanism of such regulation has been characterized. However, detailed analysis of the regulatory mechanism controlling Cml biosynthesis is still lacking. RESULTS: In the present study, several promoters from the cml cluster were fused to the reporter gene gusA. Reporter protein activity and Cml production were assayed in the wild-type strain with and without ES, followed by similar experiments with the jadR1 deletion mutant. The latter gene was earlier reported to negatively control Cml biosynthesis, while serving as a positive regulator for the jad cluster. A double deletion mutant deficient in both jadR1 and the cml cluster was also constructed and used in promoter fusion studies. Analyses of the results revealed that ES activates Cml biosynthesis in both wild-type and jadR1 deletion mutant, while Cml production by the latter was ca 80% lower. CONCLUSIONS: These results contradict earlier reports regarding the function of JadR1, but correlate well with the reporter activity data for some promoters, while reaction of others to the ES is genotype-dependent. Remarkably, the absence of Cml production in the double mutant has a profound effect on the way certain cml promoters react to ES. The latter suggests direct involvement of Cml in this complex regulatory mechanism.

Ibuprofen-in-cyclodextrin-in-W/O/W emulsion - Improving the initial and long-term encapsulation efficiency of a model active ingredient.

A challenge in formulating water-in-oil-in-water (W/O/W) emulsions is the uncontrolled release of the encapsulated compound prior to application. Pharmaceuticals and nutraceuticals usually have amphipathic nature, which may contribute to leakage of the active ingredient. In the present study, cyclodextrins (CyDs) were used to impart a change in the relative polarity and size of a model compound (ibuprofen) by the formation of inclusion complexes. Various inclusion complexes (2-hydroxypropyl (HP)-ß-CyD-, α-CyD- and γ-CyD-ibuprofen) were prepared and presented within W/O/W emulsions, and the initial and long-term encapsulation efficiency was investigated. HP-ß-CyD-ibuprofen provided the highest encapsulation of ibuprofen in comparison to a W/O/W emulsion with unassociated ibuprofen confined within the inner water phase, with a four-fold increase in the encapsulation efficiency. An improved, although lower, encapsulation efficiency was obtained for the inclusion complex γ-CyD-ibuprofen in comparison to HP-ß-CyD-ibuprofen, whereas α-CyD-ibuprofen had a similar encapsulation efficiency to that of unassociated ibuprofen. The lower encapsulation efficiency of ibuprofen in combination with α-CyD and γ-CyD was attributed to a lower association constant for the γ-CyD-ibuprofen inclusion complex and the ability of α-CyD to form inclusion complexes with fatty acids. For the W/O/W emulsion prepared with HP-ß-CyD-ibuprofen, the highest encapsulation of ibuprofen was obtained at hyper- and iso-osmotic conditions and by using an excess molar ratio of CyD to ibuprofen. In the last part of the study, it was suggested that the chemical modification of the HP-ß-CyD molecule did not influence the encapsulation of ibuprofen, as a similar encapsulation efficiency was obtained for an inclusion complex prepared with mono-1-glucose-ß-CyD.

Assessment of capillary anion exchange ion chromatography tandem mass spectrometry for the quantitative profiling of the phosphometabolome and organic acids in biological extracts.

Metabolic profiling has become an important tool in biological research, and the chromatographic separation of metabolites coupled with mass spectrometric detection is the most frequently used approach for such studies. The establishment of robust chromatographic methods for comprehensive coverage of the anionic metabolite pool is especially challenging. In this study, the development of a capillary ion exchange chromatography (capIC) - negative ESI tandem mass spectrometry (MS/MS) workflow for the quantitative profiling of the phosphometabolome (e.g., sugar phosphates and nucleotides) is presented. The chromatographic separation and MS/MS conditions were optimized, and the precision of repetitive injections and accuracy in terms of error percentage to true concentration were assessed. The precision is excellent for a capillary flow system with an average CV% of 8.5% for a 50-fmol standard injection and in the lower 2.4-4.4% range for higher concentrations (500-7,500 fmol). The limit of detection (LOD) ranges from 1 to 100 nM (5-500 fmol injected on column), and the limit of quantitation (LOQ) ranges from 1 to 500 nM (5-2,500 fmol injected on column). A fast gradient method with the injection of 50% methanol in water between analytical samples is needed to eliminate carry-over and ensure optimal re-equilibration of the column. Finally, the quantitative applicability of the system was tested on real biological matrices using the constant-volume standard addition method (SAM). Extracts of the human kidney Hek293 cell line were spiked with increasing concentrations of standards to determine the concentration of each metabolite in the sample. Forty-four metabolites were detected with an average uncertainty of 4.1%. Thus, the capIC-MS/MS method exhibits excellent selectivity, sensitivity and precision for the quantitative profiling of the phosphometabolome.

Quantitative analysis of amino and organic acids by methyl chloroformate derivatization and GC-MS/MS analysis.

Alkyl chloroformates are known for their ability to produce mixed anhydrides, and they have found use as versatile derivatization reagents for gas chromatographic (GC) separation of amino- and organic acids. Triple-quadrupole mass spectrometers are excellent detectors for high sensitive and selective analysis. Here, we describe a methyl chloroformate (MCF) GC-MS/MS method for the quantitative analysis of metabolites containing amino- and/or carboxylic groups. The method covers over 60 metabolites with quantitation limits down to the low picomole range injected on column, and any metabolite with amino- and/or carboxylic acid functional groups that yield a stable and volatile MCF derivative can be included in the method. Absolute quantitation can be achieved by including a stable isotope-coded derivatization agent (d3-MCF) and deuterated alcohol solvent (e.g., d4-methanol). As the carboxylic and amino groups are differently labeled, the former from the solvent methanol while the latter from MCF, this approach can also be used to identify a number of amino and carboxylic groups in unknown analytes in an extract.

Periodate oxidation of polysaccharides for modification of chemical and physical properties.

A limited degree (typically 1-20%) of periodate oxidation of polysaccharides may give rise to derivatives with entirely altered chemical and physical properties. Notably, the ring opening caused by periodate leads to the formation of highly flexible 'hinges' in otherwise rather semiflexible or rigid structures. This review highlights selected examples with the main focus on periodate oxidation of alginates, chitosans, hyaluronan, scleroglucan/schizophyllan, and cellulose.

A study of the chain stiffness and extension of alginates, in vitro epimerized alginates, and periodate-oxidized alginates using size-exclusion chromatography combined with light scattering and viscosity detectors.

A series of alginates isolated from the stem and leaf of a brown algae (Laminaria hyperborea), bacterial mannuronan, in vitro epimerized mannuronans, and periodate oxidized alginates were analyzed by size-exclusion chromatography (SEC) combined with online multiangle laser light scattering (MALS) and viscometry (collectively abbreviated SMV). Selected samples were also analyzed off-line using low-angle laser light scattering and capillary viscometry. Excellent agreement between the two methods was obtained for properly purified samples. In contrast, abnormal results were obtained for some industrial samples due to the presence of particulate material. Naturally occurring alginates and in vitro epimerized mannuronans were found to obey essentially the same RG-M and [eta]-M relations, and hence, the same Mark-Houwink-Sakurada (MHS) equations (valid for I = 0.10 M): 20 000 g/mol < M < 100 000 g/mol, [eta] = 0.0054 .M(1.00); 100 000 g/mol < M < 1 000 000 g/mol, [eta] = 0.071 .M(0.89). Application of the wormlike chain model to the [eta]-M data obtained by SMV yielded persistence lengths (q) of 15 nm for all alginates at an ionic strength of 0.17 M. Intrinsic viscosities corresponding to infinite ionic strength were estimated on the basis of Smidsrød's B-parameter, and the wormlike chain model then yielded q = 12 nm. Periodate oxidized alginates showed, in contrast, a pronounced decrease in persistence length with increasing degree of oxidation, reaching values below 4 nm at 44% oxidation. Periodate oxidation also resulted in some depolymerization, even in the presence of a free-radical scavenger.