Behavioral effects of bovine lactoferrin administration during postnatal development of rats

We tested the hypothesis that rats consuming bovine lactoferrin (bLf) during postnatal development would show better performance of stressful tasks during adolescence. In the first study, we orally administered bLf (750 mg/kg) once daily between postnatal days 16–34. Rats then underwent a battery of behavioral tests: open field (forced exploration of risky environment), light–dark emergence (voluntary exploration of risky environment), baited holeboard (working and reference memory), food neophobia (preference for familiar versus novel food), forced swim (test for antidepressant efficacy), and shuttle-box escape (learning to escape footshock). bLf-supplemented rats showed less exploration of the risky environment, greater preference for the familiar food odor, and faster escape responses. The effect of bLf on forced-swim behavior depended on sex: immobility increased for males and decreased for females. In the next study, we replaced the forced-swim test with an escape-swim test in which rats learned to use a visual cue to locate an escape platform, and we tested the dose response of bLf on this and the shuttle-box escape test, with subjects receiving vehicle or bLf at 500, 1,000, or 2,000 mg/kg. Under this modified testing battery, improvement of escape from footshock was not observed at any dose. However, males, but not females, showed a significant dose-dependent effect of bLf on acquisition of the water-escape task. On average, males receiving a higher dose mastered the task 20–25 % sooner than rats receiving a lower dose or vehicle. These results offer preliminary evidence that bLf supplementation during development can improve subsequent cognitive performance during stress.     

Production of sophorolipid biosurfactant by Pichia anomala

Biosurfactant production by Pichia anomala PY1, a thermotorelant strain isolated from fermented food, was examined as grown in media containing various carbon and nitrogen sources. The optimal conditions for biosurfactant production included 4% soybean oil as carbon source at pH 5.5 at 30 degrees C for 7 d. Under these conditions, the surface tension of the medium decreased to 28 mN/m with oil displacement measured at 69.43 cm(2). Comparative studies of biosurfactant production in media containing glucose or soybean oil were performed. The biosurfactants obtained were isolated and purified by chromatographic methods. The molecular weights of samples were further investigated by mass spectrometry. In medium containing glucose, biosurfactants of molecular weights of 675, 691, and 707 were obtained, while those isolated from medium containing soybean oil were of molecular weights of 658, 675, and 691. These results reveal that sophorolipid compounds containing fatty acids of C20 and C18:1 were produced from both media.     

Development of poly(ethylene glycol) conjugated lactoferrin for oral administration

Lactoferrin (LF) is an iron-binding glycoprotein that possesses multifunctional biological activities. Recent reports from clinical trials suggest that LF is potentially effective as a therapeutic protein against cancer and gangrene. However, pharmaceutical proteins such as LF are unstable in vivo. Therefore, to improve stability, we developed mono-PEGylated bovine LF (20k-PEG-bLf) with branched 20 kDa (2 x 10 kDa) poly(ethylene glycol) (PEG). We examined in vitro activities such as iron binding, IL-6 cell based assay, and resistance to a proteolytic enzyme in artificial gastric fluid. The 20k-PEG-bLf protein was fully active in iron binding and exhibited 69.6 +/- 2.9% (mean +/- S.E., n = 6) of the original anti-inflammatory activity. The proteolytic half-life increased 2-fold over that of unmodified LF. In vivo pharmacokinetic analyses were performed to examine absorption from the intestinal epithelium and serum clearance. Direct administration of 20k-PEG-bLf (30 mg/kg) into rat stomachs demonstrated that the amount of absorption from the intestinal tract increased approximately 10-fold relative to unmodified LF. Intravenous injection of the protein (1 mg/kg) revealed that 20k-PEG-bLf prolongs serum half-life by approximately 5.4-fold, and that the area under the curve (AUC) was increased approximately 9.2-fold compared to that of unmodified LF. PEGylation improved the physical and pharmacokinetic properties of bovine LF. This is the first report on the use of bioconjugation of LF for the development of a promising oral pharmaceutical agent.     

Interaction of lactoferrin with ceruloplasmin

When added to human blood serum, the iron-binding protein lactoferrin (LF) purified from breast milk interacts with ceruloplasmin (CP), a copper-containing oxidase. Selective binding of LF to CP is evidenced by the results of polyacrylamide gel electrophoresis, immunodiffusion, gel filtration, and affinity chromatography. The molar stoichiometry of CP:LF in the complex is 1:2. Near-uv circular dichroism spectra of the complex showed that neither of the two proteins undergoes major structural perturbations when interacting with its counterpart. K(d) for the CP/LF complex was estimated from Scatchard plot as 1.8 x 10(-6) M. The CP/LF complex is found in various fluids of the human body. Upon injection into rat of human LF, the latter is soon revealed within the CP/LF complex of the blood plasma, from where the human protein is substantially cleared within 5 h.     

Integrated sophorolipid production and gravity separation

A novel method for the integrated gravity separation of sophorolipid from a fermentation broth has been developed, enabling removal of a sophorolipid phase of either higher or lower density than the bulk fermentation broth, while cells and other media components are recirculated and returned to the bioreactor. The capability of the separation system to recover an enriched sophorolipid product phase was demonstrated on three sophorolipid producing fed batch fermentations using Candida bombicola, giving an 11% reduction in fermenter volume required whilst maintaining sophorolipid production. Sophorolipid recoveries of up to 86% (280 g) of the total produced over a whole fermentation were achieved at an enrichment of up to 9. Furthermore, the broth viscosity reduction achieved by removal of the sophorolipid phase enabled a 34% reduction in mixing power to maintain the same dissolved oxygen level by the end of the fermentation, with a 9% average reduction over the course of the fermentation. Fermentation duration could be extended to 1023 h, allowing production of 623 g sophorolipid from 1 l initial batch volume. These benefits could lead to a substantial decrease in the cost of sophorolipid production, making high volume applications such as enhanced oil recovery economically feasible.     

Interaction of calmodulin with lactoferrin.

Calmodulin, as a major intracellular calcium-binding protein, regulates many Ca(2+)-dependent enzymes and plays an important role in a wide spectrum of cellular functions of the eukaryotes. Interaction between calmodulin and human lactoferrin, a 78 kDa protein with antibacterial properties, was found in the presence of Ca2+ using (i) a method for the detection of calmodulin binding proteins with biotinylated calmodulin, (ii) affinity chromatography on an agarose-calmodulin column with subsequent detection by an enzyme-linked immunosorbent assay (ELISA). The binding of calmodulin to lactoferrin blocked the ability of lactoferrin to agglutinate Micrococcus lysodeikticus.     

Oral administration of lactoferrin increases hemoglobin and total serum iron in pregnant women.

Iron deficiency anemia (IDA) during pregnancy continues to be of world-wide concern. IDA is a risk factor for preterm delivery and subsequent low birth weight, and possibly for poor neonatal health. Iron supplementation in pregnancy is a widely recommended practice, yet intervention programs have met with many controversies. In our study, 300 women at different trimesters of pregnancy were enrolled in a trial of oral administration of ferrous sulfate (520 mg once a day) or 30% iron-saturated bovine lactoferrin (bLf) (100 mg twice a day). Pregnant women refusing treatment represented the control group. In this group hemoglobin and total serum iron values measured after 30 d without treatment decreased significantly, especially in women at 18-31 weeks of pregnancy. In contrast, after 30 d of oral administration of bLf, hemoglobin and total serum iron values increased and to a greater extent than those observed in women treated orally for 30 d with ferrous sulfate, independently of the trimester of pregnancy. Unlike ferrous sulfate, bLf did not result in any side effects. These findings lead us to hypothesize that lactoferrin could influence iron homeostasis directly or through other proteins involved in iron transport out of the intestinal cells into the blood.     

Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture

To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process.     

Enzymatic synthesis of a galactopyranose sophorolipid fatty acid-ester**

Sophorolipid lactones produced by Candida bombicola were deacetylated and ring opened with sodium methoxide to their corresponding methyl esters. The methyl esters, after re-acetylation with vinyl acetate using an immobilized lipase, were transesterified with 1,2-3,4-di-O-isopropylidene-d-galactopyranose in tetrahydrofuran using the same lipase catalyst. The di-O-isopropylidene sophorolipid sugar esters were hydrolyzed to give the galactopyranose sophorolipid esters as the final products.     

Plasma lactoferrin in patients with neutropenia

This study examines the role of plasma lactoferrin in the assessment of neutropenia. In particular, we have studied lactoferrin as an inhibitor of granulopoiesis and as an indicator of the size of the total blood granulocyte pool (TBGP). Plasma lactoferrin concentration was determined in a heterogeneous group of 30 patients with neutropenia. Serial plasma lactoferrin levels in a patient with cyclic neutropenia correlated with the cycles of the neutrophil count. Patients with splenomegaly had a grossly elevated lactoferrin:neutrophil ratio. Most chronic idiopathic neutropenia patients had no real clinical problems and a normal plasma lactoferrin level. The results provide further evidence to support the concept that plasma lactoferrin indicates the size of the TBGP and the lactoferrin: neutrophil ratio indicates the degree of granulocyte margination. There was no evidence to suggest that lactoferrin acting as a feedback inhibitor of granulopoiesis caused neutropenia in these patients.     

Enzymatic conversion of a sophorolipid into a glucose lipid

The diacetylated lactonic sophorolipid from Candida bombolica was converted into the deacetylated acidic form by alkaline hydrolysis and subsequently treated with several glycosidases. One of these enzymes, a hesperidinase (E.C. 3.2.1.40), was most active in the specific release of one glucose molecule from the disaccharide lipid. The novel glucolipid was isolated and characterized. The surface and interfacial tension of aqueous solutions were measured and compared with the lactonic and acidic sophorolipid.     

Limited cleavage of human lactoferrin with pepsin

• 1.1. Human lactoferrin in the iron-saturated form was treated with pepsin at pH 3.0, and a fragment with a single iron-binding site was isolated in a 90% pure state.
• 2.2. Its mol. wt was near 33,000.
• 3.3. Its N-terminal residue was alanine, and its iron-binding properties were identical to those of lactoferrin.
• 4.4. The curve showing iron incorporation from lactoferrin and its fragment into reticulocytes showed a biphasic character.
• 5.5. It is concluded that lactoferrin consists of two largely independent parts, which may have arisen by a gene duplication process.

     

Microbial degradation of phenanthrene by addition of a sophorolipid mixture

The influence of sophorolipids on microbial degradation of poorly soluble phenanthrene in liquid and soil suspension culture was evaluated in the work presented. Experiments were carried out in two parts. In the first part, important basic physico-chemical characteristics of the biosurfactant and the pollutant used were determined. The critical micelle concentration (CMC) and the solubilization ratio of the biosurfactant were found to be in a good range compared with synthetic surfactants. Also, a reduction to 71% of the detectable amount of phenanthrene was measured within 4 d in soil suspension without any biotic influence. In the second part, culture experiments were done with Sphingomonas yanoikuyae, the bacterium used throughout the work presented here with the aim to assess the toxicity of the sophorolipids on these bacteria and the effect of the surfactant on biodegradation. In exponential growth tests, no toxicity up to 1 g l(-1) sophorolipids could be detected, whereas in an agar plate test, slight growth hindrance was measured at a lower concentration of 250 mg l(-1). The above mentioned data were important for planning further experiments. In the following cultivations with liquid and soil suspension media, enhancements of the biodegradation with surfactant addition were measurable. Fluorescence measurements showed that this effect was not due to an increasing biomass, but to an augmentation of bioavailability of the phenanthrene through increasing the apparent dissolved pollutant. Surfactant addition had the consequence of decreasing the residual detectable pollutant concentration (after 36 h 0.5 compared with 2.3 mg l(-1) soil suspension) and increasing the maximal degradation rate (127 instead of 80 mg l(-1) soil suspension x 10 h). Therefore, the two main problems of biological soil remediation techniques, longer process time and residual pollutants, may be solved by the use of surfactants.     

Interaction of lactoferrin and lysozyme with casein micelles

On addition of lactoferrin (LF) to skim milk, the turbidity decreases. The basic protein binds to the caseins in the casein micelles, which is then followed by a (partial) disintegration of the casein micelles. The amount of LF initially binding to casein micelles follows a Langmuir adsorption isotherm. The kinetics of the binding of LF could be described by first-order kinetics and similarly the disintegration kinetics. The disintegration was, however, about 10 times slower than the initial adsorption, which allowed investigating both phenomena. Kinetic data were also obtained from turbidity measurements, and all data could be described with one equation. The disintegration of the casein micelles was further characterized by an activation energy of 52 kJ/mol. The initial increase in hydrodynamic size of the casein micelles could be accounted for by assuming that it would go as the cube root of the mass using the adsorption and disintegration kinetics as determined from gel electrophoresis. The results show that LF binds to casein micelles and that subsequently the casein micelles partly disintegrate. All micelles behave in a similar manner as average particle size decreases. Lysozyme also bound to the casein micelles, and this binding followed a Langmuir adsorption isotherm. However, lysozyme did not cause the disintegration of the casein micelles.     

Interaction of tear lipocalin with lysozyme and lactoferrin

The regeneration of the tyrosyl radical in chemically reduced native or p-butoxyphenol-treated radical free forms of mouse ribonucleotide reductase R2 protein has been studied. Chemical reduction has been achieved by treatment with light-activated flavin compounds: deazaflavin, flavin mononucleotide, or deazaflavin with methylviologen as mediator. The admission of air to the flavin reduced mouse R2 protein results in regeneration of up to 59% of the initial tyrosyl radical contents, whereas not more than 6% could be regenerated in the p-butoxyphenol-treated form. The mixed-valent EPR signal generated in the p-butoxyphenol-treated mouse R2 protein is different from the spectrum observed after flavin reduction in the native mouse R2 protein, indicating that treatment of the protein with p-butoxyphenol results in a structural rearrangement of the diferric/radical site. The presence of 0.1 mM Fe(II) in the anaerobic protein/buffer solution significantly improves the regeneration of tyrosyl radical upon admission of air to the flavin reduced mouse R2 protein, but less to the protein treated with p-butoxyphenol.     

Activation of rainbow trout, Oncorhynchus mykiss, phagocytic cells by administration of bovine lactoferrin

The chemiluminescent response of kidney phagocytic cells was significantly increased by the oral administration of bovine lactoferrin to rainbow trout, Oncorhynchus mykiss. The phagocytic activity of kidney cells was also increased by administration of bovine lactoferrin to the fish. Activation of kidney cells was also observed in vitro with cells cultured in the presence of bovine lactoferrin.     

Bactericidal effect of lactoferrin and lactoferrin chimera against halophilic Vibrio parahaemolyticus

Infections caused by Vibrio parahaemolyticus, an halophilic member of the genus Vibrio, have increased globally in the last 5 years. Diarrhea caused by V. parahaemolyticus results from eating raw or undercooked seafood. The aim of this work was to investigate whether lactoferrin and some lactoferrin-peptides have bactericidal activity against Vibrio parahaemolyticus ATCC 17802, the pandemic strain O3:K6, and the multidrug resistant isolate 727, as well as against Vibrio cholerae strains O1 and non-O1. Whereas both peptides lactoferricin (17-30) and lactoferrampin (265-284) did not have bactericidal activity, 40 microM of lactoferrin chimera (a fusion of the two peptides) inhibited the growth of all Vibrio tested to the same extent as the antibiotic gentamicin. The cidal effect of LFchimera showed a clear concentration response in contrast to bovine lactoferrin which showed higher inhibition at 10 microM than at 40 microM. FITC-labeled LFchimera bound to the bacterial membranes. Moreover LFchimera permeabilized bacterial cells and membranes were seriously damaged. Finally, in experiments with the multidrug resistant isolate 727, sub-lethal doses of LFchimera strongly reduced the concentrations of ampicillin, gentamicin or kanamicin needed to reach more than 95% growth inhibition, suggesting synergistic effects. These data indicate that LFchimera is a potential candidate to combat the multidrug resistant pathogenic Vibrio species.     

Antimicrobial properties of lactoferrin

Milk is a vital nutritional source for the offspring of all mammals, including humans. In addition to its nutritional value, it is a rich source of proteins including lactoferrin. Lactoferrin is a truly multifunctional protein that has been studied extensively over the past decades. It is best known for its ability to bind iron, which eventually led to the discovery of its antibacterial activity. In addition, lactoferrin has demonstrated potent antiviral, antifungal and antiparasitic activity, towards a broad spectrum of species. It is also considered to be an important host defense molecule during infant development. In this review, we focus on the antimicrobial activities of lactoferrin with particular emphasis on antibacterial and antiviral activities, although its antifungal and -parasitic activity are also discussed.     

Utilization of restaurant waste oil as a precursor for sophorolipid production

Approximately 100 billion liters of oil is generated per week as waste from restaurants around the country. Because of health, environmental, and economic factors, current methods of disposal are ineffective for disposal of the restaurant oil wastes. In this study we have investigated the ability of Candida bombicola to fermentatively transform the restaurant oil waste into glycolipids called sophorolipids. Batch and fed-batch studies were carried out using oil waste as the lipid feedstock in Erlenmeyer flasks and in a fermentor. Batch fermentation in a fermentor gave the highest yield of sophorolipids of 34 g L-1. Fermentation using oleic acid as control feedstock were also carried out. Batch fermentation in the fermentor using this pure fatty acid gave a highest yield of 42 g L-1. The difference in the sophorolipid yield was attributed to the fatty acid composition of restaurant oil waste.