Affinity enrichment of bovine lactoferrin in whey

Bovine lactoferrin was enriched in various whey samples by affinity chromatography using immobilized gangliosides. Bovine gangliosides were isolated from fresh buttermilk using a combination of ultrafiltration and organic extraction. Isolated gangliosides were covalently immobilized onto controlled-pore glass beads. The immobilized matrix contained 66 micrograms of gangliosides per gram of beads. After loading the matrix with reconstituted whey protein isolate (WPI) or whey protein concentrate (WPC), the matrix was washed with sodium phosphate buffer (pH 7) followed by sodium acetate buffer (pH 4) before elution of lactoferrin with 1 M NaCl in sodium acetate buffer. From the intensities of the protein bands in SDS-PAGE, lactoferrin constituted a minimum of 40% of the total protein in the salt eluted sample. WPI, pretrated by heating and ultrafiltration, showed the highest lactoferrin purity among protein sources, while WPI (10% wt/vol) showed the highest recovery. These results show that immobilized gangliosides can be used to enrich the lactoferrin content of whey.     

Simple purification of immunoglobulins from whey proteins concentrate

We have developed a new protocol with only two steps for purification of immunoglobulins (Ig) from a protein concentrate of whey. Following this protocol, we have an 80% recovery of immunoglobulins, fairly pure. The purification was achieved by eliminating the BSA, via a strong adsorption on DEAE-agarose. Full desoprtion of the other serum proteins could be achieved without contamination with BSA. Thus, a protein solution containing only Ig and very small proteins (e.g., beta-lactoglobulins and alpha-lactalbumin) was obtained. Offering this protein mixture to a lowly activated aminated support, only Ig adsorbed on the support. It has been shown that BSA is able to interact with other proteins (including Ig and lactalbumins). This ability to form complexes with other proteins prevented the success of the direct adsorption of Ig on this mildly activated support, even although Ig should be the largest protein presented in dairy whey.     

Single step purification of lactoperoxidase from whey involving reverse micelles‐assisted extraction and its comparison with reverse micellar extraction

The extraction of lactoperoxidase (EC 1.11.1.7) from whey was studied using single step reverse micelles‐assisted extraction and compared with reverse micellar extraction. The reverse micelles‐assisted extraction resulted in extraction of contaminating proteins and recovery of lactoperoxidase in the aqueous phase leading to its purification. Reverse micellar extraction at the optimized condition after forward and backward steps resulted in activity recovery of lactoperoxidase and purification factor of the order of 86.60% and 3.25‐fold, respectively. Whereas reverse micelles‐assisted extraction resulted in higher activity recovery of lactoperoxidase (127.35%) and purification factor (3.39‐fold). The sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) profiles also evidenced that higher purification was obtained in reverse micelles‐assisted extraction as compared of reverse micellar extracted lactoperoxidase. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Protein purification from polyacrylamide gels by sonication extraction

A protein purification procedure using sonication extraction from polyacrylamide gels (PAGE), which involves identification of a particular protein band and its excision, homogenization, sonication, and subsequent passage through a Sephadex G-25 minicolumn, is reported. Our results show a high degree of recovery regardless of the nature of the protein (soluble or membrane bound) or the characteristics of the gel (SDS-PAGE, native gels, or Tricine-SDS-PAGE). The percentage of recovery was dependent on the protein concentration applied in the gel. This technique is fast, gives high yield, provides good resolution, and can be used without any specialized equipment. The method was tested with a wide variety of membranes and soluble proteins and was found to give good results and to be applicable to different studies. The amino acid sequence of one of the purified proteins (Rf 0.45 stallion ejaculated sperm protein) was determined successfully.     

Scaling-up of a batch whey fermentation by Kluyveromyces fragilis

Summary A whey fermentation by Kluyveromyces fragilis was scaled-up to a 1000-dm³ stirred fermentor, by varying the stirrer speed, the air-flow rate and the initial concentration of lactose. Its evolution was simulated by applying the same unstructured model (consisting of a microbial specific growth rate of pseudo-first order with respect to the COD concentration and constant biomass yield per unit COD removed) set up in previous experiments using 8- to 80-dm³ fermentors. Despite the great scale-up ratios, very different operating conditions, and geometric dissimilarity, a series of empirical regressions previously developed allowed approximate, but acceptable prediction of the stoichiometric and kinetic coefficients of the above mathematical model, thus confirming the capability of this model to provide a reliable basis for further scale-up of this fermentation process to a production scale.     

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.     

Production of butyric acid by batch fermentation of cheese whey withClostridium beijerinckii

Summary The effect of pH on the fermentation of butyric acid byClostridium beijerinckii using cheese whey as a substrate was studied. Maximum concentrations of the acid were produced when the pH was controlled at 5.5. Raising or lowering of pH was found to reduce the total acid formation. This particular strain ofC. beijerinckii produced insignificant amounts of butanol in all the pure culture cases investigated. A comparative study of the fermentation in a synthetic glucose medium and in cheese whey showed the whey to produce more butyric acid.     

Butanol from whey ultrafiltrate: batch experiments with Clostridium beyerinckii LMD 27.6

Summary For batch fermentations by Clostridium beyerinckii LMD 27.7 (formerly known as Clostridium butylicum) whey ultrafiltrate, glucose, lactose, and galactose were used as substrates. The aims of the experiments were to find the conditions for butanol production from whey ultrafiltrate and to compare the results with those of other substrates. The conditions necessary for butanol production were established. The mean solvent productivity found on whey ultrafiltrate fermentation was two to three times lower than that found on glucose; the overall solvent yields were comparable. Butanol production from galactose and mixtures of glucose and galactose was also possible.     

A nonchromatographic process for purification of secretory immunoglobulins from caprine whey

Secretory immunoglobulins are an important antibody class being primarily responsible for immunoprotection of mucosal surfaces. A simple, non‐chromatographic purification process for secretory immunoglobulins from caprine whey was developed. In the first process step whey was concentrated 30–40‐fold on a 500 kDa membrane, thereby increasing the purity from 3% to 15%. The second step consisted of a fractionated PEG precipitation, in which high molecular weight impurities were removed first and in the second stage the secretory immunoglobulins were precipitated, leaving a majority of the low molecular weight proteins in solution. The re‐dissolved secretory immunoglobulin fraction had a purity of 43% which could then be increased to 72% by diafiltration at a volume exchange factor of 10. Further increase of purity was only possible at the expense of very high buffer consumption. If diafiltration was performed directly after ultrafiltration, followed by precipitation, the yield was higher but purity was only 54%. Overall, filtration performance was characterized by high concentration polarization, therefore process conditions were set to low trans‐membrane pressure and moderate protein concentration. As such purity and to a lesser extent throughput were the major objectives rather than yield, since whey, as a by‐product of the dairy industry, is a cheap raw material of almost unlimited supply. Ultra‐/diafiltration performance was described well by correlations using dimensionless numbers. Compared with a theoretical model (Graetz/Leveque solution) the flux was slightly overestimated. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:642–653, 2017     

Thermodynamics of gallium complexation by human lactoferrin

Equilibrium constants for the successive binding of 2 equiv of Ga3+ to human lactoferrin have been measured by difference ultraviolet spectroscopy in 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing 5 mM bicarbonate at pH 7.4 and 25 degrees C. Ethylenediamine-N,N'-diacetic acid was used as the competing chelating agent. Values of the effective binding constants for the stated experimental conditions are log K1 = 21.43 +/- 0.18 and log K2 = 20.57 +/- 0.16. Comparison of these results with literature values for the gallium-transferrin binding constants indicates that lactoferrin binds gallium more strongly by a factor of approximately 90. The ratios of successive binding constants for the two proteins are essentially identical. A linear free energy relationship (LFER) for the complexation of gallium(III) and iron(III) has been prepared and used to estimate an iron(III)-lactoferrin binding constant for pH 7.4. The LFER prediction is compared with thermodynamic data on iron binding at pH 6.4 and gallium binding at pH 7.4. The results indicate that the ratio of iron binding constants for lactoferrin and transferrin is likely in the range of 50-90.     

Production of recombinant human lactoferrin from transgenic plants

Molecular farming provides a powerful tool for low cost production of recombinant proteins with pharmaceutical value. The use of transgenic plants has been increasingly tested as alternative system for obtaining biologically active human lactoferrin in plants. Precise selection of plant species, transformation techniques and expression cassettes, in addition to conduction of detailed glycosylation and immunogenicity studies, serves as basis of obtaining safe recombinant human lactoferrin in high concentrations for the use of pharmacy. On the other hand, expression of antimicrobial protein lactoferrin in plants is a promising opportunity for crop quality improvement by increasing plant disease resistance.     

Lactic acid production by batch fermentation of whey permeate: A mathematical model

Summary The batch fermentation of whey permeate to lactic acid was improved by supplementing the broth with enzyme-hydrolyzed whey protein. A mathematical model based on laboratory results predicts to a 99% confidence limit the kinetics of this fermentation. Cell growth, acid production and protein and sugar use rates are defined in quantifiable terms related to the state of cell metabolism. The model shows that the constants of the Leudeking-Piret model are not true constants, but must vary with the medium composition, and especially the peptide average molecular weight. The kinetic mechanism on which the model is based also is presented.Nomenclature K _i lactic acid inhibition constant (g/l) - K _pr protein saturation constant during cell growth (g/l) - K _pr protein saturation constant during maintenance (g/l) - K _s lactose saturation constant (g/l) - [LA] lactic acid concentration (g/l) - [PR] protein concentration (g/l) - [S] lactose concentration (g/l) - t time (h) - [X] cell mass concentration (g/l) - , fermentation constants of Leudeking and Piret - specific growth rate (l/h) - Y _{g, LA/S} acid yield during cell growth (g acid/g sugar) - Y _{m, LA/S} acid yield during maintenance (g acid/g sugar) - Y _x/pr yield (g cells/g protein) - specific sugar use rate during cell growth (g sugar/h·g cell) - specific sugar use rate during maintenance (g sugar/h·cell)     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Evaluation of methods for the extraction and purification of DNA from the human microbiome

Background

DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.

Methodology/Principal Findings

In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.

Conclusions/Significance

Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.     

The extraction and purification of a peptide from rat insulinoma tissue

A peptide was extracted and purified from rat insulinoma tissue which, although similar, was not identical to normal rat C peptides. The purity of the peptide, called rat insulinoma peptide (RIP), was investigated using polyacrylamide gel electrophoresis, isoelectric focusing and high-performance liquid chromatography. It appears to contain two peptides similar to each other but differing in their isoelectric points. The peptides as assessed by fast atom bombardment mass spectrometry have molecular masses in the region of 1982 Da, given a chain length of approx. 22 amino-acid residues. Evidence obtained using an established rat C peptides radioimmunoassay suggests that RIP shares a common C-terminus with rat C peptides. The antiserum produced to RIP was used to develop a radioimmunoassay using a tracer prepared by iodinating purified tyrosylated RIP.     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.