Development of a time-temperature integrator system using Burkholderia cepacia lipase

A time-temperature integrator (TTI) is a device used to show a time-temperature dependent change that reflects the temperature history and quality status of the food to which it is attached. An enzymatic TTI system based on the reaction between Burkholderia cepacia lipase and tricaprylin, which causes a pH change, was developed. The temperature dependence of the response rate of this new lipase-type TTI was modeled using the Arrhenius equation, and the estimated activation energy was calculated as 70.61±11.10 kJ/mol (±95% confidence interval). The TTI response was validated under dynamic storage conditions with independent variable temperature experiments, and a good agreement was obtained between the predicted and measured values.     

Enhancing activity and stability of Burkholderia cepacia lipase by immobilization on surface-functionalized mesoporous silicates

Burkholderia cepacia lipase was immobilized on various types of phenyl-functionalized mesoporous silicates (MPS). MPS, anchored with a phenyl group on the silica wall and with three dimensional (3D) mesoporosity, showed highest lipase adsorption capacity and best activities both in aqueous and organic reagents.     

Construction of a Rhizopus delemar genomic library and screening for direct lipase gene expression

Recombinant DNA methods were used to begin a molecular biological study of the biotechnically important lipase produced by the fungus Rhlzopus delemar. Pure, high molecular weight DNA was isolated, mildly digested with Mbo I, ligated into pBR322, and introduced into E. coli by transformation. Transformants were recovered by ampicillin selection. The frequency of insertional inactivation of tetracycline resistance in the transformants was 95%. The cloned DNA inserts ranged in size from approximately 0 to 14 kilobases (average: 4.7). Colony hybridization using fungal genomic DNA as a probe verified the presence of fungal sequences in the transformed cells. A rapid, sensitive assay for lipase production was developed and applied to a sufficient number of transformants to represent several genomic equivalents of fungal DNA. No lipase‐producing clones were detected.     

Generation of monoclonal antibodies using simplified single-cell reverse transcription-polymerase chain reaction and cell-free protein synthesis

The single-step PCR amplification of IgG Light chain (Lc) and Heavy chain (Hc) (Fd portion) from the cDNAs of a single cell was facilitated using a low concentration of cDNA-specific primers with 5' homotags in the presence of a homotag-specific primer. This method was found to be successful in generating a functional antibody with an antigen-binding activity and useful for the high-throughput generation or screening of monoclonal antibodies.     

Single-step single-molecule PCR of DNA with a homo-priming sequence using a single primer and hot-startable DNA polymerase

We have previously reported that a protein library can be constructed by directly combining PCR amplification of a single DNA molecule and cell-free protein synthesis. To specifically amplify single DNA molecules, however, two-step PCR with nested primers was used. Here we describe a simpler method for single-step amplification of a single molecule. The method involves the use of both hot-startable DNA polymerase and a DNA template that has homo-priming sequences at both ends for amplification using a single primer. These two modifications greatly decreased the possibility of formation and subsequent accumulation, respectively, of primer-dimers that inhibit the amplification of target template. In addition, a high-fidelity DNA polymerase was successfully used, resulting in the significant reduction of the accumulation of mutations during amplification.     

A simple regression method for determining the quantity of translated protein in a cell-free protein synthesis system

A simple regression method for quantifying endogenous leucine in S30 extract was developed. This method enabled the quantity of translated protein in a cell-free protein synthesis system to be exactly determined.     

Stabilization effect of zeolite on DHFR mRNA in a wheat germ cell-free protein synthesis system

The effects of zeolites and monocations on the protein synthesis in a cell-free system derived from wheat germ were investigated. M type of synthetic zeolite markedly enhanced the translation efficiency. Whereas this kind of stimulatory effect of zeolite in an Escherichia coli cell-free system resulted from a change in the salt compositions of the reaction solution with the addition of zeolite, the enhancement of protein synthesis in a wheat germ cell-free system was not due to the ion exchange reaction of zeolites. From the results of mRNA stability analysis, it was found that zeolite could stabilize the mRNA in a wheat germ cell-free protein synthesis system. The stabilization of mRNA by the simple addition of zeolites is useful for the enhancement of protein synthesis in a wheat germ cell-free system, since conventional methods to improve mRNA stability, such as the addition of nuclease inhibitor, have not been effective for a wheat germ cell-free system.     

In vitro digestibility of protein and amino acids in protein mixtures

Six protein sources, casein, field peas, peanut meal, wheat flour, rapeseed and soya bean concentrate and their blends (ratio 1:1) were subjected to in vitro enzymic digestion. Wheat flour had the lowest in vitro digestibility (30% in 6 h) while the other sources had similar digestibilities (40%). Basic and aromatic amino acids were the most readily liberated from these protein sources. Some protein combinations, such as a rapeseed/field pea blend, gave in vitro digestibilities higher than calculated from individual proteins. The type of response observed could not be predicted from either nitrogen digestibility or amino acid composition of the individual sources. The digestibility of some amino acids was modified and this could be due to varying affinity of digestive enzymes for the protein.     

In vitro effects of steroidal saponins from Yucca schidigera extract on rumen microbial protein synthesis and ruminal fermentation

In a first experiment, ground alfalfa hay and rolled barley grain were incubated in buffered ruminal fluid with and without Yucca schidigera extract (YE, 0 or 10 mg ml⁻¹). Gas and total VFA production from barley grain were increased (P < 0.05) by YE during the first 10 h of incubation; from alfalfa hay, these were reduced (P < 0.001) throughout the 24 h. Yucca extract reduced (P < 0.001) acetate/propionate ratios and ammonia concentrations, irrespective of substrate. In a second experiment, ground barley grain was incubated in a buffered suspension of mixed ruminal microbes obtained by low‐speed centrifugation of ruminal fluid. Steroidal saponins (SAP) isolated from YE were included at 0, 15, 75 or 225 µg ml⁻¹. Microbial incorporation of ¹⁵N was increased (P < 0.05) by 15 µg SAP ml⁻¹ but decreased (P < 0.05) by 225 µg SAP ml⁻¹. Gas and VFA production peaked with 75 µg SAP ml⁻¹ and were elevated (P < 0.05) relative to control in the presence of 75 µg SAP ml⁻¹. Microbial protein synthesis was increased (P < 0.05) by SAP at 15 µg ml⁻¹ and reduced (P < 0.05) by the higher concentrations. Acetate/propionate ratios were linearly reduced by SAP from 8 to 24 h incubation (P < 0.01). The effects of SAP on digestive microbes were less pronounced (P < 0.05) with barley grain digestion than with alfalfa. A YE × diet interaction was recorded. The biological activity of YE was shown to be attributable to its SAP and to be diet‐dependent. © 2000 Society of Chemical Industry. Contributions of Y Wang, T A McAllister, L J Yanke and Z Xu © Minister of Public Works and Government Services Canada 2000     

In vitro screening of probiotics and synbiotics according to anti-inflammatory and anti-proliferative effects

There is emerging evidence of the efficiency of probiotic, prebiotic and synbiotic treatments in inflammatory bowel diseases (IBDs) and one of their long-term complications, colorectal cancer (CRC). In this study, various strains of probiotic lactic acid bacteria, prebiotic glucooligosaccharides (GOS) or a synbiotic combination of the two were screened for anti-inflammatory and anti-proliferative effects in different in vitro models in the context of such diseases. To mimic IBD response to Gram negative bacteria, HT-29 cells were sensitised to inflammatory response to lipopolysaccharide (LPS) by IFNγ which increased expression of TLR4, the LPS biosensor, and were then treated by probiotics, prebiotics and synbiotics. Secreted IL-8 and activated NF-κB were monitored as inflammation biomarkers. A selection of active strains were then subjected to a second inflammatory cell culture model consisting of inflammatory activated transgenic Caco-2 cells transfected by a reporter gene under the control of NF-κB inducible promoter. Quantification of reporter gene expression allowed us to demonstrate some probiotic inhibitory properties or to confirm such characteristics in two different models. Proliferation of cancerous HT-29 cells was monitored by XTT assay. Only three probiotic strains induced a proliferation decrease, but with a lack of reproducibility. Binary or ternary probiotic associations, complemented or not by prebiotic GOS, significantly decreased proliferation, especially with a synbiotic association of Bifidobacterium breve, Lactococcus lactis and oligoalternan, a GOS. This combination was selected for the following experiments. We showed the involvement of both bacterial and carbohydrate compounds of this synbiotic in the observed effect by dose range tests. We demonstrated that this decrease in proliferation may be due to an induction of a differentiated phenotype, as shown by the up-regulation of intestinal alkaline phosphatase, a biomarker of differentiation, monitored by real-time RT-PCR in HT-29 cells treated by the selected synbiotics. Thus, this study demonstrates the ability of probiotics to exert anti-inflammatory effects and shows some anti-proliferative characteristics for a specific synbiotics. These products should be further evaluated in animal models to confirm the in vitro results.     

Stabilization of affinity-tagged recombinant protein during/after its production in a cell-free system using wheat-germ extract

We found that the affinity tag fused to the carboxyl (C-) terminal of a single-chain Fv (scFv) antibody was proteolytically degraded in a wheat germ cell-free protein synthesis system. The addition of two extra residues of glycine to the tail of the cMyc tag significantly increased the stability of the tag, suggesting that wheat endogenous carboxypeptidase(s) play a primary role in the C-terminal tag-specific degradation. In addition to the modification of the tag sequence, addition of diisopropyl fluorophosphate, which is known as an inhibitor of carboxypeptidases, prevented the cMyc tag sequence degradation. The effects of other protease inhibitors on the translation reaction and stability of the synthesized protein are also reported.     

In vitro digestibility of protein from barley and other cereals.

An in vitro method for measuring barley protein digestibility is presented. Samples were first incubated with pepsin in HCl; pancreatin was then added concomitantly with a bacteriostatic borate buffer. After TCA-precipitation, soluble nitrogen was measured. The digestion was unaffected by accumulated free amino acids. There were no free amino acids following pepsin treatment, but the essential ones were well liberated by pancreatin. Results for barley grown in the field or in pots, and for decortified barley fractions agreed with true digestibility values determined with rats. Of these samples, the field-grown barleys per se differed too little for the accuracy to be confirmed. The other cereals tested, oats, rye, maize, wheat, and rice, gave unsatisfactory results with pepsin/pancreatin, and also with pepsin, pancreatin, or pronase used separately. The ranking of the cereals according to in vitro digestibility depended on the type of enzyme and on the enzyme-to-substrate ratio.     

In vitro determination of digestible and unavailable protein in edible seaweeds

Edible seaweeds are considered a complementary source of food protein for human and animal nutrition. The physiological effects of seaweed protein depend on the degree of enzymatic digestion of protein in the small intestine and bacterial fermentation in the large intestine. The objective of this work was to estimate total, digestible, fermentable and unavailable protein in some red and brown seaweeds. Brown seaweeds Fucus vesiculosus, Laminaria digitata and Undaria pinnatifida and red seaweeds Chondrus crispus and Porphyra tenera were treated with pepsin and pancreatin to separate digestible protein. Residues containing indigestible protein were inoculated for 24 h with rat caecal droppings, and protein contents were evaluated in the non‐fermented residue. Protein content in the seaweeds ranged from 8.9 to 25% of dry matter. Digestible protein was the major protein fraction (69%) only in P tenera; in the other seaweeds, this fraction ranged from 15 to 45%. Significant amounts of unavailable protein were found in all samples (2–24%). The distribution of total protein among the three fractions, ie digestible, fermentable and unavailable protein, could yield information about the physiological and metabolic consequences of the intake of seaweed proteins. © 2002 Society of Chemical Industry     

Enzymatic synthesis of structured lipids using a novel cold-active lipase from Pichia lynferdii NRRL Y-7723

Structured lipids (SL) were synthesized by the acidolysis of borage oil with caprylic acid using lipases. Six commercial lipases from different sources and a novel lipase from Pichia lynferdii NRRL Y-7723 were screened for their acidolysis activities and Lipozyme RM IM and NRRL Y-7723 lipase were selected to synthesize symmetrical SL since recently NRRL Y-7723 lipase was identified as a novel cold-active lipase. Both lipases showed 1,3-regiospecifity toward the glycerol backbone of borage oil. The effects of enzyme loading and temperature on caprylic acid incorporation into the borage oil were investigated. For Lipozyme RM IM and NRRL Y-7723 lipase, the incorporation of caprylic acid increased as enzyme loading increased up to 4% of total weight of the substrate, but significant increases were not observed when enzyme loading was further increased. The activity of NRRL Y-7723 lipase was higher than that of Lipozyme RM IM in the temperature range between 10 and 20 °C.     

Construction of a combinatorial protein library displayed on yeast cell surface using DNA random priming method

The random DNA fragments, generated from mRNAs of Saccharomyces cerevisiae using a novel "DNA random priming method", were inserted into a surface display vector to construct a combinatorial random protein library. Its surface display was confirmed by immunofluorescence labeling of the RGS (His)6 reporter inserted, and its potential was also evaluated.     

In vitro cytomodulatory and immunomodulatory effects of bovine colostrum whey protein hydrolysates

Bovine colostrum possesses biological compounds involved in the development of the newborn. Among them, the proteins have drawn attention as a source of bioactive peptides. This study shows the in vitro cytotoxic and immunostimulatory potential of two colostrum whey protein (CWP) hydrolysates obtained by in vitro digestion with pepsin and pancreatin. MTT cell viability, apoptosis induction, polymorphonuclear proliferation and phagocytic activity assays were performed. Treatment with the hydrolysates induced a significant decrease in the viability of MDA-MB-231 cell lines due to apoptosis and also a significant increase in the proliferation of blood mononuclear cells. It could also be observed that for the RM-1 and PC-3 prostate cancer cell lines and for the two times of exposure (24 and 48 h), the hydrolysate H1 is significantly more cytotoxic than CWP. These results showed the potential of bovine CWP and its hydrolysates for the treatment of chronic diseases such as cancer.     

In vitro digestibility of original and modified protein isolate from faba beans

Protein isolate from Vicia faba (API) and 18 different partial hydrolysates of API as well as plasteins produced from them were submitted to an in vitro digestion with pepsin and pancreatic proteases at their specific pH-optimum. By means of this technique a final value of hydrolysis (49,8 +/- 3,0)% was obtained with all investigated samples, in spite of the different intensity of the preliminary hydrolysis. Two samples with a degree of preliminary hydrolysis of 35% showed with 55% and 56%, respectively, a significantly higher final value. From the results may be assumed that by means of partial hydrolysis and plastein formation only such peptides will be formed which are again hydrolyzable in vitro.