Bernard F. Gibbs

Encapsulation in the food industry: A review

Encapsulation involves the incorporation of food ingredients, enzymes, cells or other materials in small capsules. Applications for this technique have increased in the food industry since the encapsulated materials can be protected from moisture, heat or other extreme conditions, thus enhancing their stability and maintaining viability. Encapsulation in foods is also utilized to mask odours or tastes. Various techniques are employed to form the capsules, including spray drying, spray chilling or spray cooling, extrusion coating, fluidized bed coating, liposome entrapment, coacervation, inclusion complexation, centrifugal extrusion and rotational suspension separation. Each of these techniques is discussed in this review. A wide variety of foods is encapsulated--flavouring agents, acids bases, artificial sweeteners, colourants, preservatives, leavening agents, antioxidants, agents with undesirable flavours, odours and nutrients, among others. The use of encapsulation for sweeteners such as aspartame and flavours in chewing gum is well known. Fats, starches, dextrins, alginates, protein and lipid materials can be employed as encapsulating materials. Various methods exist to release the ingredients from the capsules. Release can be site-specific, stage-specific or signalled by changes in pH, temperature, irradiation or osmotic shock. In the food industry, the most common method is by solvent-activated release. The addition of water to dry beverages or cake mixes is an example. Liposomes have been applied in cheese-making, and its use in the preparation of food emulsions such as spreads, margarine and mayonnaise is a developing area. Most recent developments include the encapsulation of foods in the areas of controlled release, carrier materials, preparation methods and sweetener immobilization. New markets are being developed and current research is underway to reduce the high production costs and lack of food-grade materials.

Regulation of Cytochrome P450 by Posttranslational Modification

Cytochrome P450s are a family of enzymes represented in all kingdoms with expression in many species. Over 3,000 enzymes have been identified in nature. Humans express 57 putatively functional enzymes with a variety of critical physiological roles. They are involved in the metabolic oxidation, peroxidation, and reduction of many endogenous and exogenous compounds including xenobiotics, steroids, bile acids, fatty acids, eicosanoids, environmental pollutants, and carcinogens [Nelson, D. R., Kamataki, T., Waxman, D. J.,17 Guengerich, F. P., Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J.,18 Gunsalus, I. C., Gotoh, O. (1993) The P450 superfamily: update on new sequences, gene 19 mapping, accession numbers, early trivial names of enzymes, and nomenclature. DNA Cell 20 Biol. 12(1):1- 51.]. The development of numerous diseases and disorders including cancer and cardiovascular and endocrine dysfunction has been linked to P450s. Several levels of regulation, including transcription, translation, and posttranslational modification, participate in maintaining the proper function of P450s. Modifications including phosphorylation, glycosylation, nitration, and ubiquitination have been described for P450s. Their physiological significance includes modulation of enzyme activity, targeting to specific cellular compartments, and tagging for proteasomal degradation. Knowledge of P450 posttranslational regulation is derived from studies with relatively few enzymes. In many cases, there is only enough evidence to suggest the occurrence and a possible role for the modification. Thus, many P450 enzymes have not been fully characterized. With the introduction of current proteomics tools, we are primed to answer many important questions regarding regulation of P450 in response to a posttranslational modification. This review considers regulation of P450 in a context that describes the potential role and physiological significance of each modification.

Sweet and taste-modifying proteins : A review

The search for non-carbohydrate sweeteners from natural sources has led to the discovery of many intensely sweet-tasting substances. The occurrence of sweet-tasting proteins such as thaumatin, monellin, mabinlin and pentadin in the pulp of fruits of various rain forest species has provided a new approach to the potential treatment of diabetes, obesity and other metabolic disorders. These proteins can also be used as low calorie sweeteners to enhance and modify the taste of existing foods. Thaumatin is currently commercially available as a sweetener, flavor enhancer, additive to pharmaceuticals, chewing gum and animal feeds. It can also be secreted extracellularly by genetic modification of the yeast Saccharomyces cerevisiae. The taste-modifying proteins, miraculin and curculin, can be utilized in controlling the palatability of foods or in the development of new food products. This review discusses some properties of sweet and taste-modifying proteins discovered to date.

OmpT Outer Membrane Proteases of Enterohemorrhagic and Enteropathogenic Escherichia coli Contribute Differently to the Degradation of Human LL-37

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are food-borne pathogens that cause serious diarrheal diseases. To colonize the human intestine, these pathogens must overcome innate immune defenses such as antimicrobial peptides (AMPs). Bacterial pathogens have evolved various mechanisms to resist killing by AMPs, including proteolytic degradation of AMPs. To examine the ability of the EHEC and EPEC OmpT outer membrane (OM) proteases to degrade α-helical AMPs, ompT deletion mutants were generated. Determination of MICs of various AMPs revealed that both mutant strains are more susceptible than their wild-type counterparts to α-helical AMPs, although to different extents. Time course assays monitoring the degradation of LL-37 and C18G showed that EHEC cells degraded both AMPs faster than EPEC cells in an OmpT-dependent manner. Mass spectrometry analyses of proteolytic fragments showed that EHEC OmpT cleaves LL-37 at dibasic sites. The superior protection provided by EHEC OmpT compared to EPEC OmpT against α-helical AMPs was due to higher expression of the ompT gene and, in turn, higher levels of the OmpT protein in EHEC. Fusion of the EPEC ompT promoter to the EHEC ompT open reading frame resulted in decreased OmpT expression, indicating that transcriptional regulation of ompT is different in EHEC and EPEC. We hypothesize that the different contributions of EHEC and EPEC OmpT to the degradation and inactivation of LL-37 may be due to their adaptation to their respective niches within the host, the colon and small intestine, respectively, where the environmental cues and abundance of AMPs are different.

Interaction between MMACHC and MMADHC, two human proteins participating in intracellular vitamin B12 metabolism

The identification of eight genes involved in inherited cobalamin (Cbl) disorders has provided insight into the complexity of the vitamin B₁₂ trafficking pathway. Detailed knowledge about the structure, interaction, and physiological functions for many of the gene products, including the MMACHC and MMADHC proteins, is lacking. Having cloned, expressed, and purified MMACHC in Escherichia coli, we demonstrated its monodispersity by dynamic light scattering and measured its hydrodynamic radius, either alone or in complex with each of four vitamin B₁₂ derivatives. Using solution-phase intrinsic fluorescence and label-free, real-time surface plasmon resonance (SPR), MMACHC bound cyanocobalamin and hydroxycobalamin with similar low micromolar affinities (K(D) 6.4 and 9.8 μM, respectively); adenosylcobalamin and methylcobalamin also shared similar binding affinities for MMACHC (K(D) 1.7 and 1.4 μM, respectively). To predict specific regions of interaction between MMACHC and the proposed partner protein MMADHC, MMACHC was subjected to phage display. Five putative MMACHC-binding sites were identified. Finally, MMADHC was confirmed as a binding partner for MMACHC both in vitro (SPR) and in vivo (bacterial two-hybrid system).

A rapid method for the analysis of urinary methylmalonic acid

Abstract A method for the determination of urinary methylmalonic acid, applicable on a routine clinical basis, is described. The method is based on the isolation of a mono-tooligo-carboxylic acid fraction from urine by a series of ether extractions and the subsequent gas Chromatographie analysis of these acids as their trimethylsilyl esters and ethers. The method is simple, rapid, and quantitative, and involves no hazardous chemistry.

Identification of Peptides in Cheddar Cheese by Electrospray Ionization Mass Spectrometry

Abstract Extracts of defatted mild, medium and sharp Cheddar cheese and a commercial Cheddar cheese flavor preparation were subjected to reversed-phase high-performance liquid chromatography (RP-HPLC) for separation of peptides. Fractions were subjected to electrospray ionization mass spectrometry (ESI-MS) for molecular weight (MW) determination and mass spectrometry/mass spectrometry (MS/MS) was used for amino acid sequence determination. The combination of MW and amino acid sequence was used for peptide identification. Peptides were compared with the known sequences of casein peptides and cleavage sites were identified. These techniques resulted in the confirmation of 13 peptides from α s1 -casein, seven from β -casein and five from κ -casein. No peptides corresponding to α s2 -casein were identified.

Effects of crude enzyme of Lactobacillus casei LLG on water-soluble peptides of enzyme-modified cheese

Abstract The effects of crude enzyme extract of Lactobacillus casei ssp. casei LLG on the water-soluble peptides of enzyme-modified cheese (EMC) were studied by reverse phase HPLC and amino acid analysis. A wide range of peptidolytic activities (aminopeptidase 1615.44 unit/ml; x-prolyldipeptidyl peptidase 66–73 unit/ml; proline-iminopeptidase 38–63 unit/ml) were detected in the crude enzyme extract. Bitter enzyme-modified cheese (EMC N24) was prepared with Neutrase® 0.-5L for 24 h at 45 °C and treated with (EMC NL72) and without (EMC N96) the crude enzyme for 72 h at 35 °C. The percent peak areas of two hydrophobic peptides (peak I and peak II) in EMC N24 were increased from 1.63% to 3.65% and from 0.88% to 3.23% in EMC N96, respectively, but decreased to below the detectable range in EMC NL72. The bitterness of EMC N96 may have been related to the increase in areas of these two peaks. Based on the amino acid compositions, peak I was identified as the α sl -casein fraction 26–31(Ala-Pro-Phe-Pro-Glu-Val), and peak II as the β-casein fraction 190–192 (Phe-Leu-Leu), respectively. The results suggest that both aminopeptidase and proline-specific peptidases present in the crude extracts are responsible for degrading the hydrophobic peptides in bitter EMC.

Characterization of a purified α-amylase inhibitor from white kidney beans (Phaseolus vulgaris)

A crude extract prepared from white kidney beans (Phaseolus vulgaris) showed α-amylase inhibitory activity. Four fractions showed α-amylase inhibitory activity after the extract was subjected to reverse phase chromatography. The fraction with the highest activity was isolated and characterized. It was found to be a glycoprotein with an N-glycosylation site whose deglycosylated molecular weight is 54,847 as determined by electrospray ionization mass spectrometry (ESI-MS). Its C-terminal residues were serine, followed by alanine and tyrosine. Its binding constant was 2.8 μM at 55°C. The carbohydrate moiety was not involved in binding as its removal did not decrease the binding constant. Physiological amounts of kidney homogenate, plasma proteases, pronase or thermolysin readily hydrolysed the purified inhibitor; however, it was resistant to pepsin. Chloride ions were found to be important for full activity while calcium increased the initial rate of binding. Magnesium or sulfate ions did not have an effect on inhibition. Classical competitive inhibition of α-amylase was observed at pH 6.9.

Simple and rapid high-performance liquid chromatographic method for the determination of aspartame and its metabolites in foods

A method for the determination of aspartame (N-L-alpha-aspartyl-L-phenylalanine methyl ester) and its metabolites, applicable on a routine quality assurance basis, is described. Liquid samples (diet Coke, 7-Up, Pepsi, etc.) were injected directly onto a mini-cartridge reversed-phase column on a high-performance liquid chromatographic system, whereas solid samples (Equal, hot chocolate powder, pudding, etc.) were extracted with water. Optimising chromatographic conditions resulted in resolved components of interest within 12 min. The by-products were confirmed by mass spectrometry. Although the method was developed on a two-pump HPLC system fitted with a diode-array detector, it is straightforward and can be transformed to the simplest HPLC configuration. Using a single-piston pump (with damper), a fixed-wavelength detector and a recorder/integrator, the degradation of products can be monitored as they decompose. The results obtained were in harmony with previously reported tedious methods. The method is simple, rapid, quantitative and does not involve complex, hazardous or toxic chemistry.