T. Richardson

Activated oxygen species and oxidation of food constituents.

Activated oxygen species which may be important in initiating oxidative changes in foods include singlet oxygen, hydroxyl radical, ozone, superoxide anion (perhydroxyl radical at low pH), and hydrogen peroxide. Chemical and enzymic reactions known to occur in biological materials can generate singlet oxygen, hydroxyl radical, superoxide anion, and hydrogen peroxide. Ozone is primarily a product of photoreactions in polluted air. Reactions involving singlet oxygen, hydroxyl radical, and ozone with food constituents can ultimately yield peroxides which decompose to initiate oxidative chain reactions. Superoxide anion and hydrogen peroxide are relatively inert toward organic molecules but can decompose to produce the more reactive singlet oxygen and hydroxyl radical. Inhibition of reactions initiated by reactive oxygen species in foods should be very important in preserving the oxidative stability of foods. The generation, detection, measurement, reaction, and inhibition of reactions of active oxygen species are surveyed in this review.

Secondary structure of bovine αS1- and β-casein in solution

Abstract Circular dichroism spectra of bovine α S1 - and β-casein in neutral buffers between 190 and 250 nm indicated that both proteins had low proportions of periodic structure, but more than in the denaturing solvents, 4.5 m guanidine-hydrochloride, or 6 m urea. The addition of low concentrations of calcium chloride to the protein solutions did not alter their spectra. Between 255 and 320 nm the spectrum of β-casein was the same in neutral buffer and in denaturing solvents indicating that the aromatic chromophores were in unordered segments of the sequence. The spectrum of α S1 -casein, however, increased in intensity in going from guanidine-hydrochloride to neutral buffer to buffered calcium chloride solution. Similar changes were apparent when carboxypeptidase A-treated α S1 -casein (which loses the C-terminal tryptophan residue) was substituted for α S1 -casein suggesting that the long-wavelength spectral changes arose from conformational changes involving the tripeptide tryptophan 164-tyrosine 165-tyrosine 166. A structure prediction program using primary sequence data indicated that segments 37–40, 50–69, 75–79, 95–100, and 116–128 of α S1 -casein may be helical while segments 22–25, 106–111, and 164–168 may be extended sheet structures. Similarly segments 2–5, 19–26, 42–45, and 92–106 of β-casein may be helical while segments 79–85, 155–158, 185–192, and 206–209 may be extended sheet structures. It is notable that the peptide bonds most sensitive to the milkcoagulating enzyme, chymosin (23–24 and 24–25 in α S1 -casein, and 189–190 and 192–193 in β-casein), are in short extended sheet structures surrounded by conformations predicted to be aperiodic or reverse turn.

Sterol Oxides in Foodstuffs: A Review

The toxicological significance of oxidized cholesterol has been well documented in numerous studies. This review focuses on the analysis of dietary sterol oxides in the foodstuffs examined to date with particular emphasis on isolation and characterization techniques. Eight common oxidation products of cholesterol have been identified in certain cholesterol-rich foods subjected to oxidative stress during food processing and/or storage. These products include 25-hydroxycholesterol, α or β 5,6-epoxycholesterol, α or β 7-hydroxycholesterol, 7-ketocholesterol, cholesta-3,5-dien-7-one and cholestane-3β, 5α, 6β-triol. A limited number of studies on the biological effects of dietary phytosterol oxides indicate these products may also be of nutritional concern. Four common autoxidation products of β-sitosterol have been identified in edible oils; these include α or β 7-hydroxysitosterol, 7-ketositosterol and setosta-3,5-dien-7-one. Few quantitative data are available on the sterol oxide content of foods. Moreover, studies without apparent precautions against the artifactual formation of sterol oxides may be flawed. Additional research is necessary to adequately identify and quantify the sterol oxides which most likely exist in certain foods.

Anomalous behavior of bovine αs1- and β-caseins on gel electrophoresis in sodium dodecyl sulfate buffers

Abstract Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of M r and the mobility of the protein through the gel matrix. However, bovine caseins (especially α s1 -casein) have lower mobilities than expected on the basis of their known M r . The binding of SDS to both α s1 -casein ( M r 23,600) and β-casein ( M r 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the α s1 -casein:SDS complex was larger than the β-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both α s1- and β-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13 C NMR results showed that SDS bound to α s1- and β-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of α s1 -casein increased its mobility in SDS-gel electrophoresis, but neither modification affected β-casein mobility. These and other results indicate that the low electrophoretic velocity of α s1 -casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of α s1 -casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the α s1 -casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori , it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the M r of the protein.

Applications of Microbial Enzymes in Food Systems and in Biotechnology

Publisher Summary This chapter summarizes the applications of enzymes in food and biotechnology and suggests the future trends and applications. Selected strains of molds, bacteria, and yeasts are currently used as sources of enzymes for food processing. Aspergillus oryzae, Aspergillus niger, and Bacillus subtilis are the three most useful, well-known, and safe microbial sources for enzymes. Enzymes have great utility in food processing. For example, the processing of common foods such as bread, beer, cheese, and soft drinks requires microbial enzymes as an integral part of their manufacture. There are several advantages of enzymes in food processing over alternative physical or chemical manipulations. First, enzymes catalyze a specific action, avoiding potentially undesirable side reactions resulting from less specific processing methods, second, the extremes of pH or temperature necessary for chemical or physical treatments are avoided by using enzymes and this minimizes the side reactions as well. Many of the enzymes used in biotechnology are derived from microbial sources. Microbial enzymes are often used in analysis, such as the clinical use of fungal glucose oxidase and bacterial catalase to measure blood glucose to monitor the diabetic condition.

Identification and quantification of cholesterol oxides in grated cheese and bleached butteroil

Butteroil samples bleached with benzoyl peroxide (BP) and 17 commercial cheeses were screened for oxidized sterols by thin layer chromatography (TLC). Ungrated cheeses made from bleached milk and freshly bleached butteroil contained no detectable oxidized sterols. Oxidized sterols were detected in stored, bleached butteroils and in grated cheeses. Four major oxidation products were the isomeric 5,6-epoxycholesterols and the epimeric 7-hydroxycholesterols identified by TLC, high performance liquid chromatography (HPLC) and mass spectrometry (MS). Additional sterol oxides (tentatively identified and not quantified) present in these samples included low levels of 7-ketocholesterol and cholesta-3,5-dien-7-one. The epimeric 7-hydroxycholesterols were detected in bleached butteroils stored in air (BP-A) and nitrogen (BP-N) for 22 days at 15 C. Butteroil, after 90 days of storage at 15 C, had 30 (BP-N) and 60 (BP-A) µg total oxides/g of bleached oil and, after 1-year at −20 C, had 70 (BP-N) and 180 (BP-A) µg/g butteroil. A grated, unbleached cheese packaged in clear glass contained the most oxidized sterols (44 µg/g). Sterol oxides were not detected in bleached cream using a simulated industrial process.

Photodegradation of DNA with fluorescent light in the presence of riboflavin, and photoprotection by flavin triplet-state quenchers.

Superoxide anion was photogenerated upon illumination of nucleic acids with fluorescent light in a solution containing phosphate buffer, pH 7.8 and riboflavin. DNA was a better reducing substrate for this reaction than was RNA. A similar riboflavin-sensitized photoreaction caused single- and double-strand scissions of supercoiled PM2 DNA as detected by electrophoresis in agarose gels. None of specific scavengers or quenchers for superoxide anion and other active oxygen species prevented the DNA strand breaks. However, among the flavin triplet-state quenchers, potassium iodide, butylated hydroxyanisole, and ferricytochrome c protected the supercoiled DNA from photodegradation; butylated hydroxytoluene, alpha-tocopherol, tyrosine and hemoglobin did not have any protective effect. These results indicate that triplet-state riboflavin or a derivative formed from it participate directly in the observed riboflavin-sensitized DNA photodegradation and that active oxygen species are not directly involved.

Essential fatty acids and glucose permeability of lecithin membranes

Abstract Lecithins were isolated from egg yolk, and from internal organs of fish, essential fatty acid-deficient rats and normal rats. Fatty acid composition of each preparation was determined by gas-liquid chromatography. From each lecithin, liposomes were formed in 0.3 M glucose. Extraparticulate glucose was removed by dialysis, and the subsequent rates of glucose efflux from within the particles were determined at various temperatures. The liposomes prepared from essential fatty acid-deficient lecithin permitted a slower rate of glucose diffusion than those of normal rats and fish at all temperatures, and had a comparable rate with that of egg lecithin. Energies of activation for diffusion were 26.3 ± 1.6, 28.9 ± 5.8, 26.2 ± 3.9 and 26.7 ± 2.4 kcal/mole glucose, for lecithins from egg, essential fatty acid-deficient rats, normal rats and fish, respectively. Differences in glucose diffusion rates were largely dependent upon the degree of unsaturation of the fatty acid residues in the lecithin molecules.

Use of immobilized lactase in milk systems

Immobilization of lactase for continuous hydrolysis of lactose in milk systems offers considerable potential for the improvement of fluid dairy products. The hydrolyzed product, which contains glucose and galactose, may possess improved functional and nutritional properties. In particular, the usage of cheese whey, a by-product of cheese manufacturing, may be expanded greatly with the development of immobilized lactase technology. Although lactases occur rather widely in nature, only microbial enzymes are of commercial value. The various approaches to lactase insolubilization have been reviewed. Additionally, pertinent factors in operating an immobilized lactase reactor system have been discussed. Commercial lactose reactors are being used currently for industrial production of low-lactose skimmed milk and appear to have economic potential for processing of cheese whey.

Use of a papain superpolymer to elucidate the structure of bovine casein micelles

Abstract Micellar and soluble casein samples were treated with papain which had been cross-linked with glutaraldehyde to form a superpolymer. Unhydrolyzed portions of casein samples were fractionated on polyacrylamide gels. The stained gels were scanned with a densitometer. In both casein samples, the amount of unhydrolyzed κ-, β-, and α s- caseins decreased gradually as the reaction proceeded. The percentage of hydrolysis after 60 min of reaction was 69% in micellar-casein and 78% in soluble casein. However, none of the three casein fractions was hydrolyzed completely. Unhydrolyzed casein in both samples had approximately the same composition throughout the entire reaction. These results suggest that κ-casein does not have a specific location in the casein micelle and that the three major casein fractions are distributed unformly throughout the micelle.