Carbon-centered radicals in isolated soy proteins

ABSTRACT:  Solid-state electron paramagnetic resonance (EPR) spectroscopy of commercial samples of isolated soy proteins (ISP) revealed a symmetrical free-radical signal typical of carbon-centered radicals ( g = 2.005) ranging from 2.96 × 10¹⁴ to 6.42 × 10¹⁴ spins/g. The level of free radicals in ISP was 14 times greater than similar radicals in sodium caseinate, 29 times greater than egg albumin, and about 100 times greater levels than casein. Nine soy protein powdered drink mixes contained similar types of free radicals up to 4.10 × 10¹⁵ spins/g of drink mix, or up to 6.4 times greater than the highest free-radical content found in commercial ISP. ISP samples prepared in the laboratory contained trapped radicals similar to the levels in commercial ISP samples. When ISP was hydrated in 2.3 mM sodium erythorbate or 8.3 mM L-cysteine, frozen and dried, the level of trapped free radicals increased by about 17- and 19-fold, respectively. The ESR spectrum of defatted soybean flakes contained overlapping signals from the primary free-radical peak ( g = 2.005) and a sextet pattern typical of manganese-II. The manganese signal was reduced in the laboratory ISP and very weak in the commercial ISP.     

Effect of moisture, lipids, and select amino acid blocking agents on the formation and stability of metastable radicals in powdered soy proteins

Incremental increases in the moisture content of powdered soy protein products from 4.4% to 13.4% produced an inverse effect on the ability of soy proteins to maintain metastable free radicals. The corresponding reduction in electron paramagnetic resonance signal was not due to dielectric loss in the range of moisture contents examined. Subsequent evaluations of various treatments were conducted after drying soy proteins with molecular sieve to a water activity below 0.085 in order to minimize the influence from variations in moisture. Isolated soy protein (ISP) samples, prepared with "defatted flour" that had been further extracted with chloroform/methanol (2 : 1), had a 96% reduction in total lipids compared to the control ISP samples. The initial rate of radical accumulation in the "reduced-lipid" ISP for the first 3 wk was not significantly different from the initial rate of radical increases in the control ISP. After 3 wk, radical accumulation in the "reduced-lipid" ISP continued to increase, but at a rate that was less than the control. These findings indicate that the initial reactions contributing to the formation of metastable radicals in the powdered ISP are not strongly dependent on associated lipids. Blocking sulfhydryl groups during ISP preparation with N-ethylmaleimide did not significantly slow the rate of radical accumulation compared to the control ISP. Blocking arginine residues in ISP samples with phenylglyoxal caused an increased rate of radical accumulation for the first 4 wk. PRACTICAL APPLICATION: Levels of metastable radicals in powdered soy protein products typically range from 10 to 100 times greater than the free radicals in other food protein sources. This current research examines various compositional and treatment parameters that might be used to minimize the content of free radicals in foods containing soy proteins.     

An investigation into gamma-ray treatment of shellfish using electron paramagnetic resonance spectroscopy

BACKGROUND: Gamma irradiation is a safe method for the decontamination of shellfish, and its technical feasibility and safety have been endorsed (FAO/IAEA/WHO). In order to implement proper food‐processing regulations, the identification of radiation treatment is required. In this study, electron paramagnetic resonance (EPR) spectroscopy was employed to identify the irradiation of seven different types of commonly consumed shellfish. RESULTS: Characteristics (g factor and signal intensity) of the EPR spectra of control and irradiated samples were recorded in order to clarify the gamma‐ray treatment of shellfish. Radiation‐specific signals around g = 2.0038 ± 0.0006 (g₁) and g = 1.9996 ± 0.0002 (g₂ and g₃) were observed for all shellfish samples irradiated with absorbed doses of 3 and 6 kGy. The X‐ray diffraction (XRD) spectra of some shellfish samples were recorded to investigate their mineral profiles. CONCLUSION: The EPR spectroscopic results indicated that carbonate minerals were the source of the paramagnetic species that were produced upon irradiation, entrapped in the shellfish and showed radiation‐specific asymmetric signals whose intensities increased quantitatively with the absorbed radiation dose. The XRD analysis revealed that aragonite and calcite minerals were present in the shells of shellfish. Copyright © 2011 Society of Chemical Industry     

Oxidative effects in uninfected tissue in leaves of French bean (Phaseolus vulgaris) containing soft rots caused by Botrytis cinerea

Several markers of oxidative processes have been measured in leaves of Phaseolus vulgaris infected with Botrytis cinerea, with the specific objective of investigating changes induced by this necrotrophic pathogen in tissue remote from the lesion. There was a progressive decrease with time in the contents of ascorbic acid (AA) in apparently healthy tissues from infected plants and non‐inoculated plants grown under identical high‐humidity conditions (abiotically stressed controls), and for periods >48 h this decrease was greater in the infected plants. This decline in AA content was accompanied by an elevation in the intensity of the electron paramagnetic resonance (EPR) signal from adducts of the spin trap α‐(4‐pyridyl‐1‐oxide)‐N‐t‐butylnitrone (POBN), a destabilisation of the (monodehydro) ascorbate radical (Asc^·) signal in the presence of POBN, and an increase in the ratio of Asc^· to AA in samples studied in the absence of the spin trap. These results are consistent with a shift in redox status to more oxidising conditions in apparently healthy tissue of infected plants and indicate the prevalence of chemical processes that are distinctly different from those in uninfected plants. However, no differences in lipid peroxidation products or the single‐peak free radical and Fe(III) (g = 4.27) EPR signals were observed between these tissues distant from the lesions and those from abiotically stressed controls. In addition, the pathogen‐derived sesquiterpene toxin botrydial and a second Mn(II) EPR signal, both of which are associated with Botrytis infection, were not detected in these ‘apparently healthy’ tissues. Copyright © 2003 Society of Chemical Industry     

Lipoxygenase Independent Hexanal Formation in Isolated Soy Proteins Induced by Reducing Agents

ABSTRACT:  Compared to corresponding controls, 6.5 mM dithiothreitol (DTT) elevated headspace hexanal level over aqueous slurries of both commercial isolated soy proteins (ISP) and laboratory ISP prepared with 80 °C treatment. Further analysis revealed that lipoxygenase (LOX) activity was not detected from these ISP, indicating that LOX is not involved in the observed hexanal increase. Levels of the induced headspace hexanal over the ISP aqueous slurries were proportional to the amount of DTT added in the range of 0 to 65 mM. Subsequent systematic investigations with model systems revealed that iron was required for the reducing agent-induced hexanal formation from linoleic acid. Erythorbate, another reducing agent, can also induce hexanal formation in both ISP and model systems. As a comparison, the LOX activity and hexanal synthesis in defatted soy flour were examined. The corresponding results showed that defatted soy flour maintained high LOX activities and that hexanal synthesis in such sample was significantly inhibited by high concentration DTT (above 130 mM). Data from the current investigation demonstrate the existence of LOX independent hexanal formation induced by reducing agents in ISP and the potential requirement of iron as a catalyst.     

Pectin stabilization of soy protein isolates at low pH

The stabilizing behaviour of pectins in acidified dispersions of soy protein was studied. Objective of this work was to understand if different soy isolates and different pectins show differences in stability. The behaviour of a commercially available isolated soy protein (ISP) was compared to that of a soy protein in the laboratory. At pH 3.8 while suspensions of commercial ISP showed significant precipitation, the native SPI (prepared in the laboratory) was much more stable against precipitation. High methoxyl pectin (HMP) had better stabilization behaviour than low methoxyl pectin (LMP). A higher amount of HMP was needed to avoid protein precipitation of native SPI, compared to that used for commercial ISP. It was demonstrated that at low pH pectin forms soluble complexes with SPI through electrostatic interactions.     

Thermally Stimulated Luminescence in Powdered Soy Proteins

Heating powder isolated soy proteins (ISPs) in a N₂ environment produced thermally stimulated luminescence (TSL), in 2 major temperature regions, 50 to 250°C (region R1) and 250 to 350°C (region R2). In soy protein 7S fraction, strong TSL was detected in both regions with glow peak maximum (T_m) at 150 ± 15°C and at 300 ± 10°C. Two additional satellite or shoulder peaks were detected from the ISP and 7S protein fraction within region R1 at T_m = 90°C and T_m = 210°C. The soy protein 11S fraction produced a broad, poorly defined TSL peak in the low‐temperature region. Electron paramagnetic resonance spectroscopy data from the control ISP sample, deuterium sulfide‐treated ISP, ISP stored in either N₂ or O₂, and defatted soy flour, indicated that the trapped radicals present in ISP is associated with the production of the primary TSL peak at 150 ± 15°C. Activation energies required to release the trapped charges (for luminescence to occur) are approximately 0.70, 0.78, 1.50, and 1.8 eV for TSL at T_m = 100, 150, 200, and 300°C, respectively. The reaction mechanism that leads to the release of the trapped charges for TSL to occur followed a mixed order kinetic, between 1.5 and 1.8. The frequency factor varied between 10⁷/s and 10¹⁷/s.     

A ≥ 100000-MW Soybean Protein Fraction that Inhibits the Formation of Methanethiol and Hydrogen Sulfide in Aqueous Slurries of Isolated Soy Proteins with Added L-Cysteine

ABSTRACT: The addition of L-cysteine to aqueous slurries of commercial isolated soy proteins (ISP) increased methanethiol headspace levels by 17- to 36-fold over the control. Corresponding levels of hydrogen sulfide were about 10 to 19 times greater than methanethiol. Neither methanethiol nor hydrogen sulfide were detected when L-cysteine was added to aqueous slurries of hexane-defatted soy flour. The production of hydrogen sulfide and methanethiol in aqueous slurries of commercial ISP was inhibited by the addition of a component(s) recovered from the pH 4.6 supernatant obtained during laboratory preparation of ISP by isoelectric precipitation. The inhibitory component had a molecular weight (MW) of ≥ 100000 and an isoelectric point of about 5.9. This component was not serine acetyl transferase. Its inhibitory properties were inactivated at 70°C and diminished with elevated levels of methionine. Adding the ≥ 100000-MW soluble-proteins (from the isoelectric precipitation step) back to a nonheat-treated laboratory ISP during processing reduced the methanethiol level by 88%.     

Rheological and Thermal Properties of Extruded Mixtures of Rice Starch and Isolated Soy Protein

Many foods gain new mechanical, thermal and textural properties after being processed due to interactions between carbohydrates and proteins. This effect is characteristic for each foodstuff. The properties of extruded isolated soy protein (ISP) and rice starch were studied considering the following extrusion variables: starch proportion with respect to ISP (0–100%), pH (3–9), moisture content (20–30%) and temperature (140–180ºC). The following characteristics were measured: Water absorption index (WAI), water solubility index (WSI), glass transition temperature (T_g), melting temperature (T_m), viscosity at 90ºC and at 50ºC, storage (G′), loss modulus (G′′) and tan δ. The results indicate that the extruded starch exhibits higher WAI and WSI values than untreated starch. For extruded ISP these values are much lower than for untreated ISP. Extrudates with higher starch proportion had higher T_g and T_m values; pH has a significant effect (p<0.05), at pH 3 higher T_g values were observed, and at pH 9 higher values of T_m. The highest viscosities at 90ºC and 50ºC were observed for extrudates with a higher starch proportion and pH 9. Extruded mixtures showed a more elastic than viscous behavior and an extruded 1:1 blend of starch‐ISP exhibited the behavior of a viscous liquid.     

STORAGE STABILITY OF FERROUS IRON IN WHOLE WHEAT FLOUR NAAN PRODUCTION

Premix containing ferrous sulfate, ethylenediamine tetraacetic acid and folic acid (20.0:20.0:1.5 ppm) was used to fortify whole wheat flour stored at ambient temperature for 42 days. Naans (flat bread) were prepared from 0‐, 20‐, 40‐ and 60‐ppm ferrous iron‐fortified flour samples at weekly intervals and were analyzed for physicochemical constants and sensory evaluation. It was observed that flour containing 60‐ppm ferrous sulfate contained the highest iron residues. Total iron in flour samples showed no significant difference, while ferrous iron significantly decreased in fortified flour (0.53–3.08%) and in the naans (0.42–3.48%) because of its oxidation to ferric iron during storage. Phytic acid content decreased (0.886–0.810%) significantly during the same storage period. Iron levels affected some sensory characteristics significantly (P ≤ 0.05) including color, texture, flexibility, chewability and overall acceptability of the naans, but not taste and flavor. The sensory attributes of naans illustrated that naans containing 40‐ppm ferrous iron are more acceptable than those prepared with 60‐ppm ferrous iron.     

Effect of Various Food Additives on the Levels of 4(5)‐Methylimidazole in a Soy Sauce Model System

In this study, the effect of food additives such as iron sulfate, magnesium sulfate, zinc sulfate, citric acid, gallic acid, and ascorbic acid on the reduction of 4(5)‐methylimidazole (4(5)‐MI) was investigated using a soy sauce model system. The concentration of 4(5)‐MI in the soy sauce model system with 5% (v/v) caramel colorant III was 1404.13 μg/L. The reduction rate of 4(5)‐MI level with the addition of 0.1M additives followed in order: iron sulfate (81%) > zinc sulfate (61%) > citric acid (40%) > gallic acid (38%) > ascorbic acid (24%) > magnesium sulfate (13%). Correlations between 4(5)‐MI levels and the physicochemical properties of soy sauce, including the amount of caramel colorant, pH value, and color differences, were determined. The highest correlations were found between 4(5)‐MI levels and the amount of caramel colorant and pH values (r² = 0.9712, r² = 0.9378). The concentration of caramel colorants in 8 commercial soy sauces were estimated, and ranged from 0.01 to 1.34% (v/v).     

Formation of paramagnetic products in leaves of wheat (Triticum aestivum) as a result of ozone‐induced stress

Electron paramagnetic resonance (EPR) measurements on light‐harvested samples of wheat leaves that had been exposed to toxic ozone levels showed the production of a single‐peak free radical signal and a large increase in intensity of the Fe(III) (g = 4.3) signal compared with control samples. These changes were accompanied by a decrease in the intensity of the more complex free radical signal that is associated with a tyrosine‐based radical centre in photosystem II (PSII) protein. No qualitative changes in the Mn(II) signal were seen as a result of ozone exposure. Identification of the stress‐induced free radical is discussed in the context of previous speculations in the literature, and it is concluded that there is still insufficient evidence for a positive identification. A similar situation exists with the Fe(III) component, where the ligand(s) remain(s) unidentified, although the complex is confirmed as a mononuclear species. Copyright © 2003 Society of Chemical Industry     

ESR-Based Investigation of Radiation-Induced Free Radicals in Fresh Vegetables After Different Drying Treatments

Fresh vegetables, such as paprika, cucumber, and cabbage, were gamma-irradiated at 0, 2, and 5 kGy. Different pretreatments and selection of samples were compared to obtain improved electron spin resonance spectral features resulting in better detection of irradiation status. The non-irradiated samples exhibited a single central signal at about g₀ = 2.007), whose intensity showed a significant increase upon irradiation. However, the irradiation also produced the typical electron spin resonance spectra of the radiation-specific cellulose radicals showing two side peaks (g₁ = 2.023 and g₂ = 1.986) with a mutual distance of 6 mT. The difference in electron spin resonance responses was observed while analyzing the different parts of the same vegetable. The flesh sample gave better spectral characteristics providing clear radiation-induced electron spin resonance signals. The effect of different drying pretreatments was also evident providing the comparable or improved results when using alcoholic-extraction as an alternative to the conventional freeze-drying technique.     

EPR Study of Radicals Generated in Starch by Microwaves or by Conventional Heating

Irradiation of starch with microwaves or heating at relatively low temperature (483 K) generates radicals with anisotropic EPR signals (signal I and II) of lorentzian shape and similar g factors (g_av=2.006 and 2.007 in X and Q band, respectively). Signal I, exhibiting a doublet hyperfine structure (HFS) with A_av=1.19 mT (X band) and A_av=1.24 mT (Q band), was ascribed to a carbon radical with an unpaired electron localized at C₍₁₎ of the glucose unit, from which a hydrogen atom was abstracted. The electron interacts with the nuclear spin of the β‐hydrogen at C₍₂₎. Signal II, with g factor values similar to that of signal I but without HFS, was assigned to a radical with unpaired electron localized also at C₍₁₎ of the glucose unit from which, however, two hydrogens (α and β) were abstracted from C₍₁₎ and C₍₂₎, respectively. Signals I and II show different saturation ability in the power range 0.3–30 mW. Radicals generated in the native starch at higher temperature (503 K) exhibit more intensive EPR spectra, with dominating signal II and lower HFS constant of the signal I. The same trend of weakening of the HF interactions is observed for oxidized starch, proving that the β‐hydrogen is abstracted more easily from C₍₂₎ at higher temperature. The decay of the radicals generated by microwaves or by heating at 483 K, during storing at 293 K, occurs monotonously, according to a second order kinetics. On the other hand, the radicals formed at higher temperature (503 K) represent nonmonotonous changes with the time of storing at 293 K. Such behavior may be explained assuming nonuniform distribution of thermally generated radicals, which partially do not contribute to the signal intensity leading to the significant broadening of the EPR lines. During relaxation the radicals became better dispersed which makes them active in EPR.     

An Improved Approach to Identify Irradiated Spices Using Electronic Nose,FTIR, and EPR Spectroscopy

Changes in cumin and chili powder from India resulting from electron‐beam irradiation were investigated using 3 analytical methods: electronic nose (E‐nose), Fourier transform infrared (FTIR) spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy. The spices had been exposed to 6 to 14 kGy doses recommended for microbial decontamination. E‐nose measured a clear difference in flavor patterns of the irradiated spices in comparison with the nonirradiated samples. Principal component analysis further showed a dose‐dependent variation. FTIR spectra of the samples showed strong absorption bands at 3425, 3007 to 2854, and 1746 cm^?1. However, both nonirradiated and irradiated spice samples had comparable patterns without any noteworthy changes in functional groups. EPR spectroscopy of the irradiated samples showed a radiation‐specific triplet signal at g = 2.006 with a hyper‐fine coupling constant of 3 mT confirming the results obtained with the E‐nose technique. Thus, E‐nose was found to be a potential tool to identify irradiated spices.     

Application of electron paramagnetic resonance (EPR) spectroscopy for control of irradiated food

Four groups of foodstuffs were irradiated in a ⁶⁰Co source with doses from 0.3 to 10kGy and subsequently measured by EPR spectrometry at room temperature in air: (1) poultry bones and fins, scales and bones of carp. (2) seeds of selected fruits, (3) dehydrated mushrooms, and (4) a selected set of spices and herbs. Qualitative and, in some cases, quantitative data related to the absorbed dose of radiation were collected. In the irradiated bones from poultry and carp an asymmetric singlet (g_I = 2.0030, g_II =1.9973; ΔH_pp = 0.85 mT) was detected which was stable at room temperature and was similar to that previously found in irradiated mammalian bones. Another stable EPR signal (g₀ = 2.0024, ΔH_pp = 0.56 mT) was found in the fins and scales of carp which was about five times more intense in fins than in scales. In pips of pears irradiated with a dose of 3 kGy. a signal which was about twice as intense as the endogenous signal was recorded. A multicomponent EPR signal derived from the stones of dates differed from the endogenous signal even when a low dose (0-5 kGy) was applied. A multicomponent EPR signal is also observed in dried mushrooms irradiated with a dose of 3 kGy. In white mustard, paprika and chilli no native EPR signal exists and a radiation-induced stable EPR signal can still be observed after a period of 3 months. The radiation-induced EPR signal in black pepper was highly sensitive to moisture and disappeared, yet the native signal survived. The pilot experiments performed with irradiated stones of cherries, plums, lemons, apple pips, raspberries, cranberries, red currants, blackcurrants, gooseberries and tomatoes showed the induction of short-lived EPR signals of no practical use for the control of food irradiation.     

Electron paramagnetic resonance detection of Mexican irradiated spices

Four different spices produced in Mexico, namely black pepper, two kinds of chilli and oregano, were studied by electron paramagnetic resonance (EPR) technique for detection purposes. In all the non‐irradiated spices we tested, the same single line signal at g = 2.0041 (native signal) was registered. Irradiation induced at least two EPR signals overlapping the native signal: an intense singlet and, with the exclusion of oregano, a weak triplet with hyperfine splitting of 3 mT due to cellulose free radicals. The recombination time of the free radicals produced by irradiation was short compared with the shelf‐life of the spices, thus limiting the application of the EPR technique for detection. To extend the time when these products may be identified as irradiated, the method proposed by Yordanov and Gancheva (Appl Radiat Isot 2000; 52 :195) was applied to the spices, but with no success. Thermoluminescence tests provided correct results for all the spices, even 2 years after irradiation.     

Effect of fermented soy product on the fecal microbiota of rats fed on a beef‐based animal diet

BACKGROUND: The aim of this study was to determine whether the consumption of soy product fermented with Enterococcus faecium CRL 183, would modify the fecal microbiota of rats fed a diet containing red meat. The rats were placed in groups, distinguished by their diets. For 60 days, group I was given a standard casein‐based rodent feed and groups II–VI, the beef‐based feed. From the 30th day, groups III–VI also ingested the following products: group III, E. faecium‐fermented soy product; group IV, pure suspension of E. faecium; group V, sterilized fermented soy product; and group VI, unfermented soy product. RESULTS: Rats that ingested fermented soy product showed a slight increase in the numbers of lactobacilli (0.45 log CFU g^?1), as did the casein‐based diet group (0.47 log CFU g^?1). The fermented soy product did not cause any reduction in the number of enterobacteria or clostridia, but promoted a slight fall in the viable count of Bacteroides spp. (2.80 ± 0.20 to 2.34 ± 0.07 log CFU g^?1). CONCLUSIONS: The results indicate that the ingestion of this fermented soy product did not lead to significant changes in the fecal microbiota of the rats fed on a beef‐based diet. Copyright © 2009 Society of Chemical Industry     

From wheat sourdough to gluten‐free sourdough: a non‐conventional process for producing gluten‐free bread

Gluten‐free (GF) sourdough was prepared from wheat sourdough and analysed both in fresh (GFS) and dried forms (DGFS). The gluten content in each GF sourdough sample was <20 mg kg^?1. The dough leavening capacity and the properties of the bread samples were investigated and compared to those of bread prepared using bakery yeast (Saccharomyces cerevisiae). Two commercial rice‐based mixtures (different for the presence/absence of buckwheat flour) were used to prepare bread samples. In GFS, lactic acid bacteria (LAB) and yeasts were found in amounts corresponding to 10⁸ and 10⁷ CFU g^?1, respectively, whereas both LAB and yeasts were detected in lower amounts (about 10⁶ CFU g^?1) in DGFS. When used in bread‐making, both GFS types produced significant dough acidification and exhibited good dough development during proofing, resulting in loaves with specific volume values between 3.00 and 4.12 mL g^?1, values similar to those obtained for reference bread (3.05÷4.15 mL g^?1). The use of GFS was effective in lowering the bread staling rate during storage for up to 7 days.     

Edible Films and Coatings from Soy Protein

A method was developed by which films could be prepared from commercial isolated soy protein (ISP). ISP was treated with alkali (ATISP) to alter film orooerties. Water vapor oermeability (WVP). oxygen‘permeability (O₂P), tensile strength (TS), percent elongatidn (%E), and appearance of ISP and ATISP were compared. Alkali treatment had no effect on WVP. O₂P, and TS, gave hieher %E, and improved film appearance. Films properties were also compared at pH 6, 8, 10, and 12. In general, pH 6 gave higher WVP and O₂P and lower TS and %E; while higher pH gave lower WVP and O₂P and higher TS and %E. ATISP films could not be produced at pH 6. Film appearance generally improved with increased pH.