Changes in soybean phytate content as a result of field growing conditions and influence on tofu texture

It is known that tofu quality tends to vary among soybeans even of the same variety. Cultivation environments can affect the contents of the soybeans. Twenty-seven soybean varieties were grown in a drained paddy field and an upland field, and then their protein and phytate contents were determined using the Fourier transfer infrared spectroscopy (FT-IR) method. The phytate contents of 12 varieties were higher in the drained paddy field than in the upland field. On the other hand, the environmental factor had little effect on the protein contents. In order to determine whether the difference in phytate content affected tofu texture, the hardness of the tofu made from phytate-added soymilk was measured. The tofu texture having more phytate became softer in the range of the common coagulant concentration. We concluded that the difference in the phytate content of the soybeans among the environmental conditions is a factor that causes fluctuation in tofu quality.     

大豆豆乳和豆腐产量、品质及有关加工性状的遗传变异

采用全国各地的261个大豆品种为材料,研究豆乳和豆腐产量、品质及有关加工性状的遗传变异.结果表明豆乳和豆腐加工过程中每100g干籽粒平均生产干豆乳71.92g,生产干豆腐51.80g,损失豆渣干物质25.76g,损失乳清干物质20.12g.干豆乳蛋白质、脂肪和总糖的含量分别为51.85%、22.55%和18.42%,干豆腐蛋白质、脂肪和总糖的含量分别为46.22%、24.58%和19.36%.豆乳和豆腐产量、品质及有关加工性状在品种间的差异均达到极显著水平,变异系数都较大,从中筛选出一批优异种质.     

Decrease in the Gel Strength of Tofu Caused by an Enzyme Reaction during Soybean Grinding and Its Control

The effect of soybean grinding temperature on the firmness of tofu was investigated. The firmness of tofu decreased with increasing grinding temperature between 0°C and 50°C, this being related to the content of sulfhydryl (SH) groups in soymilk. It could, therefore, be presumed that the decrease in firmness depended upon the decrease in SH content, which was of enzymatic nature and closely correlated with the peroxide value. These facts suggested that the decrease in SH content was caused by a lipid oxidative reaction of lipoxygenases in soybean. When the soybean was ground at a low temperature and in a nitrogen atmosphere to inhibit the action of lipoxygenase, the soymilk was rich in SH content and formed firmer tofu.     

Novel method using phytase for separating soybean beta-conglycinin and glycinin

A novel method for separating soybean beta-conglycinin and glycinin from defatted soymilk by a phytase treatment was developed. Phytase was added to defatted soymilk (1000FYT/100 g of protein) at pH 6.0, and the mixture incubated for 1 h at 40 degrees C. This procedure separated beta-conglycinin and glycinin without needing a reducing agent or cooling into the soluble and insoluble fractions, respectively. Simultaneously, most of the phytate in both proteins was removed.     

Solvent extraction selection in the determination of isoflavones in soy foods

Acetonitrile is superior to acetone, ethanol and methanol in extracting the 12 phytoestrogenic soy isoflavone forms found in foods. At 53% organic solvent in water, raw soy flour, tofu, tempeh, textured vegetable protein and soy germ were evaluated for isoflavone extraction efficiency. The efficiency of acetonitrile extraction was demonstrated in mass balance evaluations of toasting of soy flour and soymilk heating.     

花生粕脂肪含量对豆腐品质的影响

为了确定适用于制备花生豆腐的原料的脂肪含量,以低温花生粕为原料,采用谷氨酰胺转氨酶（transglutaminase,TG）和高温微压煮浆（high-temperature pressure cooking,HTPC）工艺制备花生豆腐,研究不同花生粕脂肪含量（6.89%、10.12%、15.43%、20.28%和25.54%）对豆腐质构的影响,对比花生豆腐与市售大豆豆腐的耐煮性和色泽,并分析加工过程中花生蛋白的结构变化。结果表明,花生粕脂肪含量对豆腐质构有显著影响（P<0.05）,除脂肪含量为25.54%的花生粕外,其他脂肪含量的花生粕均可用于制备花生豆腐。当花生粕脂肪含量为6.89％时,可制得与市售大豆石膏豆腐的质构、耐煮性相当的花生豆腐产品,且其黄度值b（8.38）显著小于大豆石膏（15.86）和卤水（16.56）豆腐（P<0.05）,具有色泽优势。而对于花生蛋白结构变化的分析表明,当花生粕脂肪含量相对较低（6.89%~20.28%）时,TG对花生蛋白的作用效果较好,进一步施加HTPC处理可使花生蛋白完全变性,并且通过形成疏水相互作用和二硫键以维持豆腐凝胶结构。研究结果为花生豆腐的加工提供了技术指导和理论支撑。     

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy

Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectroscopy suffers from several drawbacks, the most severe being nonproportionality of the observed absorbance with the protein concentration and spectral distortions that vary from protein to protein and from sample to sample. In this study, we show (1) that the nonproportionality is due to adsorption of the protein on the ATR crystal surface; (2) that the contribution of the crystal-adsorbed protein can easily be taken into account, rendering the corrected absorbance proportional to the protein concentration; (3) that the observed variable base line distortions, likely due to changes in the penetration depth of the light beam in solutions with the refractive index that depends on the protein concentration, can be easily eliminated; and (4) that ATR IR spectra thus corrected for protein adsorption and light penetration can be used to properly analyze the secondary structure of proteins in solution.     

Immobilization of phytase on epoxy-activated Sepabead EC-EP for the hydrolysis of soymilk phytate

In this work, an active phytase concentrated extract from soybean sprout was immobilized on a polymethacrylate-based polymer Sepabead EC-EP which is activated with epoxy groups. The immobilized enzyme exhibited an activity of 0.1 U/g of carrier and activity yield of 64.7%. The optimum temperature and pH for the activity of both free and immobilized enzymes were found as 60 °C and pH 5.0, respectively. The immobilized enzyme was more stable than free enzyme in the range of pH 3.0–8.0 and more than 70% of the original activity was recovered. Both the enzymes completely retained nearly about 84% of their original activity at 65 °C. The K_m and V_max values were measured as 5 mM and 0.63 U/mg for free enzyme and 12.5 mM and 0.71 U/mg for immobilized enzyme, respectively. Free and immobilized soybean sprout phytase enzymes were also used in the biodegradation of soymilk phytate. The immobilized enzyme hydrolysed 92.5% of soymilk phytate in 7 h at 60 °C, as compared with 98% hydrolysis observed for the native enzyme over the same period of time. The immobilization procedure on Sepabead EC-EP is very cheap and also easy to carry out, and the features of the immobilized enzyme are very attractive that the potential for practical application is considerable.     

Interaction of Protein Particles with Lipids in Soybean Milk

The protein in soybean milk exists as 11S and 7S globulins, and the particles formed from them. The lipid content and composition in the protein fractions and effects of defatting on the form of the protein particles were investigated. The size-distribution of protein particles in both raw and heated soybean milk (soymilk) was not influenced by defatting with hexane, but the number of large particles were slightly increased. The protein particles from raw and heated soymilk samples contained 60% and 3% of the total lipid, respectively. Almost all neutral lipid in the particles of raw soymilk was moved to a floating fraction by heating, but a half of the phospholipids was retained in the particles. The protein components from the hexane-defatted meal were similar to those from whole meal, but those from the C-M-de-fatted meal contained remarkably little β-conglycinin. C-M-de-fatting (Removal of polar lipids) caused a reduction in the particulate fraction, and the addition of phospholipids (lecithin) promoted the formation of protein particles.     

Redox and spectral properties of flavodoxin from Anabaena 7120

We report here on the spectrophotometric and electrochemical properties of the flavodoxin from Anabaena 7120 and compare these properties with those of flavodoxins that have been studied previously. Molar absorption coefficients have been determined for all three oxidation states of this protein, at various wavelengths. For oxidized flavodoxin, molar absorption coefficients for the absorption maxima at 464 and 373 nm were 9200 and 8500 M-1 cm-1, respectively. Reduction by the first electron produced a neutral blue semiquinone which exhibited an absorption maximum at 575 nm. The molar absorption coefficients at 575 nm were 200 M-1 cm-1 for the oxidized form, 5100 M-1 cm-1 for the semiquinone form, and 250 M-1 cm-1 for the hydroquinone form. Redox potentials have been determined, in the pH range of 6.0 to 8.5, for both electron transfers. At pH 7.0, the midpoint potential values for the first and second electron transfers were -0.196 and -0.425 V, respectively. We determined that the first electron transfer is pH dependent and that a proton transfer accompanies this one electron transfer. It was also determined that the second electron transfer is pH independent in the pH range of 6.0 to 8.5.     

Mechanisms of the structural changes in erythrocyte membranes under the action of a shift in pH]

The effect of pH shifts (5--10) on erythrocyte "ghosts" has been studied using the IR spectroscopy. The process of regulation has been shown to take place within pH 7.15--8 range involving both protein (transition testified by absorption band changes 1520, 1540, 1635, 1645, 1585, 1696, 3305 CM-1) and lipid membrane components testified by absorption band splits 1745, 1470, 1380, 720 AND 1242 cm-1. The transition from a lesser to a higher regulating state of membrane structure happens unevenly, since the above changes take place within the narrow pH 7.15--8 interval, near the homeostase limit, being never observed at pH 5--7.15 or 8--10. It is supposed that the increase in regulation of both protein, phospholipid and lipid membrane fields is of phasic nature which presumably constitutes the basis for pH action mechanism as a regulation factor.     

Improvement in cell‐bound phytase activity of Pichia anomala by permeabilization and applicability of permeabilized cells in soymilk dephytinization

Aims: Whole cell permeabilization of Pichia anomala to ameliorate the cell‐bound phytase activity and usability of permeabilized cells in dephytinization of soymilk. Methods and Results: The cells of P. anomala were subjected to permeabilization using the surfactant Triton X‐100 to overcome the permeability barrier and prepare whole cell biocatalysts with high phytase activity. The statistical approach, response surface methodology (RSM) was used to optimize the operating conditions for permeabilization. The treatment of cells with 5% Triton X‐100 for 30 min resulted in c. 15% enhancement in cell‐bound phytase activity. The shrinkage of protoplast was observed, although cell viability and phytase stability were not significantly altered. The free as well as immobilized permeabilized cells hydrolysed soymilk phytate, and the latter could be reused over four consecutive cycles. Conclusions: Whole cell permeabilization of P. anomala using Triton X‐100 led to enhancement in cell‐bound phytase activity. The viability and integrity of yeast cells were not significantly affected because of permeabilization. The permeabilized P. anomala cells effectively dephytinized soymilk, and the permeabilized cells immobilized in alginate could be reused because of sustained phytase activity. Significance and Impact of the Study: This is the first report on the use of permeabilized yeast cells for mitigating phytate content of soymilk. Alginate entrapment of permeabilized P. anomala allows reuse of cells for soymilk dephytinization, thus suggesting a potential application in food industry.     

Monitoring the pH dependence of IR carboxylic acid signals upon Q(B)- formation in the Glu-L212 --> Asp/Asp-L213 --> Glu swap mutant reaction center from Rhodobacter sphaeroides

In the photosynthetic reaction center (RC) from the purple bacterium Rhodobacter sphaeroides, proton-coupled electron-transfer reactions occur at the secondary quinone (QB) site. Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations in the 1770-1700 cm-1 spectral range that are sensitive to 1H/2H isotopic exchange. With respect to the native RC, a novel protonation pattern for carboxylic acids upon QB photoreduction has been identified in the Glu-L212 --> Asp/Asp-L213 --> Glu mutant RC using light-induced FTIR difference spectroscopy (Nabedryk, E., Breton, J., Okamura, M. Y., and Paddock, M. L. (2004) Biochemistry 43, 7236-7243). These carboxylic acids are structurally close and have been implicated in proton transfer to reduced QB. In this work, we extend previous studies by measuring the pH dependence of the QB-/QB FTIR difference spectra of the mutant in 1H2O and 2H2O. Large pH dependent changes were observed in the 1770-1700 cm-1 spectral range between pH 8 and pH 4. The IR fingerprints of the protonating carboxylic acids upon QB- formation were obtained from the calculated double-difference spectra 1H2O minus 2H2O. These IR fingerprints are specific for each pH, indicative of the contribution of different titrating groups. In particular, the 1752 cm-1 signal indicates that Glu-L213 protonates upon QB- formation at pH >or= 5, whereas the 1746 cm-1 signal indicates protonation of Asp-L212 even at pH 4. An unidentified carboxylic acid absorbing at approximately 1765 cm-1 could be the proton donor between pH 8 and 5. The observation that in the swap mutant there are several uniquely behaving carboxylic acids shows that electrostatic interactions occurring between them are sufficiently modified from the native RC to reveal their IR signatures.     

Romanowsky dyes and Romanowsky-Giemsa effect. 2. Eosin Y, erythrosin B, tetrachlorofluorescein, spectroscopic characterization of pure dyes, association of eosin Y

Analytically pure samples of the Romanowsky dyes eosin y, erythrosin b and tetrachlorofluorescein are prepared. DC of the dye samples shows no contaminations. We measured the absorption spectra of the dye dianions in alkaline aqueous solution and of the dye acids in 95% ethanol at very low dye concentrations. The molar extinction coefficients of the long wavelength absorption of the monomeric dye species are determined (Table 1). The extinction coefficients may be used for standardisation of dye samples. The absorption spectra of eosin y in aqueous solution are dependent on concentration. Using a new very sensitive method it was possible to identify two association equilibria from the concentration dependency of the spectra. Dimers are formed even in very dilute solutions, at higher concentrations tetramers. The dissociation constant of the dimers D in monomers M at 293 K, pH = 12, is K21 = 2,9 X 10(-5) M; of the tetramers Q in dimers D K42 = 2,4 X 10(-3) M. From the experimental spectra of eosin solutions at various concentrations, pH = 12, and the equilibrium constants K21, K42 the absorption spectra of the pure monomers, dimers and tetramers are calculated. M has one long wavelength absorption band, VM = 19300 cm-1, epsilon M = 1,03 X 10(5) M-1 cm-1; D also one absorption band, VD = 19300 cm-1, epsilon D = 1,74 X 10(5) M-1 cm-1; Q two absorption bands, VQ1 = 19100, VQ2 = 20200 cm-1, epsilon Q1 = 1,65 X 10(5), epsilon Q2 = 1,96 X 10(5) M-1 cm-1. The absorption spectrum of the dimers is discussed by quantum mechanics.     

Purification and Properties of a Phytate-degrading Enzyme from <Emphasis Type="Italic">Pantoea agglomerans</Emphasis>

A periplasmatic phytate-degrading enzyme from Pantoea agglomerans isolated from soil was purified about 470-fold to apparent homogeneity with a recovery of 16% referred to the phytate-degrading activity in the crude extract. It behaved as a monomeric protein with a molecular mass of about 42 kDa. The purified enzyme exhibited a single pH optimum at 4.5. Optimum temperature for the degradation of phytate was 60°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K_M = 0.34 mmol/l and k_cat = 21 s^-1 at pH 4.5 and 37°C. The enzyme exhibited a narrow substrate selectivity. Only phytate and glucose-1-phosphate were identified as good substrates. Since this Pantoea enzyme has a strong preference for glucose-1-phosphate over phytate, under physiological conditions glucose-1-phosphate is its most likely substrate. The maximum amount of phosphate released from phytate by the purified enzyme suggests myo-inositol pentakisphosphate as the final product of enzymatic phytate degradation.     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

EXAFS analysis of the pH dependence of the blue-copper site in amicyanin from Thiobacillus versutus

The room temperature Cu K-edge EXAFS (extended X-ray absorption fine structure) spectrum of reduced and oxidized amicyanin, the blue copper protein from Thiobacillus versutus, was measured at low and high pH. The data interpretation was partly based on independent NMR evidence for the occurrence of a ligand histidine protonation at low pH (pKa = 6.9) in the reduced protein. In the oxidized protein two nitrogen-donors (from two histidines; Cu-N distances 1.95-2.01 A and 1.86-1.89 A) and a sulfur-donor (from a cysteine; Cu-S distance 2.11-2.13 A) were identified and the coordination appears independent of pH. Upon reduction at high pH the Cu-S bond and one of the Cu-N bonds lengthen slightly (from 2.11 to 2.19 A and from 2.01 to 2.18 A, respectively). Upon lowering of the pH one of the N-donors of the Cu in reduced amicyanin disappears from the Cu EXAFS and a second S-donor (from a methionine) becomes visible at 2.41 A from the Cu. The Debye-Waller factors are compatible with a Cu-N vibrational stretch frequency in the range of 150-250 cm-1 and one greater than 285 cm-1, and a Cu-S vibrational stretch frequency of about 150 cm-1 (Cu-Smet; reduced amicyanin at low pH) and one in the range of 230-800 cm-1 (Cu-Scys).     

The phytate and mineral content of some cereals,cereal products,legumes, legume products,snack bars,and nuts available in New Zealand

Analyses for phytate by an indirect precipitation method and for the minerals calcium (Ca), zinc (Zn), iron (Fe), copper (Cu), and manganese (Mn) by atomic absorption spectrophotometry were carried out on 100 foods available in New Zealand. Foods with 1% phytate (dry weight basis) included untoasted muesli, rolled oats, wheat germ, wheat bran, soybean, and some soy products. Most breads contained between 0.35 and 0.60% phytate; legumes on average had 0.62% phytate, as did snack bars. There was a wide variation in Ca and Zn contents: There was a tenfold variation in Ca content among the legume products, whereas there was a seventyfold variation in Zn content among the cereals. The phytate: Zn molar ratio, which is presumed to indicate the biovailability of Zn, was above 20∶1 for two-thirds of the cereals and almost all of the snack bars; it was above 15∶1 for one-third of the breads, almost all of the legumes, and half of the legume products. These high phytate: Zn molar ratios, as well as some Ca: phytate molar ratios above 6∶1, indicate that there might be a reduced biovailability of Zn in many of the foods analyzed in this study.     

Effects of phytate reduction,fat extraction,and level of Ca on Ca and Zn bioavailability

The effect of phytate reduction, fat extraction, and addition of Ca in the form of skim or whole milk on Ca and Zn bioavailability from a breakfast containing bran muffins was studied in vitro and in vivo. Ca and Zn solubility were measured after in vitro simulated peptic and pancreatic digestion under pH conditions resembling those in the human GI tract. Absorption of Ca and Zn was measured in rats fed test meals tagged with 45Ca and 65Zn. Radioactivity in the femur and liver relative to levels found in rats injected with the isotopes served as criteria for 45Ca and 65Zn absorption, respectively. In vitro solubility was significantly depressed by the presence of phytate and inversely correlated with the phytate:Zn and the (phytate)(Ca):(Zn) ratios. Ca solubility was enhanced by extraction of fat and markedly increased by reduction in both fat and phytate. None of these effects was seen in vivo. However, 65Zn absorption was significantly decreased by fat extraction and phytate reduction although this treatment increased in vitro Zn solubility. Possible reasons for these discrepancies are discussed.     

The interaction of the vanadyl(IV) cation with phytic acid

The interactions of VO²⁺ with phytate to form both soluble and insoluble complexes, have been studied by electronic absorption spectroscopy. A soluble 1∶1 VO²⁺: phytate complex is formed at pH <1. At higher pH-values insoluble complexes are produced. Two different solid complexes, obtained respectively at pH=2 and 4, were isolated and characterized. The maximal bonding ratio of VO²⁺: phytate was found to be 4, on the basis of a pH binding profile.