Extraction and composition of rice endosperm glutelin

“Crude” glutelin was prepared from milled rice (Oryza sativa) flour by sequential extraction of the albumin-globulin fraction with 0.5 M NaCl and prolamin with 70% ethanol-0.6% β-mercaptoethanol. The solvent, 0.5% sodium dodecyl sulphate (SDS)-0.6% β-mercaptoethanol, extracted 91% of the endosperm glutelin without gelatinizing starch granules, whereas chaotropic solvents such as urea and guanidine caused extensive gelatinization. The S-cyanoethyl glutelin (Ce-glutelin) prepared by SDS extraction of the “crude” glutelin (9.5% protein) of IR480-5-9 rice gave three major subunits with MW 38000, 25000 and 16000 in the ratio 2:1:1 as determined by SDS polyacrylamide gel electrophoresis. A similar preparation from “crude” glutelin of a lower protein containing rice had the corresponding subunits in the ratio of 16:3:1. The MW 38000 subunit was unique to glutelin and was not present in C3-albumin-globulin or prolamin; the subunits were only partially purified by SDS Sephadex G-150 gel-filtration. The C3-glutelin was also prepared from a crude glutelin-prolamin preparation from IR480-5-9 by NaOH extractions followed by precipitation at pH 10 and ethanol extraction of the precipitate (C3-glutelin). This preparation had the same three major subunits and in the same ratio as C3-glutelin prepared by the SDS method. The subunits of the former preparation were separated by carboxymethyl Sephadex C-50 chromatography; the MW 38000 subunit eluted between pH 6.2–8.5, the MW 25000 in an impure state at pH values above 9, and the MW 16000 subunit was eluted at pH 8.6—9.2. Amino acid composition of the Ce-glutelin preparations were similar to each other. The MW 38000 and 16000 subunits had lower lysine contents than whole C3-glutelin, whereas the MW 25000 subunit had a higher lysine content.     

Properties of prolamin in mature and developing rice grain

A procedure was developed for preparing lipid- and phenol-free prolamin directly from IR480-5-9 milled rice (Oryza sativa L.).The preparation consisted mainly of one protein band on analytical and SDS-polyacrylamide disc gel electrophoresis with subunit MW of 17000 and a minor fraction with subunit MW 23000. The prolamin eluted as a single peak on SDS-Sephadex G-75 gel filtration and on DEAE-cellulose column chromatography. Prolamin was poor in lysine, histidine, cystine, and methionine but rich in glutamic acid, tyrosine and proline. In dehulled developing grain of two different rices, changes in the aminogram of the prolamin fraction coincided with the start of endosperm protein body synthesis and the appearance from 7 days after flowering of a second prolamin subunit with MW 23 000.     

The role of cotyledon cell structure during in vitro digestion of starch in navy beans

Studies on the physico-chemical, microstructural characteristics and in vitro (under simulated gastric and small intestine conditions) starch digestibility of navy beans were carried out. The microstructure of raw and cooked beans observed through scanning electron microscopy (SEM) showed the presence of hexagonal or angular shaped cotyledon cells (50-100 μm size) containing starch granules with a size ranging between 10 and 50 μm. The extent of starch hydrolysis (%) after 120 min of in vitro gastro-intestinal digestion differed between whole navy beans (∼60%) and milled bean flour and bean starch (85-90%) after they were cooked under similar conditions. Starch hydrolysis (%) increased significantly when the cotyledon cells in the cooked whole navy beans were disrupted using high pressure treatment (French press). The storage of freshly cooked whole beans resulted in a lower (40-45%) starch hydrolysis whereas re-heating increased the same to 70-80% during in vitro small intestinal digestion. The SEM pictures of cooked navy bean digesta after different intervals of in vitro gastric and small intestinal digestion showed that the cotyledon cell structure is maintained well throughout the digestion period. However cotyledon cells appear shrunken and developed wrinkles during in vitro digestion. Particle size analysis of cooked bean paste taken before and after the in vitro gastro-intestinal digestion showed similar particle size distributions.     

Fate of malathion and chlorpyrifos methyl in rough rice and milling fractions before and after parboiling and cooking.

Long-grain rough rice treated with malathion (14 ppm) or chlorpyrifos methyl (Reldan) (6 ppm or 12 ppm) was sampled after 1,6, and 12 wk. Samples from each treatment were processed raw or were parboiled with fresh steeping water, once-used, and twice-used steeping water. Three replicates of rough rice and of each milling fraction were preserved, and three of milled rice were cooked. Chemical residues were measured on rough rice, hulls, brown rice, bran, milled rice, and cooked rice. Parboiling reduced residues on rough rice and hulls but tended to increase residues in the other fractions. Residues of Reldan in bran were substantially increased by parboiling. Doubling the amount of Reldan applied to rough rice approximately doubled the residues found in the milling fractions. Small amounts of the protectants survived all processing including cooking. Residues of malathion in cooked rice averaged about 0.016 ppm in nonparboiled and 0.013 ppm in parboiled rice. Residue of Reldan in cooked rice was commensurate with the amount applied to rough rice. When applied to rough rice at 6 ppm, residues of Reldan in cooked rice averaged 0.05 ppm in nonparboiled rice and 0l.065 ppm in parboiled rice. When applied to rough rice at 12 ppm, residues in cooked rice averaged .053 ppm in nonparboiled rice and 0.15 ppm in parboiled rice. Legal tolerances were not exceeded in any milling fraction. Reuse of the steeping water had little or no effect on residues.     

Properties of a major α-globulin of rice endosperm

Globulin was isolated from milled rice (Oryza sativa, line IR480-5-9) by 5% NACl extraction and was precipitated by (NH₄)₂SO₂ or by dialysis against water. The extract was purified by repeated isoelectric precipitation at pH 4.5. The major globulin fraction (40%) exhibited one band by electrophoresis at pH 4.5 but two bands at pH 8.3. Similarly, one sharp peak was shown by sedimentation corresponding to 1.41S (α-globulin) in acetic acid (pH 2) and NaOH (pH 11.7) but a broad asymmetric peak was obtained at pH 6.7, 8.3 and 8.9. Gel filtration of the α-globulin at pH 6.5 exhibited 2 proteins with MW 20 000 and 98 000. The results suggest a pH dependent aggregation phenomenon. The two proteins could not be separated by DEAE-cellulose chromatography. SDS-polyacrylamide electrophoresis of α-globulin revealed one subunit with MW 18 000. This α-globulin is poorer in lysine and histidine but richer in cystine, methionine, arginine, tyrosine and glutamic acid than whole milled rice proteinfa]Taken part from the M.S. thesis of AAP from the University of the Philippine at Los Baños (1977).     

Some Physico-Chemical Properties of Non-Waxy Paddy Rice Starch in Niigata Prefecture

Fifteen samples of milled rice had protein contents ranging from 5.53 to 7.83% and amylose contents ranging from 17.6 to 22.1%. Eating and cooking qualities of cooked rice gave gross palatability indicies ranging from 100 to 61, cohesiveness values by balance method from 64 to 9, and starch-iodine color test values of milled rice from 0.297 to 0.574. Pasting characteristics of a 8% suspension of rice starch gave maximum viscosity values ranging from 550 to 380 Brabender Unit (B.U.), temperature at maximum viscosity from 82° to 92.5°C, break down values from 260 to 20B.U., and set back values from ?0.5 to 60B.U. Good correlations were found between cooking and eating qualities of rice and rheological property of starch, and between rheological property and amylose content of starch, respectively.     

Accessing the reproducibility and specificity of pepsin and other aspartic proteases

The aspartic protease pepsin is less specific than other endoproteinases. Because aspartic proteases like pepsin are active at low pH, they are utilized in hydrogen deuterium exchange mass spectrometry (HDX MS) experiments for digestion under hydrogen exchange quench conditions. We investigated the reproducibility, both qualitatively and quantitatively, of online and offline pepsin digestion to understand the compliment of reproducible pepsin fragments that can be expected during a typical pepsin digestion. The collection of reproducible peptides was identified from > 30 replicate digestions of the same protein and it was found that the number of reproducible peptides produced during pepsin digestion becomes constant above 5–6 replicate digestions. We also investigated a new aspartic protease from the stomach of the rice field eel (Monopterus albus Zuiew) and compared digestion efficiency and specificity to porcine pepsin and aspergillopepsin. Unique cleavage specificity was found for rice field eel pepsin at arginine, asparagine, and glycine. Different peptides produced by the various proteases can enhance protein sequence coverage and improve the spatial resolution of HDX MS data. This article is part of a Special Issue entitled: Mass spectrometry in structural biology.     

FRACTIONATION OF PEPSIN : I. ISOLATION OF CRYSTALLINE PEPSIN OF CONSTANT ACTIVITY AND SOLUBILITY FROM PEPSINOGEN OR COMMERCIAL PEPSIN PREPARATIONS. II. PREPARATION OF A LESS SOLUBLE FRACTION. III. SOLUBILITY CURVES OF MIXTURES OF THE SOLUBLE AND INSOLUBLE FRACTIONS. IV. PREPARATION OF HIGHLY ACTIVE PEPSIN FROM PEPSINOGEN

1. Solubility curves of crude pepsin preparations indicate the presence of more than one protein. 2. One of these proteins has been isolated and crystallized and found to have constant activity and constant solubility in several solvents. 3. The solubility measurements are complicated by the unstable nature of the protein and the fact that in certain solvents the solubility of the protein is markedly affected by the presence of non-protein nitrogen decomposition products while in others this is not the case. 4. A more insoluble protein has been prepared of lower solubility and lower activity, as measured by hemoglobin digestion. The activity, as measured by the digestion of other proteins, is about the same as the more soluble fraction. This insoluble fraction does not have constant solubility. 5. Mixtures of the insoluble and the soluble fractions give preparations having rounded solubility curves typical of solid solutions and resembling very closely those of the original preparation. 6. A small amount of pepsinogen and pepsin from pepsinogen has been separated which has nearly twice the enzymatic activity on hemoglobin as does the most active pepsin previously isolated.     

Concentrated Protein Body Product Derived from Rice Endosperm as an Oral Tolerogen for Allergen-Specific Immunotherapy—A New Mucosal Vaccine Formulation against Japanese Cedar Pollen Allergy

The endoplasmic reticulum-derived type-I protein body (PB-I) from rice endosperm cells is an ideal candidate formulation for the oral delivery of bioencapsulated peptides as tolerogens for allergen-specific immunotherapy. In the present study, PBs containing the deconstructed Japanese cedar pollen allergens Cryptomeria japonica 1 (Cry j 1) and Cry j 2 were concentrated by treatment with thermostable α-amylase at 90°C to remove the starch from milled rice powder, which resulted in a 12.5-fold reduction of dry weight compared to the starting material. The modified Cry j 1 and Cry j 2 antigens in this concentrated PB product were more resistant to enzymatic digestion than those in the milled seed powder despite the absence of intact cell wall and starch, and remained stable for at least 10 months at room temperature without detectable loss or degradation. The high resistance of these allergens could be attributed to changes in protein physicochemical properties induced by the high temperature concentration process, as suggested by the decreased solubility of the antigens and seed proteins in PBs in step-wise-extraction experiments. Confocal microscopy showed that the morphology of antigen-containing PB-Is was preserved in the concentrated PB product. The concentrated PB product induced specific immune tolerance against Cry j 1 and Cry j 2 in mice when orally administered, supporting its potential use as a novel oral tolerogen formulation.     

THE PRESENCE OF A GELATIN-LIQUEFYING ENZYME IN CRUDE PEPSIN PREPARATIONS

A protein fraction has been isolated from crude pepsin preparations which is about 400 times as active as crystalline pepsin in the lique-faction of gelatin. The activity as measured by the digestion of casein, edestin or egg albumin is less than that of crystalline pepsin. It is more resistant to alkali than the crystalline pepsin.     

水稻米粒延伸性的遗传剖析

以籼稻ZYQ8与粳稻JX17为亲本的DH群体作为研究材料,考察DH群体及双亲的米粒延伸率相关性状,并使用该群体的分子连锁图谱进行QTL分析.共检测到14个与稻米延伸性有关的QTL,包括2个粒长QTL、7个饭粒长QTL和5个米粒延伸率QTL,分别位于第1、2、3、5、6、7、10、11和12染色体.所有QTL的LOD值介于2.26～9.25,分别解释性状变异的5.31%～17.21%.在第3染色体上的G249～G164、第6染色体上的G30～RZ516和第10染色体上的G1082～GA223区间同时检测到控制饭粒长和米粒延伸率的QTL.米粒延伸性受多基因控制,Wx基因与位于第6染色体上的qCRE-6的G30～RZ516区间相近,对米饭的延伸性具重要影响.     

Gastric pH Distribution and Mixing of Soft and Rigid Food Particles in the Stomach using a Dual-Marker Technique

Mixing of a particle-laden material during peristaltic flow in the stomach has not been quantified in vivo. Gastric mixing plays a key role in the digestion process; it determines the availability of acid and enzymes to individual food particles and controls the length of time particles will spend in the antral region, where they are subjected to mechanical breakdown from antral contraction waves. Solid particle mixing has been quantified using a dual-indigestible marker technique (TiO₂ and Cr₂O₃) in soft (cooked brown and white rice) and rigid (raw and roasted almonds) particle meals fed to growing pigs. All meals consisted of two portions. Each portion was separately marked by one of the two indigestible markers, and the portions were sequentially fed to the pigs. At time periods ranging from 20 to 720 min after completion of the meal, ten intragastric chyme samples were taken from each pig to determine the marker concentration and pH value. Gastric pH was not homogeneous throughout the stomach and varied over time, with differences observed between soft and rigid meals (p?<?0.0001). The total percentage of each meal that was mixed was calculated using a statistically-based mixing index (M). White rice had the greatest amount of mixing, becoming 94 % mixed after 480 min of digestion compared to 72 % mixing for brown rice. Rigid particles underwent a slower mixing process and only arrived at 65 and 71 % mixing after 720 min for raw and roasted almonds, respectively. Meal composition plays a role in the overall meal mixing during gastric digestion.     

Kinetics of whole serum and prepurified IgG digestion by pepsin for F(ab')2 manufacture

An alternative route for the production of polyclonal F(ab')(2) fragments that might be adopted for the facile preparation of antivenoms is assessed in this work. The method involves the digestion of whole serum by free pepsin, which results in reduction of the number of processing steps commonly in use, because it avoids the initial purification of IgG's prior to their proteolytic cleavage by the enzyme. Digestion kinetics of whole serum and caprylic acid prepurified IgG using free pepsin were monitored with SDS-PAGE followed by densitometric analysis and antigen binding activity assay of the digested samples. It was observed that with equal units of pepsin activity, caprylic acid prepurified IgG was digested more rapidly than whole serum but that the overall retention of antigen binding activity was significantly greater in the latter case. The estimated first-order digestion rate parameters were 11.8 and 4.42 microM min(-)(1) for pure IgG and whole serum, respectively. The K(m) value obtained for whole serum digestion was 33 microM and that for pure IgG digestion was 43.5 microM. Calibration with undigested whole serum and pure IgG samples of known concentrations was performed using SDS-PAGE followed by image analysis. A linear relationship was observed between the protein concentration and the respective band intensity within the range of concentrations investigated (0.63-31.2 microM IgG concentration). This technique proved to be relatively rapid, reproducible, and more precise than size-exclusion chromatography as a result of its F(ab')(2)/IgG resolving power. Staining and destaining protocols were reproduced in terms of staining and destaining times, volumes added, and compositions. Furthermore, all digestion experiments were performed in duplicate sets to monitor the extent of variation of the digestion kinetic parameters measured by this method. The results obtained from this technique confirm and quantify previous observations that pepsin digestion of whole serum is slower and easier to control than digestion of pure IgG and results in higher recovery of antigenic binding activity.     

Microbiological evaluation of processed rice consumed in Japan

Samples of processed rice from four different brands showed counts of mesophilic aerobic bacteria from 1 × 10² to 5 × 10³ for milled rice and from 1 × 10⁶ to 6.8 × 10⁶ cells g^–1 for brown rice. Rice seeds contaminated by milling had extensive counts including faecal coliforms. In cooked rice, no microbial growth was noted during 4 days at room temperature (spring season, 15–20 °C) and at least 2 weeks at 4 °C. No contamination was detected in cooked rice packs. A rapid and highly reliable procedure for detection of microorganisms in cooked rice is proposed.     

Isolation of collagen from the skins of Ehlers-Danlos syndrome-affected dogs by acetic acid extraction and pepsin digestion

Collagen was isolated by acetic acid extraction in the presence of protease inhibitors and also by pepsin digestion from the skins of dogs affected with the Ehlers-Danlos syndrome and the skins on non-affected dogs. The collagen preparations isolated by acetic acid extraction from the Ehlers-Danlos syndrome-affected dog skin contained a greater proportion of alpha-chains than the collagen preparations from the normal dog skin. When the collagen from the Ehlers-Danlos syndrome-affected dog skin was reduced with NaBH4 before heat denaturation, and electrophoresis, there was a greater proportion of beta-chains present. The collagen isolated from the normal dog skin was not affected by the NaBH4 reduction. Collagen preparations isolated by pepsin digestion from both the Ehlers-Danlos syndrome-affected dog skin and the non-affected dog skin contained the same quantity of alpha- and beta-chains. In addition, collagen from both affected and non-affected dog skins isolated by pepsin digestion contained 10-11% type III collagen as determined by the interrupted sodium dodecyl sulfate polyacrylamide gel electrophoresis method. Pepsin digestion of the collagens isolated by acetic acid extraction in the presence of protease inhibitors from the skins of affected and non-affected dogs eliminated the differences between the alpha:beta ratios of the affected and non-affected collagen preparations.     

Confocal laser scanning microscopy of dextran-rice starch mixtures

The microstructure of rice starch and dextran-rice starch mixtures was studied using confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). Surface pores and channels of rice starch were looked for. Channels could not be found in rice starch granules after reaction with 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA). Fluorescein-labeled dextran was mixed with rice starch in order to locate hydrocolloid molecules in hydrocolloid-rice starch mixtures. The results showed that FITC-dextran (ave. Mw 4000; FD4) penetrated into raw rice starch granules, with the degree of penetration varying from granule to granule. FD4 could penetrate throughout cooked rice starch granules. FITC-dextran with an average Mw of more than 10,000 could not penetrate either raw or cooked rice starch granules.     

Chemical Characteristics of Grains of Rice (Oryza sativa L.) Cultivated in Saline Media of Varying Ionic Composition

Two varieties of rice (Oryza sativa L.), Pokkali (salt-tolerant)and IR5929-12-3 (salt-sensitive), were grown in media of varyingionic composition namely chloride-sulphate (with sulphate dominant),sulphate-chloride (with chloride dominant) and chloride. Eachsalinity type had four different levels (expressed as electricalconductance) 2, 4, 6 and 8 dS m^–1. Amylose content ofthe endosperm, gelatinization temperature of the endosperm starch,brown rice protein content and the Na content of the hull andmilled rice showed that different salinity types affect thesecharacteristics differently. The greater Na accumulation inthe hull than in the milled rice in both varieties is presumedto be an adaptive response to salinity. Key words: Salinity, ionic compositions, rice grain.     

Control of foreign polypeptide localization in specific layers of protein body type I in rice seed

Key message

Prolamin–GFP fusion proteins, expressed under the control of native prolamin promoters, were localized in specific layers of PB-Is. Prolamin–GFP fusion proteins were gradually digested from outside by pepsin digestion.

Abstract

In rice seed endosperm, protein body type I (PB-I) has a layered structure consisting of prolamin species and is the resistant to digestive juices in the intestinal tract. We propose the utilization of PB-Is as an oral vaccine carrier to induce mucosal immune response effectively. If vaccine antigens are localized in a specific layer within PB-Is, they could be protected from gastric juice and be delivered intact to the small intestine. We observed the localization of GFP fluorescence in transgenic rice endosperm expressing prolamin–GFP fusion proteins with native prolamin promoters, and we confirmed that the foreign proteins were located in specific layers of PB-Is artificially. Each prolamin–GFP fusion protein was localized in specific layers of PB-Is, such as the outer-most layer, middle layer, and core region. Furthermore, to investigate the resistance of prolamin–GFP fusion proteins against pepsin digestion, we performed in vitro pepsin treatment. Prolamin–GFP fusion proteins were gradually digested from the peripheral region and the contours of PB-Is were made rough by in vitro pepsin treatment. These findings suggested that prolamin–GFP fusion proteins accumulating specific layers of PB-Is were gradually digested and exposed from the outside by pepsin digestion.

     

Effect of pepsin on partially purified pig gastric mucus and purified mucin

Partially purified native-pig gastric mucus and purified pig gastric mucin, prepared by column chromatography and caesium chloride (CsCl) density-gradient ultracentrifugation, were subjected to pepsin digestion. The products of peptic digestion were chromatographed on Sepharose CL-2B, and fractions were assayed for carbohydrate by the periodic acid-Schiff reaction. The polymeric gastric mucin in the purified mucin samples was readily degraded by pepsin. In sharp contrast, the polymeric mucin in the partially purified mucus was relatively resistant to pepsin digestion. In 45 min, pepsin degraded 40% of the polymeric mucin in the purified samples, whereas it produced no significant degradation (less than 10%) in the partially purified mucus samples. In partially purified gastric mucus, treated with CsCl but not fractionated by ultracentrifugation, digestion with pepsin was also slow and incomplete. This showed that differences in susceptibility between partially purified and purified preparations are not due to the chaotropic effects of CsCl. In addition, the recombination of low-density nonmucin fractions in CsCl ultracentrifugation with the mucin also resisted pepsin digestion. Finally, we have shown that the low-density fractions in mucus exhibited a strong inhibitory effect of peptic activity in vitro. We conclude that under our experimental conditions, pepsin has little effect on partially purified mucus, and our findings indicate an inhibitor of peptic digestion is present in native gastric mucus. It is likely, but unproven, that this inhibitor is a noncovalently bound lipid present in the low-density fraction.     

Composition of cell wall preparations of rice bran and germ

Cell walls of petrol-defatted non-waxy IR32 rice bran and germ were prepared by protein removal with 0.5% SDS—0.6% β-mercaptoethanol, heating the residue to 80°, and destarching with Bacillus licheniformis α-amylase. A waxy rice, IR29, had a similar cell wall composition as IR32. Principal wall sugars were arabinose, xylose, and glucose. The 0.5 M sodium or potassium hydroxide and 8 M urea preferentially extracted arabinose-, xylose- and uronic acid-rich polysaccharides but 6 M sodium hydroxide—0.81 M boric acid extracted mannose-rich polysaccharides. DEAE-cellulose BO₃^3? chromatography of the 0.5 M sodium hydroxide extracts gave fractions of similar arabinose— xylose ratios. Proteins in the cell wall preparations had only 0.4–1.6% hydroxyproline, and were bound mainly to polysaccharides, based on disc gel electrophoresis. The preparations were autofluorescent in UV and rich in phenols, mainly ferulic acid. The cell wall preparations and their 8 M urea fractions had a softening effect on defatted waxy starch aqueous gel at 0.2–2% of the starch.