Aggregation of peptides during hydrolysis as a cause of reduced enzymatic extractability of soybean meal proteins

With the purpose of analyzing the size and composition of enzyme-unextractable proteins in differently heat-treated soybean meals, a selection of extractants was screened for their ability to extract these proteins from enzyme-unextractable residues. The largest effects were obtained with urea, urea plus beta-mercaptoethanol, and dilute alkali; the latter extracted up to 87% of the enzyme-unextractable protein. Gel permeation chromatography indicated that a large proportion of the extracted material was of high molecular weight. However, the combined results from gel electrophoresis, LC-MS, and MALDI-ToF MS showed that the extracted protein material was composed of aggregated peptides. The largest aggregates were observed in the enzymatic residues originating from meals heat-treated at high humidity. Extracted aggregates were fully degraded upon subsequent proteolytic treatment.     

Lunasin and Bowman-Birk protease inhibitor concentrations of protein extracts from enzyme-assisted aqueous extraction of soybeans

Lunasin and Bowman-Birk protease inhibitor (BBI) are two soybean peptides to which health-promoting properties have been attributed. Concentrations of these peptides were determined in skim fractions produced by enzyme-assisted aqueous extraction processing (EAEP) of extruded full-fat soybean flakes (an alternative to extracting oil from soybeans with hexane) and compared with similar extracts from hexane-defatted soybean meal. Oil and protein were extracted by using countercurrent two-stage EAEP of soybeans at 1:6 solids-to-liquid ratio, 50 °C, pH 9.0, and 120 rpm for 1 h. Protein-rich skim fractions were produced from extruded full-fat soybean flakes using different enzyme strategies in EAEP: 0.5% protease (wt/g extruded flakes) used in both extraction stages; 0.5% protease used only in the second extraction stage; no enzyme used in either extraction stage. Countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes was used as a control. Protein extraction yields increased from 66% to 89-96% when using countercurrent two-stage EAEP with extruded full-fat flakes compared to 85% when using countercurrent two-stage protein extraction of air-desolventized, hexane-defatted soybean flakes. Extruding full-fat soybean flakes reduced BBI activity. Enzymatic hydrolysis reduced BBI contents of EAEP skims. Lunasin, however, was more resistant to both enzymatic hydrolysis and heat denaturation. Although using enzymes in both EAEP extraction stages yielded the highest protein and oil extractions, reducing enzyme use to only the second stage preserved much of the BBI and Lunasin.     

Physicochemical Properties of Rice Endosperm Proteins Extracted by Chemical and Enzymatic Methods

Rice proteins are nutritional, hypoallergenic, and healthy for human consumption. Efficient extraction with approved food‐grade enzymes and chemicals are essential for commercial production and application of rice protein as a functional ingredient. Rice endosperm proteins were isolated by alkali, salt, and enzymatic methods and evaluated for extractability and physicochemical properties. Alkali (RP_A) and salt (RP_S) methods extracted 86.9 and 87.3% of proteins with 65.9 and 58.9% yield, respectively. The enzymatic methods with Termamyl (RP_ET) and amylase S (RP_EA) extracted 85.8 and 81.0% proteins with 85.2 and 86.2% yield, respectively. Enthalpy values of RP_A (1.79 J/g), RP_S (1.22 J/g), RP_ET (nondetectable), and RP_EA (0.17 J/g), determined by differential scanning calorimetry, demonstrated that the varying level of denaturation of proteins depends on the method of extraction. Surface hydrophobicity data supported this observation. Alkali‐ and salt‐extracted proteins had higher solubility and emulsifying properties than those of enzyme‐extracted proteins. Comparatively, more favorable protein composition, lower surface hydrophobicity, higher solubility, and a lower degree of thermal denaturation of alkali‐ and salt‐extracted proteins contributed to higher emulsifying and foaming properties than those of enzyme‐extracted proteins; therefore, alkali‐ and salt‐extracted proteins can have enhanced functional use and a potential starting material for preparing tailored rice protein isolates.     

Composition of proteins in okara as a byproduct in hydrothermal processing of soy milk

Protein quality, based on its subunit composition, in okara obtained as a byproduct during hydrothermal cooking of soy milk was assessed. The composition of 7S and 11S protein fractions was correlated with the physicochemical properties of protein in okara produced from six soybean varieties. The basic 7S globulin (Bg7S) and 11S protein were two main proteins in okara. Investigated soybean genotypes produced okara with mainly acidic A(5) and basic B(1,2,4) polypeptides of 11S proteins. Soybean 11S content was not an indicator of okara protein recovery or extractability. Of all tested relationships, extractable soluble protein content of okara was influenced only by soybean Bg7S (r = 0.86; p < 0.05) and its light subunit contents (r = 0.93; p < 0.05). Okara protein recovery depended on Bg7S heavy subunit content in soybeans (r = 0.81; p < 0.05). The high quantity of vegetable protein in okara (around 35%) and very high protein extractability (around 85%) qualify this byproduct for potential application in food preparation as a functional ingredient.     

Protein extraction from heat-stabilized defatted rice bran. 1. Physical processing and enzyme treatments

Physical processing with or without enzyme treatments on protein extraction from heat-stabilized defatted rice bran (HDRB) was evaluated. Freeze-thaw, sonication, high-speed blending, and high-pressure methods extracted 12%, 15%, 16%, and 11% protein, respectively. Sonication (0-100%, 750 W), followed by amylase and combined amylase and protease treatments, extracted 25.6-33.9% and 54.0-57.8% protein, respectively. Blending followed by amylase and protease treatment extracted 5.0% more protein than the nonblended enzymatic treatments. High-pressure treatments, 0-800 MPa, with water or amylase-protease combinations, extracted 10.5-11.1% or 61.8-66.6% protein, respectively. These results suggest that physical processing in combination with enzyme treatments can be effective in extracting protein from HDRB.     

Wheat gluten phenolic acids: occurrence and fate upon mixing

Ferulic acid (FA, 4.9-17.7 microg/100 mg), sinapic acid (SA, 1.4-3.5 microg/100 mg), and traces of p-coumaric acid and vanillic acid were detected after saponification of six wheat glutens from industrial and pilot-scale origins. FA and SA occurred mostly as soluble-bound and insoluble-bound forms according to their extractability by acetone/methanol/water (7:7:6, v/v/v). The major part of FA (50-95%) was found in the unextractable fraction, whereas SA was mostly extractable (64-85%). The carbohydrate contents of the glutens were determined also after acid hydrolysis. The highest levels of glucose, arabinoxylan, and FA were obtained from the unextractable fractions of the pilot-scale extracted glutens, probably in relation with a lower efficiency of washing during extraction compared to industrial processes. On the other hand, SA compounds were in similar concentrations in all samples, suggesting their involvement in specific interactions during gluten protein agglomeration. Saponification of the soluble-bound phenolic acids released mainly glucose, whereas a beta-glucosidase treatment had no effect. FA and SA extractability, especially that of soluble-bound ones, decreased strongly in overmixed gluten/water doughs. These low molecular weight conjugates of phenolic acids could be involved in the dough breakdown phenomenon.     

Stepwise extraction of proteins and carbohydrates from soybean seed

The stepwise hot water extraction of soybeans, which were extractions in a series of procedures of whole soybean seeds, dehulled and sliced ones, and pressed ones carried out by autoclaving, was investigated to study the localization in the seed and their characteristics. The characteristics of each extraction were studied by HPLC, SDS-PAGE, components analysis, microscopic observation, and effect for some enzymes. Carbohydrates were easier to extract than protein. In the extractions, the ratio of uronic acid per total sugar was constantly about 0.3. A comparison of these extracts, soybean milk, extraction from defatted soybean meal, and soybean milk residues was also carried out, and the characteristics and the localization were investigated. Mid-sized proteins in soybean milk were easy to extract. However, hardly any high molecular weight proteins or high molecular weight carbohydrates were extracted. The proteins and carbohydrates were considered to be localized in the middle lamella and in the protein and/or oil bodies of the cell, and the proteins and carbohydrates were gradually extracted through seed and cell breaking. Gelation was observed only in the boiled extracts from whole seeds. Pepsin and trypsin digests of the high molecular weight protein had inhibitory activity against the angiotensin I converting enzyme.     

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.     

亚麻籽分离蛋白结构表征及其性能分析

为实现亚麻籽饼粕中分离蛋白的综合加工利用,该文以冷榨亚麻籽饼粕为原料,对其进行脱胶脱脂处理,采用超声辅助水提法分别提取亚麻籽分离蛋白和脱胶脱脂亚麻籽分离蛋白,并对理化性质、结构及功能特性进行分析比较。结果表明,冷榨亚麻籽饼粕和脱胶脱脂亚麻籽饼粕中蛋白质质量分数分别为37.52%±0.04%、37.47%±0.02%。采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定亚麻籽分离蛋白和脱胶脱脂亚麻籽分离蛋白的分子质量,显示10～55 kDa之间有明显谱带,其中水溶性低分子质量的白蛋白（10、14、15及17 kDa）和盐溶性高分子质量的球蛋白（30、33、35、40、45及55 kDa）谱带最为明显。氨基酸的种类各检测到17种,含有丰富的必需氨基酸和非必需氨基酸。傅里叶变换红外光谱测定的2种蛋白质的二级结构稳定性一般;由扫描电镜和X-射线衍射可知亚麻籽分离蛋白比脱胶脱脂亚麻籽分离蛋白的微观孔隙率低,结构中都较缺乏结晶度或有序排列。2种蛋白的两亲性与大豆分离蛋白相比,亲水/油特性突出,亲油性是大豆分离蛋白的2倍多。通过对不同pH（2～11）和盐离子浓度（0～1.25 mol/L）下溶解度、起泡性、泡沫稳定性、乳化活性及乳化稳定性的测定,显示两者具有良好的碱溶性,脱胶脱脂亚麻籽分离蛋白的起泡性、乳化活性及乳化稳定性均优于亚麻籽分离蛋白,而泡沫稳定性恰好相反,该研究结果有利于拓宽2种分离蛋白在健康食品领域中的应用前景,为食品中的应用提供有益参考和数据支撑。     

Evaluation of the Functionality of Five Different Soybean Proteins in Hot‐Press Wheat Flour Tortillas

Five different soybean protein sources were added to wheat flour to increase the protein content by 15–25%, and the resulting composite flours were optimally processed into hot‐press tortillas in a pilot plant. The rheological properties of composite flours were evaluated with the farinograph, alveograph, and other wheat quality tests. Tortilla‐making qualities of the control and soybean‐fortified flours were evaluated during dough handling, hot pressing, and baking. The resulting tortillas were tested in terms of yield, physical and chemical parameters, sensory properties, color, and objective and subjective texture. The soybean‐fortified tortillas had increased yields because of the higher dough water absorption and enhanced essential amino acid scores. Among the five different soybean proteins, the defatted soybean flour (SBM1) with the lowest fat absorption index and protein dispersibility index (PDI) and the soybean concentrate produced the best fortified tortillas. The protein meals with high PDI and relatively lower water absorption index (SBM3 and SBM4) produced sticky doughs, lower alveograph P/L values, and defective tortillas. All soybean proteins produced higher yields of tortillas with an enhanced protein quality and amount of dietary fiber.     

Cellulase and protease preparations can extract pectins from various plant byproducts

The use of protease and cellulase preparations to extract pectins from plant byproducts (chicory, cauliflower) was investigated. Different enzymatic preparations were characterized by their activities toward proteins, cellulose, and pectins. These preparations were then tested regarding pectin extraction, and extraction conditions (nature and concentration of enzyme, incubation time) were optimized. Enzymatic and acidic extractions were compared and also combined in sequential extractions. This study shows that it is possible to extract pectins by using cellulases and proteases. Enzymes can extract pectins with a higher yield ( approximately 35%) than acid (approximately 28%) but enzyme-extracted pectins have a smaller molar mass (300,000 g/mol) than acid-extracted pectins (500,000 g/ mol). Different hypotheses are tested and discussed to explain this mass difference.     

大豆蛋白限制性酶解模式与产品胶凝性的相关性

为了改善大豆蛋白的胶凝性,对大豆浓缩蛋白、大豆分离蛋白进行限制性酶解处理,并考察相应产品的蛋白酶酶解模式与胶凝性变化的相关性。该研究以蛋白质的水解度为指标,通过中性蛋白酶、胰蛋白酶的酶解作用,水解大豆浓缩蛋白、大豆分离蛋白至蛋白质水解度(DH)为1%、2%,考察酶性质、蛋白质的DH对产品胶凝性影响,并利用SDS-凝胶电泳进行确认。结果表明:大豆浓缩蛋白经中性蛋白酶、胰蛋白酶的酶解后,产品胶凝性均显著下降;大豆分离蛋白经中性蛋白酶的酶解后,产品胶凝性在DH为1%时增加,但在DH为2%时下降;大豆分离蛋白经胰蛋白酶酶解后,胶凝性显著改善。SDS-凝胶电泳确认,蛋白质的酶水解模式和水解度不同是导致产品胶凝性产生不同变化的原因。     

Preparation of Rice Endosperm Protein Isolate by Alkali Extraction

Rice endosperm extraction conditions were optimized by response surface methodology. The optimum alkali extraction conditions were pH 11.0 at 40°C for 3 hr with 8:1 solvent‐to‐solid ratio. The maximum protein yield was 43.1% at these conditions. As the extraction pH was increased from 9.0 to 12.0, protein extractability and content increased but the solubility and emulsifying properties of the extracted protein decreased. The extracted protein was recovered by either isoelectric precipitation (IEP) or ultrafiltration (UF). Ultrafiltering the supernatant with a 5‐kDa hollow fiber membrane concentrated the protein from 1.8 to 16% (dry basis) and the resulting solution was spray‐dried to produce a protein concentrate (RP_UF) with 71% protein. Although RP_IEP contained higher protein (86%) than RP_UF (71%), RP_UF showed higher solubility and emulsifying properties. The solubility of RP_UF was higher (37%) than RP_IEP (15%). RP_UF also demonstrated higher emulsion activity (0.414) and stability (22.4 min) compared with the emulsion activity (0.282) and stability (15.5 min) of RP_IEP. Higher solubility and the soluble nonprotein components of RP_UF contributed to higher emulsifying properties than RP_IEP. The UF provided milder extraction conditions with improved emulsifying properties than conventional IEP.     

Extraction and Film Properties of Kafirin from Coarse Sorghum and Sorghum DDGS by Percolation

The high cost of kafirin and zein restricts their use for bioplastic and food applications. Effective, simple, and rapid kafirin/zein isolation processes are required. Here a percolation‐type aqueous ethanol solvent extraction process from coarse meals (grits) and coarse sorghum distillers dried grains and solubles (DDGS) for kafirin and zein isolation employing a low ratio of extractant to meal (2.5:1) was investigated, which is potentially applicable in the grain bioethanol industry. Postextraction filtration times were more than twice as fast using coarse meals compared with fine flours. Washing the meals prior to extraction to remove starch improved protein preparation purity to 73–85% compared with 68–72% for unwashed meals. Hence, no subsequent filtration or centrifugation step is required to clean up the kafirin/zein solution prior to solvent evaporation. With a single extraction step, kafirin/zein yields were 48% (protein basis) for DDGS and 53–70% for washed sorghum/maize meals. Cast films were used as a model bioplastic system to evaluate extracted kafirin/zein functional properties. DDGS kafirin films had rough surfaces but had the lowest water uptake and in vitro digestibility, owing to heat‐induced disulfide crosslinking during DDGS processing. Extraction by percolation using coarse meal/DDGS has potential to improve kafirin/zein viability.     

Isolation and enzymatic digestion of body complex of soybean seed

The body complex of the soybean seed (BCSS) was isolated from the single cells (27.2%) by a sequential procedure of autoclaving with water, cellulase digestion for the primary cell wall, pectinase digestion for the secondary cell wall, and defatting with hexane washing. Its characteristics were then investigated. The defatted BCSS (DBCSS) consisted of protein (76.5%) and mannose-rich carbohydrates (3.2%). Screening of the food-processing protease for the digestion of DBCSS was carried out, and a kind of alkaline protease was selected. The inner protein of DBCSS was easily extracted with 0.1 M sodium carbonate buffer, pH 10, and the insoluble shell of the body complex (SDBCSS) was left. SDBCSS consisted of hydrophobic amino acid-rich protein. SDBCSS was easily digested by the selected alkaline protease. SDBCSS was dissolved by boiling with sodium dodecyl sulfate-mercaptoethanol, and it was found to consist of a protein of approximately 3 kDa. The high enzymatic digestion including the selected protease for soybean seed and defatted soybean meal was carried out; both were extracted and digested with a yield of >99.5%. The final indigestible residue was found as paired hexagonal and filamentous organs of the soybean cells.     

Extraction of green labeled pectins and pectic oligosaccharides from plant byproducts

Green labeled pectins were extracted by an environmentally friendly way using proteases and cellulases being able to act on proteins and cellulose present in cell walls. Pectins were isolated from different plant byproducts, i.e., chicory roots, citrus peel, cauliflower florets and leaves, endive, and sugar beet pulps. Enzymatic extraction was performed at 50 degrees C for 4 h, in order to fulfill the conditions required for microbiological safety of extracted products. High methoxy (HM) pectins of high molar mass were extracted with three different enzyme mixtures. These pectins were subsequently demethylated with two pectin methyl esterases (PMEs), either the fungal PME from Aspergillus aculeatus or the orange PME. It was further demonstrated that high molar mass low methoxy (LM) pectins could also be extracted directly from cell walls by adding the fungal PME to the mixture of protease and cellulase. Moreover, health benefit pectic oligosaccharides, the so-called modified hairy regions, were obtained after enzymatic treatment of the residue recovered after pectin extraction. The enzymatic method demonstrates that it is possible to convert vegetable byproducts into high-added value compounds, such as pectins and pectic oligosaccharides, and thus considerably reduce the amount of these residues generated by food industries.     

Effects of moist storage and different drying temperatures on the extractability of iron,copper, manganese,and zinc in soil samples

Abstract

The objective of this study was to investigate the effect of different pretreatments on the extraction of cationic micronutrients [iron (Fe), copper (Cu), manganese (Mn), and zinc (Zn)] from four different soils. Samples were either stored in the field‐moist state for seven days before drying or dried immediately. Three drying treatments were used: air‐dried (72 hours), oven‐dried at 40°C (48 hours), or oven‐dried at 105°C (24 hours). Micronutrients were then extracted with 0.1N HC1 or diethenetriamine‐pentacetic acid (DTPA). Storage, drying temperature, extractant, and soil type all influenced micronutrient extractability. In general, a higher temperature increased the level of micronutrient extracted. However, the reverse effect was also observed. The effect of storage was variable and probably depended on the sample moisture content. We conclude that the results from routine analysis and experimentally determined indices can only be compared if soil samples are subjected to the same pretreatments. Hence, rigorous standardization of the sample preparations is imperative for accurate determination of plant‐available micronutrients.     

Soyasaponins resist extrusion cooking and are not degraded during gut passage in Atlantic salmon (Salmo salar L.)

The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.     

Growth rate of bacteria is affected by soil texture and extraction procedure

Effects of soil texture on the extraction efficiency of bacteria from soils and on biosynthetic activity of the extracted bacteria were studied. Bacterial extracts were prepared from three soils of different texture by homogenization (ultrasonication and mixing) or by homogenization-centrifugation at different speeds. Bacterial biosynthetic activity was estimated using thymidine and leucine incorporation techniques. In each step of the extraction procedure, a higher extractability of bacteria was obtained in finer soils than in coarse soil. Also cell-specific growth rates of bacteria were higher in the finer soils than in the coarse soil. However, in all soils, the extracted bacteria always had significantly lower cell-specific thymidine and leucine incorporation rates than the bacteria in soil slurries and thus did not represent so well the bacterial growth in the original soils. The total declines in cell-specific incorporation rates caused by the extraction were larger in fine soil (96-98%) than in coarse soil (90%), but bacteria in the coarse soil were more responsive to only minor intervention. The homogenization-centrifugation method eliminated the differences in bacterial biosynthesis found when working with soil slurries. Therefore, we recommend using of soil slurries or, optionally, soil suspensions to compare bacterial biosynthetic activity among soils of different textures.     

Protein Extraction from Triticale Distillers Grains

Triticale is being actively explored as a feedstock for bioethanol production in Western Canada. Triticale distillers grains, an important coproduct of the bioethanol industry, are used mainly as animal feed. This study aims to develop methods of protein extraction from triticale distillers wet grains and distillers dried grains with solubles. Osborne fractionation showed low protein extractability because excessive protein denaturation occurred during sample preparation. Five methods were used to extract proteins: pH shifting, 60% ethanol, alkaline‐ethanol solution, glacial acetic acid, and enzyme‐aided extraction. Extracts obtained with the alkaline‐ethanol and glacial acetic acid methods showed comparatively higher protein contents (≈61–65%) when compared with the other extraction methods (≈35–57%). Enzyme‐aided extraction with Protex 6L yielded 75–82% protein at a content of 43–57%, depending on the types of raw materials. Establishing methods of protein extraction from triticale distillers grains would facilitate further studies on new uses of triticale proteins.