Characterization of Oat Endoproteinases that Hydrolyze Oat Globulins

During the germination of oats, the major seed storage proteins (globulins) are hydrolyzed by endoproteinases. We have used two methods to characterize these endoproteinases. A qualitative PAGE method that used oat globulins as gel‐incorporated substrates was used to determine which enzymes hydrolyzed the globulins. The proteolytic hydrolysis products were studied by hydrolyzing the globulins in vitro with the endoproteinases and analyzing the products by SDS‐PAGE. Class‐specific proteinase inhibitors were used to show that the globulin hydrolyzing enzymes were cysteine‐class proteinases. The proteinases were active at pH 3.8. Using the gel analysis method, a little activity was present at the beginning of seed germination, but the major activity only appeared on the sixth day of germination. Extracts from four‐day germinated oats contained cysteine proteinases that hydrolyzed the globulins in vitro to form a polypeptide of intermediate size (MW ≈34,500). Cysteine proteases from an eight‐day germinated sample totally hydrolyzed the globulins in <1 hr. Very little hydrolysis occurred at pH 6.2, the pH of germinated oats endosperm tissue. The fact that hydrolysis occurred quickly at pH 3.8 implies that there is probably pH compartmentalization within the endosperm, with some areas of the seed having a low pH value where the globulins can be degraded.     

Polymorphism of Aspartic Proteinases in Resting and Germinating Barley Seeds

Both resting and germinated barley seeds (Hordeum vulgare L. ‘Morex’) contain aspartic endopeptidase activities, and the activities increase during germination. We have extracted and partially purified aspartic endopeptidases from both resting seeds and green malt (four-day germinated barley). Six aspartic proteinase activities were found in resting barley seeds while only four activities were detected in green malt. All of the aspartic proteinases had similar pH activity optima (pH 3.5–4.5) and pI values (≈4.5). The purified green malt aspartic proteinases selectively digested a group of barley seed proteins, postulated to serve as defensive proteins, that are coded by the amylase-trypsin inhibitor super gene family. The aspartic proteinases that bound to a pepstatin A affinity column at pH 4.5 cross-reacted with antiserum raised against aspartic proteinases purified from barley seed. However, those that did not bind the affinity column also did not cross-react with the antiserum, indicating that there are two distinct groups of aspartic proteases in germinating barley.     

Qualitative Zymographic Studies of Major Proteinases from Malted Sorghum (Sorghum bicolor L.) and Effects of Cultivar

Proteolysis during cereal germination is vital both to seedling growth and the success of commercial malting and brewing. In this study, proteinases in proteolytic extracts from seeds and germinated grains of 11 Botswana sorghum cultivars were analyzed and partially characterized by one‐dimensional electrophoresis on SDS‐PAGE gels containing incorporated gelatin. Proteinase polymorphism was detected in both germinated and ungerminated sorghum grains. Fifteen distinct proteinase bands, with M_r values of 27,000–100,000 were detected in sorghum malt extract, while ungerminated sorghum displayed a maximum of four bands (M_r ≈ 78,000–100,000). Band numbers and identity varied markedly according to cultivar. More proteinase bands were detected at pH 4.6, than at pH 6.2 and 7.0, suggesting pH optima considerably below neutrality. Cysteine‐proteinases constituted a higher proportion (9 of 15) of the detected sorghum malt proteinases and were most detectable at pH 4.6. Multiple representatives were also detected for both serine‐ and metallo‐proteinases, although these were more active at pH 6.2 and 7.0. 1‐10 Phenanthroline inhibited malt metallo‐proteinase more strongly than EDTA, suggesting that these enzymes were most probably zinc‐dependent. Aspartyl‐proteinases were not detected, probably because of the substrate employed. Results indicate that the sorghum proteinase system is complex.     

Differences Between Sensory Profiles and Development of Rancidity During Long‐Term Storage of Native and Processed Oat

Changes in the sensory attributes, lipid composition, and amounts of volatile and phenolic compounds of native and processed (germinated and dried) crushed oat were followed during a 12‐month storage period. The influence of the chemical attributes on the sensory profiles of oats was analyzed by statistical multivariate techniques (PLS regression). During the storage period, significant changes in the sensory profiles of the native and processed oat groats were observed. The stability of oat groats was significantly increased through germination and subsequent drying because the chemical changes causing rancidity and bitterness developed more slowly in the processed oat when compared with the native oat. In native oat, the most intensive changes due to deteriorat ion had already occurred after one month of storage, whereas in processed oat, these changes were perceived considerably later. Stored oat that had deteriorated was evaluated as being musty and earthy in odor and bitter and rancid in flavor. The accumulation of free fatty acids and volatile compounds related to lipid oxidation were closely correlated with the development of the undesired sensory attributes described above. The total amount of phenolic compounds, as well as the volatile aromatic and branched chain compounds derived mainly from protein degradation, showed a significant relationship with favorable sensory attributes such as roasted odor and flavor. Lipid oxidation occurred during the storage and was observed both in the polar and in the nonpolar lipid classes of native oat, whereas in the processed oat, these changes were nonsignificant. Photo‐oxidation of acylated fatty acids may significantly contribute to the development of volatile lipid oxidation products during storage.     

Proteolytic Enzymes in Germinating Rye Grains

The proteolytic activities during rye (Secale cereale L. ‘Humbolt’) grain germination were monitored using in‐solution methods and one‐ and two‐dimensional PAGE with gels that contained incorporated substrate proteins. The total proteolytic activity increased during the first three days of germination, but not after that. The proteinase activity was measured at pH 3.8, 6.0, and 8.0 in the presence and absence of class‐specific proteinase inhibitors. This indicated that enzymes from all four proteinase classes were present during the germination process. Germinated rye grain contained mainly aspartic and cysteine proteinase activities that are especially active at pH 3.8. Serine‐ and metallo‐proteinases were less abundant. Overall, the pattern of hydrolysis was very similar to that observed during barley and wheat germination.     

Development of a Germination Process for Producing High β‐Glucan,Whole Grain Food Ingredients from Oat

Germination can be used to improve the texture and flavor of cereals. However, germination generally causes breakdown of β‐glucans, which is undesirable with respect to the functional properties of β‐glucan. Our aim was to assess possibilities of germinating oat without substantial loss of high molecular weight β‐glucan. Two cultivars, hulled Veli and hull‐less (naked) Lisbeth were germinated at 5, 15, and 25°C and dried by lyophilization or oven drying. Elevated germination temperatures led to an increase in Fusarium, aerobic heterotrophic bacteria, Pseudomonas spp., lactic acid bacteria, enterobacteria, and aerobic spore‐forming bacteria. Therefore, the germination temperature should be kept low to avoid excessive growth of microbes. Of the samples germinated at 15°C, only one contained low amounts of the Fusarium toxin deoxynivalenol (52 μg/kg). Germination led to the breakdown of β‐glucans, but the decrease in the molecular weight of β‐glucan was initially very slow. A short germination schedule (72 hr, 15°C) terminated with oven drying was developed to produce germinated oat with retained β‐glucan content. Compared with the native oat, 55–60% of the β‐glucan could be retained.     

Comparison of Endoproteinases of Various Grains

Two‐dimensional isoelectric focusing (IEF) × PAGE gels were used to compare the endoproteolytic (gelatinase) activities of germinated barley with those of bread and durum wheat, rye, triticale, oat, rice, buckwheat, and sorghum. Barley was used as the standard of comparison because its endoproteinase complement has been studied previously in the greatest detail. The characteristics of the grain proteases were appraised from their migration patterns and by how they were affected by pH levels. All of the germinated grains contained multiple enzyme activities and their separation patterns and pH levels were at least similar to those of barley. The proteinases of the bread and durum wheats, rye, oat, and sorghum were most similar to those of barley, whereas the other grains provided more varied patterns. The rice and buckwheat proteinases developed much more slowly than those of the other grains. The activity patterns of the triticale resembled those of the parents, wheat and rye, but the triticale contained many more activities and higher overall proteolytic activities than any of the other species. These results should be applied to scientific or commercial procedures with caution because grains contain potent endogenous proteinase inhibitors that could inactivate some of these enzymes in various tissues or germination stages.     

小芸木种子生理特性及萌发影响因素的初步研究

对分布于滇南热带雨林的小芸木种子的萌发生理进行初步研究 ,结果表明 :小芸木种子是一种顽拗性种子 ,含水量为 4 8%～ 50 %的种子在 2 0、 2 5和 30℃发芽率达 90 %以上 ;含水量在 4 5%以下时 ,发芽率在 63%以下 ;种子含水量降低至 2 5.78% ,胚含水量降至 1 3.4 4%时 ,均完全失去发芽力。种子有休眠特性 ,轻度脱水、流水冲洗可以打破休眠。种子在 2 0、2 5、30℃恒温条件下 ,发芽率较高且较快 ,而在 1 2、4 0℃和室温 (白天 2 4～ 35℃ ,夜间 1 6～ 2 4℃ )时发芽率低且较慢。随着含水量的降低 ,种子萌发时对温度和光照的敏感性升高 ,低温对种子萌发的抑制作用加强 ;用 0 .0 1 %的高锰酸钾溶液处理含水量为 4 5.0 4 %的种子 ,在 2 0、 2 5、 30℃种子萌发率可提高 1 5%～ 60 %     

Protein metabolism in rice seedling

The relationship between protein synthesis and degradation in germinating rice seed were studied with protein synthetic inhibitors. Both DNP and 8-AG inhibited the degradation of glutelin, the major storage protein in rice seed, while the inhibitors had no direct effect in the activity of rice seed proteinllse in vitro. The prevention can be partly ascribed to the Inhibition of proteinase synthesis because the inhibitors depressed the increase in proteinase activity during germination. When DNP treatment was started at the onset of germination, the degradation of glutelin in the endosperm was seriously inhibited and the endosperm remained rigid over 9 days of incubation at 30°C. In contrast, the inhibition was less efficient clent when the treatment was started in the later stage. It is suggested that the degradation of the storage protein in rice seed depends on the synthetic process of the hydrolytic enzymes which increase during germination. disintegrate the compartmentation of the endosperm and allow the storage proteins to come in contact with the existing proteinases     

Effect of aluminium and calcium on growth of wheat seedlings and germination of seeds

Two separate experiments were conducted to investigate the aluminium (Al) and calcium (Ca) effects on wheat seedling growth and on seed germination. Wheat (Tritcum aestivum L, cs Yangmai No. 5) seedlings were grown for a 15‐day period and treated with 0.5 mM Al with low Ca (1 mM Ca) or high Ca (5 mM Ca). The growth of seedlings was signficantly inhibited by Al. Supplement of Ca improved the growth of Al‐treated plants, increased dry matter weight of plant and leaf area, and decreased shoot/root ratio. This showed that Ca ameliorated Al toxicity in wheat. In experiments on seed germination, Al concentrations less than 2 mM in the germinating medium had little or no visible effect on length of shoot and root of germinating seed. The germinating rate of seed was not affected significantly by Al, when Al concentrations lower than 5 mM Al. The addition of 3 mM Ca did not increase the length of shoot and root and germination rate of seeds. Both pretreatments with 6 mM Ca and 1 μM GA had no significant effect on the length of shoot and root and amylolytic activity of Al‐treated germinating seeds. No significant differences were found in the total amylolytic activity in Al‐treated and control seeds two days and five days after germination. The results of Al and Ca effects on seedlings and seed germination showed that Al‐toxicity on germinating seeds was different from on seedling growth. The high concentrations of Al inhibit growth of roots and shoots of germinating seeds by other toxicity mechanism rather than interaction of Al with Ca and mobilization of carbohydrate reserves.     

Plant seed cystatins and their target enzymes of endogenous and exogenous origin

Cystatins are protein inhibitors of cysteine proteinases of the papain family, and those of animal origin have long been studied from medical and physiological aspects. In the meantime, oryzacystatin cloned from rice seeds in 1987 was recognized as the first well-defined cystatin of plant origin. Cloning studies followed to disclose various plant cytstatins including those of corn and soybean origin, their similarities to and differences from animal cystatins being analyzed in detail. Plant seed cystatins are now understood as factors controlling germination by inhibition of endogenous cysteine proteinases. They can also recognize insect midgut proteinases as exogenous target enzymes to control. This paper discusses chemical and phytophysiological relationships between cystatins and their targets.     

Intravarietal level of aluminum resistance in cereal crops

The relative growth parameters of seedlings of some oat (Avena sativa L.) and barley (Hordeum vulgare L.) varieties from different intravarietal seed fractions in the absence or presence of 1–2 mM aluminum (Al) sulfate were investigated in several experiments using the solution‐paper culture technique. There were statistically significant differences in Al‐resistance level of seeds, which differ by its weight, place on ear, place, and year of reproduction and rate of germination. The data suggest that the level of Al resistance/susceptibility is not determined by genotype exactly, but may be modified by some endogenous chemical and biochemical factors (for example, content of seed storage proteins, inhibitors of germination, natural plant growth regulators, etc.). Such non‐uniformity of seeds within cultivars may be used for investigation of plant Al‐resistance mechanisms.     

Application of Oat Oil in Breadbaking

Lipids, especially polar lipids, can improve loaf volume, grain and texture, and delay staling in bread. Oats (Avena sativa L.) are rich in total and polar lipids. We have investigated the effect of oat lipids in a bread formulation on loaf volume, appearance, and bread staling. Oat oil was fractionated into polar and nonpolar fractions by water‐degumming. Crude oat oil and shortening (at 3%) increased loaf volume by ≈11% over the zero lipid formulation. The polar lipid fraction increased loaf volume by nearly the same amount when added at only a 0.5% level. The addition of 3% crude oat oil or 0.7% oat oil polar fraction significantly delayed bread firming and starch retrogradation; the difference between oat lipids and shortening was more evident at the end of a four‐day storage period. Oat lipids had a stronger relative effect on bread from a weak flour (10% protein) than from a strong flour (14% protein). The effects of oat oil in the bread formulation could be related to the amphipathic character of polar lipids in oats that enables them to interact with starch, proteins, and other bread components.     

Optimizing Conditions for Experimental Oat Dehulling

The determination of groat percentage in experimental oat breeding lines requires the dehulling of oats. Here, we report the results of our efforts to optimize dehulling conditions so that the most accurate and reliable result can be obtained. Hand dehulling was always reliable and accurate, however, it was the most time‐consuming and tedious of the methods studied. Two mechanical methods of oat dehulling, compressed‐air dehulling and impact dehulling, also frequently provided reliable results, however, results were strongly influenced by dehulling conditions. Optimal dehulling conditions represented compromises between unfavorable extremes. Correct aspiration strength was critical to accurate groat percentage determination. We have found that a secondary aspiration is highly desirable after compressed‐air dehulling to remove hulls remaining with the groats after dehulling. Also, increased mechanical stress on oats as exerted either by the number of passes through the impact dehuller, or by the air pressure in the compressed‐air dehuller, resulted in higher dehulling efficiency, but increased groat breakage as well. Dehulling efficiency decreased as moisture increased from 7.5 to 15%, but increased as moisture was further increased to 30%. In contrast, groat breakage with impact dehulling decreased as moisture increased from 7.5 to 30%. A new equation for groat percentage calculation has been introduced where the mass of hulled oats remaining after dehulling is subtracted from the mass of the original oat sample, so that poor dehulling efficiency does not influence the groat percentage.     

小麦与油菜种子萌发对酸雨胁迫的反应研究

用pH2.0、2.5、3.0、3.5、4.0、5.0模拟酸雨处理小麦和油菜种子试验结果表明,pH2.0时小麦和油菜种子不发芽,pH2.5时只有小麦异状发芽,pH≥3.0时小麦和油菜种子发芽率、发芽势、发芽指数、活力指数均与pH值显著正相关,小麦异状发芽率则随pH值上升而降低。小麦和油菜的吸水值、呼吸速率、贮藏物质运转效率也与pH值显著正相关,小麦贮藏物质消耗率与pH值显著正相关,而油菜与pH值显著负相关;小麦和油菜的根、芽长抑制指数均与pH值显著负相关,且小麦抗酸雨胁迫能力强于油菜。     

Thermostability of Barley Malt Proteases in Western Canadian Two‐Row Malting Barley

During the malting process, barley is germinated via a carefully controlled procedure so that its components are degraded to sugars, amino acids, and other low molecular weight compounds that can be used for subsequent fermentation. One of the most important of these processes is the hydrolysis of proteins into peptides and amino acids. During seed germination, proteases hydrolyze insoluble reserve proteins into soluble peptides that are subsequently hydrolyzed into free amino acids. During kilning, green malt is initially air dried at 40–60°C, and then the temperature is gradually increased to 85–95°C. Although most proteases are denatured during kilning, the malt contains a small proportion of heat‐stable protease enzymes able to further break down protein in the subsequent mashing process. In this study, protocols were developed and standardized to measure the activity of different proteases. These protocols were then used to study protease thermostability in Canadian two‐row spring malting barley lines. We found a wide range in protease activity under controlled conditions. Upon heat treatment, several lines exhibited significant protease thermostability. These thermostable enzymes were purified by ammonium sulfate precipitation and Sephadex columns for further study.     

Degradation of HMW Glutenins During Wheat Sourdough Fermentations

Bakeries use sourdoughs to improve bread properties such as flavor and shelf life. The degradation of gluten proteins during fermentation may, however, crucially alter the gluten network formation. We observed changes that occurred in the HMW glutenins during wheat sourdough fermentations. As fermentation starters, we used either rye sourdough or pure cultures of lactobacilli and yeast. In addition, we incubated wheat flour (WF) in the presence of antibiotics under different pH conditions. The proteolytic activities of cereal and sourdough‐derived proteinases were studied with edestin and casein. During sourdough fermentations, most of the highly polymerized HMW glutenins degraded. A new area of alcohol‐soluble proteins (≈30.000 MW) appeared as a result of the proteolytic breakdown of gluten proteins. Very similar changes were observable as WF was incubated in the presence of antibiotics at pH 3.7. Cereal and sourdough‐derived proteinases hydrolyzed edestin at pH 3.5 but showed no activity at pH 5.5. An aspartic proteinase inhibitor (pepstatin A) arrested 88–100% of the activities of sourdough enzymes. According to these results, the most active proteinases in wheat sourdoughs were the cereal aspartic proteinases. Acidic conditions present in sourdoughs create an ideal environment for cereal aspartic proteinases to be active against gluten proteins.     

Effect of Germination and UV‐C Radiation on the Accumulation of Flavonoids and Saponins in Black Bean Seed Coats

In the present study, we evaluated UV‐C radiation and germination treatments as an approach to increase the concentration of bioactive molecules in black bean seed coats. Black beans were germinated for 20 h under UV‐C radiation. Germination rate was higher in seeds radiated with UV‐C light compared with the control (nonirradiated seeds). Flavonoid content was increased twofold in seed coats of beans germinated for 10 h under UV‐C compared with the control. Quercetin‐3O‐glucoside was the major flavonoid identified in stressed seed coats. Furthermore, the application of UV‐C radiation during germination increased the content of soyasaponin Af, Ba, and αg, and it induced the de novo biosynthesis of soyasaponins (phaseoside I, soyasaponin deacetyl Af, and soyasaponin deactyl Ah) not present in the control. Germination of black beans under UV‐C radiation was an effective and simple approach to increase the concentration of bioactive molecules in black bean seed coats.     

NaCl胁迫下玉米种子萌发过程中超弱光子辐射的变化

为了揭示盐胁迫下萌发种子超弱光子辐射的生物学意义,研究了NaCl胁迫下萌发玉米种子超弱光子辐射的变化规律。结果表明,在对照组的玉米种子萌发过程中,种子鲜质量和自发光子辐射逐渐增长,种子鲜质量和自发光子辐射的变化呈现正相关(相关系数r为0.9614);在50、100和150mmol/L的NaCl胁迫下萌发的玉米种子鲜质量与自发光子辐射也呈现正相关(相关系数r分别为0.9582、0.9406和0.9389),NaCl胁迫对萌发过程中种子鲜质量和自发光子辐射的增长都有抑制作用,NaCl浓度越大,抑制作用越强。研究还发现,NaCl胁迫会导致萌发过程中玉米种子延迟光子辐射中的初始光子数、相干时间和积分强度变小,并且呈现出强度效应。研究结果为揭示盐胁迫下萌发种子超弱光子辐射的生物学意义,开发基于作物耐盐性评价和种质资源鉴定等方面的活体无损检测新技术提供参考。     

The Identification of Finnish Oat Cultivars (Avena sativa L.) by the Use of SDS-PAGE of the Avenins in Homogeneous and Gradient

Abstract

The Finnish oat cultivars were identified by homogeneous and gradient SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis on the basis of 7–14 avenin bands. REM (relative electrophoretic mobility) values of the avenins among the Finnish oat cultivars were determined by gradient SDS-PAGE to calculate the PH% (pattern homology percentage). Most of the cultivars (58%) had a PH% of over 75%, which indicates quite a. high degree of similarity between the cultivars. Homogeneous SDS-PAGE was used in addition to gradient SDS-PAGE to compare the electrophoregrams of the cultivars by the gel separation systems. Resolution of the avenins was better by homogeneous than by gradient SDS-PAGE. Ten oat cultivars out of the 28 tested could be identified individually in homogeneous SDS-PAGE, as opposed to three which were identifiable by gradient SDS-PAGE.