Production of Proteases by Fusarium Species Grown on Barley Grains and in Media Containing Cereal Proteins

The production of proteases by the cereal plant pathogens Fusarium culmorum, F. graminearum and F. poae was followed through seven days of cultivation. The fungi were grown in mineral and in gluten culture media, and on autoclaved barley grains. The proteolytic activities of each sample were analysed at pH 2·2, 5·0 and 8·0 and the pH optima of the most active proteases were determined. All of the fungi grown in the gluten medium produced proteases that were active at pH levels between 6 and 10 and were most active at about pH 9·0. Fusarium poae also produced acid protease(s) with pH optima between 3.0 and 3.5 when grown in the gluten medium. No protease activity was detected in the cultures that were grown in the mineral medium, except that a small amount was formed after the glucose substrate was depleted. When grown on the barley grain medium the Fusarium species produced protease activities that were similar to the neutral and alkaline ones present in the gluten cultures, but no pH 2·2 protease activity was detected. The alkaline proteases had some characteristics that were similar to those of chymotrypsin.     

Bread Supplemented with Amaranth (<Emphasis Type="Italic">Amaranthus cruentus</Emphasis>): Effect of Phytates on <Emphasis Type="Italic">In Vitro</Emphasis> Iron Absorption

The objective of the present study was to evaluate the effect of the bread supplemented with whole amaranth flour (0, 20 and 40%) on iron bioavailability using Caco-2 cells model. The phytate and lower myo-inositol phosphates content in in vitro bread digests were measured by high pressure liquid chromatography. The breads made with amaranth showed significant increase of soluble phytates levels (up to 1.20 μmol/g in dry matter for the 40% of substitution) in comparison with controls, which have not detectable values. A negative correlation among phytate and Fe availability was found when increased levels of amaranth. Ferritin concentration was found 2.7- and 2.0-fold higher (P < 0.05) in cultures exposed to 20% and 40% of amaranth formulated bread samples, respectively, compared to control bread. The soluble phytate/Fe molar ratio explained the whole amaranth flour-mediated inhibitory effect associated to the limitation of available Fe; however, the use up to 20% of amaranth in bread formulation appears as a promising strategy to improve the nutritional value of bread, as indicated by the ferritin concentrations quantified in cell cultures. Higher proportion of amaranth flour increased Fe concentration although there was not detected any increase in Fe uptake.     

Effects of Cultivar and Environment on β-(1,3)-(1,4)- -glucan Content and Acid Extract Viscosity of Spanish Barleys

The acid extract viscosities and β-glucan contents of ten two- and six-rowed barley cultivars grown at seven locations in three consecutive years in Spain were studied in the present work. The viscosities varied from 2·4 to 24·8 centistokes (cSt) and the mean value was 6·4 cSt. The average β-glucan content of barleys determined by HPLC was 3·5% with a range of 1·9–5·5%. Significant differences were found in both β-glucan content and acid extract viscosity between different cultivars, locations and years. The β-glucan contents and viscosities of winter cultivars were higher than those of spring. Cvs. Barbarrosa and Hatif de Grignon were the genotypes with the highest values for both parameters, while cv. Beka had the lowest viscosity and β-glucan content. Environmental factors influenced both parameters. The acid extract viscosities of barleys were correlated negatively with the amount of precipitation (r=−0·754;P<0·05). Barleys grown in wet and rainy areas (Girona and La Coruña) had lower viscosity values.     

Protein Inhibitors ofAlpha-amylase in Mature and Germinating Grain of Rye (Secale cereale)

The activities of endogenous (R-type) and exogenous acting (D-type) protein inhibitors ofalpha-amylase and the activities ofalpha- and total amylase were determined in milling fractions of rye. High D-type amylase inhibitor activities were detected in the embryo (255 IU/g) and in the endosperm fraction (64·9 IU/g), low inhibitor activities were found in the aleurone layer fraction (25·9 IU/g). The highest R-typealpha-amylase inhibitor activity was found in the aleurone layer fraction (32·6 IU/g), and the lowest value in the epidermis containing fraction (5·0 IU/g). The D- and R-typealpha-amylase inhibitor activities varied with growing conditions. D-type amylase inhibitor activities were found to be high in those samples which grew under drought conditions and low in samples cultivated under wet and cool weather. Higher R-typealpha-amylase inhibitor activities were found in rye genotypes cultivated under wet conditions and lower values under dry weather. There were small variations inalpha-amylase inhibitor activities between sprout-stable and sprout-sensitive rye genotypes. The D- and R-typealpha-amylase inhibitor activities of all varieties were stable during 72 h of germination. Similar soil conditions will therefore lead to differentialalpha-amylase inhibitor activities depending on weather conditions during growth.     

Study of the effect of hydrothermal process conditions on pasta quality

The effect of hydrothermal treatment on the pasting, hydration properties and colour quality of commercial fresh pasta were studied following an Instantaneous Controlled Pressure Drop treatment. This hydrothermal procedure involves a physical modification at high temperature (<144 °C) and restricted moisture content (<30%) during a very short time (15–40 s) followed by a rapid pressure drop to a vacuum (50 mbar). Two process variables (steam pressure level and processing time) were investigated using response surface methodology. Steam pressure level had the greatest effect on hydration and pasting parameters. Increased pressure resulted in elevation of mass ratio between cooked and uncooked pasta (W_i/W₀)_{100 °C} and reduction of for all treated pasta. The optimum cooking time of untreated pasta was 7 min (W/W₀=2.4), whereas for the same ratio it was less than 1 min for pasta treated at pressures ranging between 2.5 and 3.5 bar. The viscosity at 10 min (V₁₀) increased linearly when the pressure level increased. V₁₀ was 146 cP for untreated pasta whereas it reached 2659 cP for pasta treated at 3.9 bar (144 °C) for 25 s. The cold paste viscosity and setback viscosity decreased when the pressure varied from 1 to 3.9 bar.     

Differences in conformational stability of barley alpha-amylase isozymes 1 and 2. Role of charged groups and isozyme 2 specific salt-bridges

Barley alpha-amylase isozymes 1 (AMY1) and 2 (AMY2) have 80% sequence identity but possess different physico-chemical properties. By incubation in the range 37–85 °C T₅₀ is 75.2 °C of AMY1 and 79.2 °C of AMY2. While AMY2 is also most stable in urea at pH 6.7, [urea]₅₀ being 8.2 M compared to 7.9 M for AMY1, AMY1 has highest stability in urea below pH 6 or in the presence of NaCl. Moreover AMY1 is most stable in guanidinium chloride. Charge screening thus destabilises AMY2 but stabilises AMY1. Isozyme sequence comparison suggests that AMY1 lacks four of the 20 salt-bridges identified in the crystal structure of AMY2. The four residues that differ comprise Lys67_AMY2 and Asp267_AMY2, forming salt-bridges on the surface of the catalytic (β/α)₈-barrel (domain A), and Glu96_AMY2 and His344_AMY2 that participate in charged networks between domain A and the small domain B and the C-terminal domain, respectively. Four corresponding AMY2 mimics A68K; D97E; Q269D; N346H were made in AMY1 by site-directed mutagenesis. While D97E and Q269D have slightly improved stability compared to AMY1 wild-type, N346H and, under certain conditions, A68K are destabilised. The four mutants show 22–176% activity (k_cat/K_m) toward 2-chloro-4-nitrophenol β- -maltoheptaoside and amylose DP17 and 43–117% activity for insoluble starch.     

Thermostability variation in alleles of barley beta-amylase

Thermostability assays in conjunction with IEF and molecular mapping were used to identify three beta-amylase alleles (Bmyl-Sd1, -Sd2L, -Sd2H) in cultivated barley and an additional allele (Bmy1-Sd3) in an accession of wild barley Hordeum vulgare ssp. spontaneum. The four forms of beta-amylase exhibit different rates of thermal inactivation in barley extracts. This variation was shown to persist after the proteolytic processing of the enzyme that occurs during germination. Three forms of beta-amylase representing the range of thermostabilities were purified and shown to have T₅₀ temperatures of 56·8°C for the Sd2L enzyme, 58·5°C for the Sd1 enzyme, and 60·8°C for the Sd3 beta-amylase from wild barley. Analysis of the relationship between beta-amylase thermostability and fermentability, i.e. the yield of fermentable sugars obtained from starch hydrolysis during brewing in 42 commercial malt samples suggests that increased thermostability results in more efficient starch degradation. Screening for specific beta-amylase alleles is proposed as a method for increasing fermentability in malting barley.     

Developmental Expression of Amylases During Barley Malting

Amylase activity and qualitative changes in amylase isoenzymes as a function of barley seedling age were investigated in 10 Brazilian barley cultivars. All cultivars showed few isoenzymes in early germination. An increase in general activity ensued in the following days when new isoenzymes were detected and those already observed since early germination had their activity increased. All cultivars disclosed increase in amylase activity until the third or fourth day of germination. Some cultivars maintained this high activity until the last day analysed. Other cultivars presented a decrease in activity in the fifth or sixth day. No electrophoretic pattern or allelomorph responsible for a higher amylase activity were detected. Beta -amylase activity was always superior to alpha -amylase activity. High beta -amylase activity was already observed on the second day of germination while alpha -amylase activity began to increase only from the third day on. The results obtained suggest that, at least for the cultivars analysed, there is a high general amylase activity around the fourth day of germination, indicating that germination could stop at this moment, ensuring that hydrolitic enzyme activity required in the brewing process is met. Beta -amylase was lightly correlated with diastatic power (r=0·565) but no correlation was observed between alpha -amylase and diastatic power (r=-0·128), or neither betweenalpha - and beta -amylase with malting quality (r=0·153 andr =−0·348, respectively). These results indicate that beta -amylase activity in barley grains, more than alpha -amylase, can be a good predictor of diastatic power.     

Changes in soluble carbohydrates, lipid peroxidation and antioxidant enzyme activities in the embryo during ageing in wheat grains

Soluble sugar contents and antioxidant enzyme activities were measured in the embryo of wheat (Triticum aestivum L., cv Charger) seeds throughout their storage under two conditions (45 °C and 100% relative humidity (RH), and 30 °C and 75% RH) to investigate whether grain deterioration during ageing was related to lipid peroxidation resulting in a decrease in the efficiency of the antioxidant defence system, and in changes in sugar metabolism. The half-viability period (P₅₀) was only 6 d at 45 °C and 100% RH against about 3.75 months at 30 °C and 75% RH. However, sensitivity of grains to accelerated ageing carried out at 45 °C and 100% RH increased during their pre-storage at 30 °C and 75% RH. Loss of grain viability at 45 °C and 100% RH was associated with an accumulation of H₂O₂ which was concomitant with a progressive decrease in catalase (CAT) and superoxide dismutase (SOD) activities, and with an increase in glutathione reductase (GR) activity. However, malondialdehyde content did not change, suggesting that there was no lipid peroxidation during such an ageing. In return, ageing of grains at 30 °C and 75% RH was not associated either with strong changes in CAT, SOD and GR activities or with an accumulation of H₂O₂. When there was no correlation between CAT, SOD and GR activities and seed viability, there was a linear correlation between CAT activity and seed vigour evaluated by the T₅₀ during ageing at 45 °C and 100% RH. Sugar metabolism in wheat embryo largely depended on ageing conditions. Loss of grain viability at 45 °C and 100% RH was associated with a marked decrease in sucrose (Su) and a slight increase in raffinose (Ra), and subsequently in the Ra/Su ratio. At 30 °C and 75% RH, seed ageing was associated with an increase in both sugars but with no strong increase in Su/Ra ratio. Our results suggest that ageing was associated with various mechanisms depending on the conditions of ageing, and that accelerated ageing at 45 °C and 100% RH was not the only model to consider in order to understand the mechanisms involved in seed deterioration.     

Water uptake during cooking of dry beans (Phaseolus vulgaris L.)

Water uptake during cooking of ten dry bean (Phaseolus vulgaris L.) varieties was investigated. Water uptake during early stages of cooking was characteristic of the variety. Although the optimal cooking times varied widely (52–85 min), all the beans absorbed similar amounts of water when cooked for their optimal times. Cooking times were significantly correlated with W_{20 min} (r=–0.92) and hardness index (r=0.76) of beans. Most other physical characteristics excepting the surface area of beans were generally unrelated to theW _{20 min},W _opt, and cooking times. No significant correlation was observed between phytate content and cooking times of beans. On cooking for their respective optimal times, all varieties absorbed nearly 1.5 times their weight of water and attained a moisture content of about 65% (wet basis).     

Comparative Enzyme Kinetics of Two Allelic Forms of Barley (Hordeum vulgare L.) Beta -amylase

The barley (Hordeum vulgare L.) varieties, Franklin and Schooner, contain two different allelic forms of beta -amylase (EC 3.2.1.2) encoded on chromosome 4H by the Bmy 1-Sd1 and Bmy 1-Sd2L alleles, respectively. The corresponding enzymes, referred to as Sd1 and Sd2L, were purified from both mature barley grain and germinated barley (green malt), and their physical and kinetic properties studied. Approximately 4 kDa were cleaved from both Sd1 and Sd2Lbeta -amylases after germination. The K_mvalue for green malt beta -amylase was less than that of mature grain beta -amylase for both varieties when potato starch was used as a substrate, although V_maxwas similar. This indicated that proteolysis after germination increased the affinity of beta -amylase for potato starch. No significant kinetic differences were observed between beta -amylase from mature grain and green malt of the two barley varieties when amylose (degree of polymerisation 100 and 18) and maltopentaose were used as substrates. Kinetic differences were also observed between the two allelic forms of beta -amylase. Sd1 beta -amylase from green malt exhibited a lower K_mvalue for potato starch than Sd2L beta -amylase, demonstrating that at non-saturating starch concentrations Sd1 beta -amylase is better able to hydrolyse starch than Sd2L beta -amylase. As the degree of polymerisation of the substrates decreased from approximately 740 (potato starch) to 5 (maltopentaose), the K_mvalues for beta -amylase increased, whereas V_maxvalues decreased. Maltose, the hydrolytic product of beta -amylase, was found to be a weak competitive inhibitor of both Sd1 and Sd2L green malt beta -amylases with respect to potato starch and amylose. Taken together the kinetic observations for bet a-amylase suggest that the allelic differences and C-terminal proteolysis might be exploited to improve the efficiency of starch hydrolysis during the mashing stage of the brewing process.     

Affinity Chromatography with Immobilised Endoxylanases Separates TAXI- and XIP-type Endoxylanase Inhibitors from Wheat (Triticum aestivum L.)

An affinity-based purification procedure allowed the resolution of two distinct groups of endoxylanase inhibitors with different molecular structures and endoxylanase specificities from wheat wholemeal. The first group comprises the so-called Triticum aestivum L. Endoxylanase inhibitor (TAXI)-type proteins which are of approx. M_r 40 000 and occur in two different molecular forms. These inhibitors were removed from a concentrated cation exchange chromatography fraction from wheat wholemeal on a Bacillus subtilis endoxylanase affinity column. The second group of structurally different endoxylanase inhibitors, the so-called xylanase inhibiting protein (XIP)-type, of approx.M_r 29 000–32 000, with pI values varying between 8·8 and 9·2, was purified from the unbound fraction from the B. subtilis endoxylanase affinity column by chromatography on an Aspergillus niger endoxylanase affinity column followed by gel permeation chromatography. The XIP-type inhibitors are not active against the B. subtilis endoxylanase and were consequently not retained on the B. subtilis endoxylanase column. Further analysis of the XIP-type proteins by high-resolution cation exchange chromatography, SDS-PAGE and iso-electrofocusing, revealed several forms. They had similar endoxylanase specificities and N-terminal amino acid sequences.     

Alpha-amylase/subtilisin Inhibitor Levels in Australian Barleys

An enzyme-linked immunosorbent assay (ELISA) was used to determine the bifunctional alpha-amylase/subtilisin inhibitor (BASI) content of barley grain from 11 cultivars grown in six diverse locations in Australia. The inhibitor ranged from 119 to 254 μg/g in 57 barley samples. Genotype had a significant (P<0·05) effect on BASI content but there was no effect due to environment. Total protein varied independently of BASI and was influenced by environment and genotype. BASI content was higher (P<0·05) in malting barley than in feed barley and was correlated positively (r=0·29;P<0·05) with alpha-amylase activity in corresponding malts. The ELISA used monoclonal and polyclonal antibodies raised against purified BASI. In immunoblot analysis the monoclonal antibody showed high specificity for the inhibitor in barley and also detected the inhibitor in wheat. Low levels of inhibitor (mean 3·2 μg/g) were found in 12 Australian wheat cultivars using the ELISA developed for barley. The assay had a linear working range of 5–50 ng/mL with a detection limit of 2 ng/mL. Reproducibility between assays was good (CV=4·9%) but mean recoveries were high, ranging from 116–129% when purified inhibitor was added to barley extracts. The ELISA may have useful applications in brewing research and barley breeding programmes.     

The Role of Dextrins in the Stickiness of Bread Crumb made from Pre-Harvest Sprouted Wheat or Flour Containing ExogenousAlpha-Amylase

Dextrins were extracted in water from bread made from pre-harvest sprouted wheat or standard flour supplemented with exogenousalpha-amylases. The dextrins were separated by gel permeation chromatography and the dextrin content (% of crumb weight) determined for different degree of polymerisation (DP) size classes; DP 1–2, DP 3–10, DP 11–50, DP 51–200 and DP >200. There were significant correlations between the dextrin content in each size class and crumb stickiness (r=0·84–0·91, 22 df ). The most significant correlation (r=0·96) was between total dextrin content and crumb stickiness. Addition of dextrins of various DP ranges from various sources to standard flour produced bread with sticky crumb. Again, the degree of stickiness was generally related to the amount of total dextrin in the crumb and not to size distribution of dextrins. In this instance, extensive enzymic hydrolysis of starch was not necessary to produce sticky crumb; the dextrins caused crumb stickiness directly. Addition of dextrins to reconstituted gluten–starch flour produced bread with unexpectedly low dextrin levels and correspondingly low stickiness scores. It is concluded that, to produce sticky crumb, high levels of dextrin of any size are necessary in the crumb; a sticky mass is produced when dextrins dissolve in the excess «free» water that is normally «bound» to starch, gluten and other insoluble components of bread crumb.     

Improved viscoelastic zein–starch doughs for leavened gluten-free breads: Their rheology and microstructure

Gluten-free bread was prepared from commercial zein (20 g), maize starch (80 g), water (75 g), saccharose, NaCl and dry yeast by mixing above zein's glass transition temperature (T_g) at 40°C. Addition of hydroxypropyl methylcellulose (HPMC, 2 g) significantly improved quality, and the resulting bread resembled wheat bread having a regular, fine crumb grain, a round top and good aeration (specific volume 3.2 ml/g). In model studies, HPMC stabilized gas bubbles well. Additionally, laser scanning confocal microscopy (LSCM) revealed finer zein strands in the dough when HPMC was present, while dynamic oscillatory tests showed that HPMC rendered gluten-like hydrated zein above its T_g softer (i.e. |G*| was significantly lower). LSCM revealed that cooling below T_g alone did not destroy the zein strands; however, upon mechanical impact below T_g, they shattered into small pieces. When such dough was heated above T_g and then remixed, zein strands did not reform, and this dough lacked resistance in uniaxial extension tests. When within the breadmaking process, dough was cooled below T_g and subsequently reheated, breads had large void spaces under the crust. Likely, expanding gas bubbles broke zein strands below T_g resulting in structural weakness.     

Factors affecting early screening of tomatoes for monogenic and polygenic resistance to Fusarium wilt

Tomatoes (cv. Bari-RVF-SMD) monogenically (I gene) resistant to Fusarium wilt (Fusarium oxysporum f.sp. lycopersici) were not affected by inoculum dilutions (10¹, 10³ and 10⁶ conidia/ml), temperature regimes (22°C and 26°C) and plant age (two and four leaves), whereas the phenotypic expression of susceptible and polygenically resistant tomatoes (cvs Rutgers and Marglobe) was conditioned by these factors. Rutgers tomatoes were found to possess a higher level of resistance than Marglobe. Early screening (33 days after inoculation) of resistant Rutgers plants could be made on plants inoculated at the two-leaf stage with an inoculum dilution of 10³ conidia/ml and grown at 22°C or 26°C. Thirty-three days after inoculation, Marglobe tomatoes inoculated at the two- and four-leaf stages with inoculum dilutions of 10³ and 10⁶ conidia/ml and grown at 22°C or 26°C proved to be susceptible. The following first-order interactions were found to be statistically significant: cultivar × inoculum dilution, cultivar × temperature, cultivar × plant age, inoculum dilution × temperature and inoculum dilution × plant age.     

Fatty acid composition of lipids of Australian oats

A method using methanolic sulphuric acid as transmethylating reagent was developed for determining the fatty acid composition of lipids of oats. The method was optimised for reaction conditions and applied to the determination of the fatty acid composition of lipids of a number of varieties of Australian oats grown in several locations. Thirteen fatty acids were detected with oleic, linoleic and palmitic acids comprising more than 95% of the total fatty acids. Total lipid content of the oats was positively related to the proportion of stearic (r=0·32) and oleic (r=0·81) acids and negatively correlated with the proportion of palmitic (r=−0·64), linoleic (r=−0·39) and linolenic (r=−0·65) acids. Significant positive correlations were found between total lipid content and absolute content of the major fatty acids (r=0·670·98), except for linolenic acid (r=0·12). Environment had significant effects on fatty acid composition, but variety was the controlling factor. The broad sense heritability estimated from individual plot ranged from 69 to 73% and that from the average of three replications and eight locations ranged from 94 to 98% for the major fatty acids. It is possible to improve fatty acid composition of oats by breeding procedures.     

Beta-amylase degradation by serine endoproteinases from green barley malt

Proteolytic degradation of barley proteins is examined in green (unkilned) malt and germinating seeds from Hordeum vulgare L. cv. Harrington. Zymographic analysis of the Harrington green malt extracts using commercial preparations of barley beta-amylase incorporated as a proteolytic substrate in 2-D SDS gels shows multiple proteolytic activities. A developmental study shows that the several green malt beta-amylase-degrading activities appear at around day 2 of germination. The several activities appear to increase and decrease through 7 days of germination in a coordinated fashion. Gels treated with class-specific proteinase inhibitors show that serine-class proteinase activities are responsible for barley beta-amylase degradation seen on the zymograms. Western blot analysis also shows that proteolytic enzymes recovered from 1-D electrophoretic gels degrade barley beta-amylase, and that the degradation is inhibited by PMSF. This is the first demonstration that malt proteinases are capable of degrading important metabolic enzymes in germinating barley, and the first postulated physiological role for the serine class proteinases in barley malt.     

Purification and Characterization of a Glutenin Hydrolysing Proteinase from Wheat Damaged by the New Zealand Wheat Bug, Nysius huttoni

A glutenin hydrolysing enzyme (bug proteinase), present in New Zealand wheat damaged by Nysius huttoni, was purified 50000-fold by anion exchange, hydrophobic interaction, immobilized metal ion affinity and gel filtration chromatography. The enzyme had an apparent M_r of 14·1k as determined by gel filtration chromatography. SDS-PAGE showed a major protein band of M_r 30k and six minor bands of M_r 13·2-28·5k, none of which was a glycoprotein. Isoelectric focusing revealed two major enzyme active bands (pI 9·6 and 9·2) and three minor activity bands (pI 9·9, 8·8 and 8·2). IEF showed no protein contaminants in the most purified sample. The enzymes had optimum activity at pH 8·9 and 45°C. The activity was stable in the pH range 4·5-11 and at 50°C for 20 min at pH 8·9. The bug proteinase was shown to be a serine proteinase by inhibition with phenylmethylsulphonyl fluoride and potato proteinase inhibitors (POT-IC and POT-ID). Thirty other proteinaceous serine proteinase inhibitors did not inhibit the enzyme. Bread baking with partially purified enzyme produced loaves with the poor quality characteristics of loaves made with bug-damage wheat.     

Fusarium Species Synthesize Alkaline Proteinases in Infested Barley

Barley (Hordeum vulgare L.) that is infested with Fusarium head blight (FHB, ‘scab’) is unsuitable for malting and brewing because it may contain mycotoxins and has unacceptable malting quality. Fungal proteinases are apparently often involved in plant-microbe interactions, where they degrade storage proteins, but very little is known about the enzymes that the fungi produce in the infected grain. We have shown previously that one plant pathogenic fungus, Fusarium culmorum, produced subtilisin- and trypsin-like enzymes when grown in a cereal protein medium. To establish whether these proteinases were also synthesized in FHB-infested barley in vivo, field-grown barley was infested as the heads emerged. Extracts were prepared from the grain as it developed and matured and their proteolytic activities were measured with N-succinyl-Ala-Ala-Pro-Phe p -nitroanilide and N-benzoyl-Val-Gly-Arg p -nitroanilide. The heavily infested barleys contained both subtilisin- and trypsin-like activities. These enzymes reacted with antibodies prepared against each of the two F. culmorum proteinases, indicating that those produced in the laboratory cultures and in the field-infested barley were the same. The presence of these proteinases correlated with the degradation of specific buffer-soluble proteins in the infested grains. These enzymes readily hydrolyzed barley grain storage proteins (C- and D-hordeins) in vitro. The presence of these Fusarium proteinases in the barley indicates that they probably play an important role in the infestation, but exactly how and when they function is not clear.