Modulation of physicochemical and conformational properties of kidney bean vicilin (phaseolin) by glycation with glucose: implications for structure-function relationships of legume vicilins

The structure-function relationships of plant oligomeric globulins are still not fully recognized. The present work investigated the influence of glycation with glucose (at 1:50 and 1:100 protein/sugar molar ratios; incubation periods of 2.5, 5.0, and 10.0 h) on the physicochemical and conformational properties of kidney bean vicilin (phaseolin), with the aim of understanding the structure-function relationships of legume vicilins. Protein solubility (PS), surface charge (isoelectric point) and hydrophobicity (H0), and secondary, tertiary, and/or quaternary conformations, as well as the emulsifying activities (emulsifying activity and emulsion stability indices, EAI and ESI) were evaluated. The 2.5 h incubation period of glycation led to least PS and highest H0, and after that, the PS and H0, on the contrary, gradually changed with increasing incubation period. The glycation increased the α-helix content and highly ordered secondary structures (α-helix+β-strand), as evidenced by far-UV circular dichroism (CD) spectroscopy. Combined analyses of differential scanning calorimetry, intrinsic emission fluorescence, and near-UV CD spectroscopy indicated that phaseolin underwent a tertiary conformation unfolding and subsequent rearrangement process (to form a new tertiary conformation), whereas the quaternary conformational flexibility progressively increased upon increasing degree of glycation. The conformation rearrangement was more distinct at the 1:100 molar ratio than at the 1:50 counterpart. The glycation at 5.0 and 10.0 h periods considerably increased the EAI, but only at the 1:50 molar ratio was the ESI progressively increased with the incubation period. These results confirmed that besides surface properties (e.g., PS and H0), the flexibility in tertiary and/or quaternary conformations played a major role in the emulsifying properties of glycated vicilins. The findings would have important implications for understanding the structure-function relationships of legume oligomeric globulins, thus providing a direction to further improve the surface-related functional properties of these proteins.     

Preparation and characterization of papain-modified sesame (Sesamum indicum L.) protein isolates

Defatted sesame meal ( approximately 40-50% protein content) is very important as a protein source for human consumption due to the presence of sulfur-containing amino acids, mainly methionine. Sesame protein isolate (SPI) is produced from dehulled, defatted sesame meal and used as a starting material to produce protein hydrolysate by papain. Protein solubility at different pH values, emulsifying properties in terms of emulsion activity index (EAI) and emulsion stability index (ESI), foaming properties in terms of foam capacity (FC) and foam stability (FS), and molecular weight distribution of the SPI hydrolysates were investigated. Within 10 min of hydrolysis, the maximum cleavage of peptide bonds occurred as observed from the degree of hydrolysis. Protein hydrolysates have better functional properties than the original SPI. Significant increase in protein solubility, EAI, and ESI were observed. The greatest increase in solubility was observed between pH 5.0 and 7.0. The molecular weight of the hydrolysates was also reduced significantly during hydrolysis. These improved functional properties of different protein hydrolysates would make them useful products, especially in the food, pharmaceutical, and related industries.     

Some nutritional and functional properties of defatted wheat germ protein

Defatted wheat germ protein (DWGP) was isolated by alkaline extraction at pH 9.5 and subsequent isoelectric precipitation at pH 4.0, and its nutritional and functional properties were studied. The results showed that the amino acid content of defatted wheat germ was as high as 26.793 g/100 g, and the contents of eight essential amino acids were all relatively high. The isoelectric point of DWGP was 4.0. When pH >6.0, the DWGP had high solubility with a nitrogen solubility index of 70%. The emulsifying activity and emulsifying stability of DWGP were similar to those of bovine serum albumin and a little higher than those of casein. DWGP had good foaming capacity, but its foaming stability (FS) was not very good. However, the FS of DWGP can be improved through physical, chemical, or enzymatic methods. Moreover, DWGP had excellent water retention (WR); especially at pH 8.0 and a temperature of 70 degrees C, the WR of DWGP was the highest at 229.4%. DWGP offers is a potential source of functional protein isolate for possible food applications.     

Functional Properties of 7s Globulin Extracted from Cowpea Vicilins

The seed of cowpea (Vigna unguiculata L.) is rich in protein and the amino acid profiles of the meal are suitable for human dietary products, but little is known about the structure and chemical properties of the protein extracted from this legume. This study determined the functional properties of two selected cowpea cultivars and their solubility, emulsifying capacity, surface hydrophobicity, and thermal stability. Seeds of red and black cowpea were surface sterilized and the 7s globulin was isolated and purified using column chromatography with Sephacryl S‐300 (Hi‐Prep 26/60) gel column. Also, SDS‐PAGE and protein structure were analyzed using biochemical procedures. At high ionic strength (μ = 0.5), cowpea 7s globulin fraction exhibited better solubility for a wide range of pH levels, higher emulsifying capacities, and greater thermal stability than those obtained at low ionic strength (μ = 0.08). The lowest solubility was observed at pH 5.3–6.4 at the low ionic strength. Emulsifying capacities at high protein concentration were greater when compared with low protein concentration. T_m values of black cowpea globulin fraction were higher than those of red cowpea globulin fraction, whereas the surface hydrophobicity of the globulin fraction in red cowpea was larger than that in black cowpea.     

Physicochemical properties of native and recombinant mungbean (Vigna radiata L. Wilczek) 8S globulins and the effects of the N-linked glycans

We have previously cloned and characterized the cDNAs of three isoforms of the 8S globulin of mungbean, expressed the major 8Salpha isoform in Escherichia coli, and purified and successfully crystallized it (Bernardo, A. E. N.; Garcia, R. N.; Adachi, M.; Angeles, J. G. C.; Kaga, A; Ishimoto, M.; Utsumi, S.; Tecson-Mendoza, E. M. J. Agric. Food Chem. 2004, 52, 2552-2560). Herein, we report the physicochemical and emulsifying properties of the native 8S and recombinant 8Salpha globulin or vicilin. The circular dichroism spectra analysis of the native 8S and recombinant 8Salpha globulins revealed that the recombinant 8Salpha formed a secondary structure close to that of the native 8S. Further, gel filtration analysis showed that 8Salpha was able to assemble into trimers. The native 8S and recombinant 8Salpha globulins were soluble at pH 3.4 and at pH 7.4-9.0 at low ionic strength, mu = 0.08. Interestingly, the native 8S was more soluble at pH 7.0 and pH 7.4 than the recombinant 8Salpha at mu = 0.08. Both forms were very soluble at pH 3.4-9.0 at high ionic strength, mu = 0.50. The native form exhibited a higher T(m) (69.2, 79.5, and 83.8 degrees C) than the recombinant form (65.6, 71.6, 77.5 degrees C) at mu = 0.1, 0.2, and 0.5, respectively. The recombinant form was found to have greater surface hydrophobicity than the native form. There was little difference in the emulsifying ability between the native 8S and 8Salpha at pH 3.4 and pH 7.6. The results indicate that the presence of N-linked glycans is not essential in the assembly and stable conformation of the mungbean vicilin. However, the N-linked glycans might have contributed to the higher solubility at low ionic strength, greater thermal stability, and decreased surface hydrophobicity of the native vicilin as compared to the recombinant 8Salpha. On the other hand, the N-linked glycans showed little effect on the emulsifying ability of the protein.     

Amaranth (Amaranthus hypochondriacus) vicilin subunit structure

The 7S-globulin fraction is a minor component of the amaranth storage proteins. The present work provides new information about this protein. The amaranth 7S-globulin or vicilin presented a sedimentation coefficient of 8.6 ± 0.6 S and was composed of main subunits of 66, 52, 38, and 16 kDa. On the basis of mass spectrometry (MS) analysis of tryptic fragments, the 52, 38, and 16 kDa subunits presented sequence homology with sesame vicilin, whereas the 66 kDa subunit showed sequence similarity with a putative vicilin. Several characteristics of the 66 kDa subunit were similar to members of the convicilin family. Results support the hypothesis that the 7S-globulin molecules are composed of subunits coming from at least two gene families with primary products of 66 and 52 kDa, respectively. According to the present information, amaranth vicilin may be classified into the vicilin group that includes pea, broad bean, and sesame vicilins, among others.     

Physicochemical and Functional Properties of Protein Isolates Obtained from Several Pea Cultivars

The goal of this research was to investigate the physicochemical and functional properties of protein isolates obtained from several pea cultivars grown at two locations in Canada. The functionalities of the pea protein isolates (PPIs) were then compared with those of commercial food protein ingredients derived from milk, egg, pea, soy, and wheat. Six pea cultivars (Agassiz, CDC Golden, CDC Dakota, CDC Striker, CDC Tetris, and Cooper) were collected from two years over two locations in Saskatchewan (Canada). Samples were evaluated for composition, surface properties, and functional properties. All PPIs had protein levels of ≈91% (db) and isolate and protein yields of ≈18 and ≈72%, respectively. Cultivars exhibited legumin/vicilin ratios from 0.36 (Agassiz) to 0.79 (CDC Golden). Differences among cultivars as well as significant cultivar × environment interactions were found only for maximum intrinsic fluorescence (195–267 arbitrary units), solubility (63–75%), and foaming capacity (167–244%). No differences owing to either cultivar or environment were observed for surface charge (zeta potential = approximately –24 mV), oil holding capacity (≈3.2 g/g), foam stability (≈75%), or emulsion stability (≈96%). Relative to the commercial isolates, PPIs prepared under laboratory conditions behaved most similarly to soy isolates, with the exception of solubility. Whey and egg were superior in solubility and foaming properties, whereas wheat and the commercial pea protein product were significantly lower in nearly all of the functionality tests. Based on their oil holding properties, the laboratory‐prepared PPIs may serve as good meat extenders. The findings also suggest that pea processors may not need to specify either the cultivar or the environment when acquiring raw material, thus creating advantages in their feedstock sourcing.     

甘薯热变性蛋白限制性酶解产物的乳化特性研究

采用5种酶(Alcalase 2.4L, As1.398, Neutrase, Pepsin, Trypsin)对甘薯热变性蛋白(SPHP)进行限制性酶解。将各酶解产物离心后分别取上清和沉淀测定和观察其乳化液的乳化颗粒平均粒径(D_4,3)、乳化活性指数(EAI)、乳化稳定性指数(ESI)、乳化液的微观结构和表观黏度。结果显示:酶解产物上清和沉淀中蛋白的溶解度均有增加,但沉淀增加的幅度小于上清。SPHP的D_4,3是71.96μm,而酶解产物上清和沉淀乳化液的D_4,3均减小,且上清的D_4,3小于沉淀的。在5种酶解产物中,Pepsin酶解物上清的D_4,3最小,为14.94μm。SPHP酶解后上清的乳化颗粒大小较为均一,且沉淀的乳化颗粒酶解前后变化不大。SPHP的EAI为11.21m2/g,酶解产物上清和沉淀的EAI均有显著提高(P<0.05),其中Pepsin酶解物上清的EAI最高为70.32m2/g。此外,酶解产物上清和沉淀乳化液的ESI增大。与沉淀相比,5种酶解产物的上清具有较低的表观黏度,且酶解产物上清和沉淀的乳化液均呈剪切变稀的非牛顿流体特性。     

Purification and characterization of a cysteine protease inhibitor from chum salmon (Oncorhynchus keta) plasma

A cysteine protease inhibitor (CPI) in chum salmon ( Oncorhynchus keta) plasma (CSP) was detected after performing inhibitory activity staining against papain under nonreducing condition. The CPI was purified from CSP by affinity chromatography with a yield and purification ratio of 0.94% and 30.36-fold, respectively. CSP CPI had a molecular mass of 70 kDa based on the results of SDS-PAGE and Sephacryl S-100 gel filtration. CSP CPI was a glycoprotein based on the periodic acid-Schiff (PAS) staining of the SDS-PAGE gel and classified as a kininogen. CSP CPI was stable in the pH range of 6.0-9.0 with maximal stability at pH 7.0. CSP CPI presented thermal stability at temperatures below 50 degrees C and exhibited maximal activity at temperatures of 20-40 degrees C. CSP CPI was determined to be a noncompetitive inhibitor against papain, with an inhibitor constant (Ki) of 105 nM.     

Functional Properties of Rice Bran Protein Isolate at Different pH Levels

The functional properties of proteins from Tarom and Shiroodi cultivars were determined and compared with technological aspects of food and nutraceutical applications. Shiroodi has higher protein content than Tarom, and the yields of protein obtained were 72.88 and 66.36%, respectively. Nitrogen solubilities of rice bran protein of Tarom were more than Shiroodi at all pH levels. In addition, higher solubility was found in acidic or alkaline conditions. Although the rice bran proteins had lower emulsifying properties than bovine serum albumin, they had similar foaming properties in comparison with egg white. Tarom isolates had a significantly higher solubility, emulsifying property, and foaming stability and greater surface properties than Shiroodi isolates. The results showed the surface hydrophobicities of rice bran protein were greater than casein and ovalbumin and lower than other proteins such as bovine serum albumin. Water and oil absorption capacities were 1.03 and 1.66 for Tarom and 87.3 and 75.3 for Shiroodi, respectively. The bulk densities of Tarom and Shiroodi were also 0.55 and 0.53 g/mL, which make them suitable for weaning food and other industrial applications. As a result, these rice bran proteins showed higher hydrophobicity than that of other rice bran protein varieties as well as more functionality. Thus, they have good potential in the food and pharmaceutical industries.     

Characterization of pea vicilin. 1. Denoting convicilin as the alpha-subunit of the Pisum vicilin family

Vicilin, a major globulin protein of pea that has been described as "extremely heterogeneous in terms of its polypeptide composition", was extracted from pea flour under alkaline conditions and subsequently fractionated by salt under acid conditions. This procedure induced the separation of vicilin into two fractions, which, after purification, were called vicilin 1 degrees and vicilin 2 degrees. Vicilin 2 degrees was seen on SDS-PAGE to contain the third globulin protein of pea, convicilin (a band at approximately 70 kDa). Vicilin fractions were thus characterized using gel electrophoresis, differential scanning calorimetry, circular dichroism, and pH-dependent solubility in order to determine whether the convicilin should in fact be considered as a third separate globulin protein of pea. On the basis of the results obtained it was concluded that this distinct polypeptide of the Pisum vicilin gene family should be further denoted as a subunit of the salt extractable protein vicilin. The definition of vicilin heterogeneity should therefore be extended to acknowledge the possible oligomeric inclusion of the 70 kDa polypeptide that is here denoted as the alpha-subunit.     

Comparison of physicochemical properties of 7S and 11S globulins from pea, fava bean, cowpea, and French bean with those of soybean--French bean 7S globulin exhibits excellent properties

Legume seeds contain 7S and/or 11S globulins as major storage proteins. The amino acid sequences of them from many legumes are similar to each other in the species but different from each other, meaning that some of these proteins from some crops exhibit excellent functional properties. To demonstrate this, we compared protein chemical and functional properties (thermal stability, surface hydrophobicity, solubility as a function of pH, and emulsifying properties) of these proteins from pea, fava bean, cowpea, and French bean with those of soybean as a control at the same conditions. The comparison clearly indicated that the 7S globulin of French bean exhibited excellent solubility (100%) at pH 4.2-7.0 even at a low ionic strength condition (mu = 0.08) and excellent emulsion stability (a little phase separation after 3 days) at pH 7.6 and mu = 0.08, although the emulsions from most of the other proteins separated in 1 h. These results indicate that our assumption is correct.     

Purification and biochemical characterization of insoluble acid invertase (INAC-INV) from pea seedlings

Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the β-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.     

高压微射流压力对大豆蛋白-大豆多糖体系功能性质的影响

为提升大豆分离蛋白（soy protein isolate,SPI）的功能性质,该文引入大豆可溶性多糖（soybean soluble polysaccharides,SSPS）,构建大豆分离蛋白-大豆可溶性多糖体系（SPI-SSPS）,研究动态高压微射流（dynamic high-pressure microfluidization,DHPM）处理对SPI-SSPS功能特性的影响。分别采用0,60,100,140和180 MPa的 DHPM压力处理SPI-SSPS,探究不同压力对SPI-SSPS起泡特性、乳化特性、溶解性、粒度分布和表面疏水性的影响。结果表明,DHPM处理能提高SPI的溶解性和起泡特性,且SSPS的存在能显著提高DHPM对SPI功能性质的改善效果（P<0.05）。100和60 MPa的DHPM处理能使SPI-SSPS呈现较高的起泡能力和起泡稳定性,分别为未处理样品的1.2和2.4倍。140 MPa的DHPM处理使SPI-SSPS溶解性较强,为未处理样品的1.8倍。然而,DHPM处理会显著降低SPI-SSPS的乳化特性、粒径和表面疏水性（P<0.05）。随着处理压力的增加,SPI-SSPS的粒度和表面疏水性逐渐降低,在180MPa的DHPM处理下SPI-SSPS具有较小的粒径和较低的荧光强度。综上所述,DHPM结合SSPS改性技术可用于改善SPI的功能性质（如溶解性、起泡性）,促进SPI在食品工业的应用。该文的研究结果可为SPI的功能性质改性提供参考。     

Stabilization of oil-in-water emulsions by beta-lactoglobulin-polyethylene glycol conjugates

The disulfide bonds of beta-lactoglobulin (beta-lg) were modified by oxidative sulfitolysis to generate beta-lgSO(3). The native protein (beta-lg) and the modified protein (beta-lgSO(3)) were conjugated to activated polyethylene glycol (PEG) to generate beta-lgPEG and beta-lgSO(3)PEG, respectively. Oil-in-water (o/w) emulsions containing 1% beta-lg or beta-lg conjugates were prepared at pH 2.8, 5.0, and 7.0. Emulsion droplet diameters and zeta potentials were measured. For the same emulsifier, emulsion droplet diameters decreased when emulsion pH increased. Zeta potentials of emulsion droplets increased with pH for beta-lg and beta-lgSO(3). Zeta potentials of beta-lgPEG and beta-lgSO(3)PEG approached zero, suggesting that the protein molecule was covered by PEG chains. Accelerated and 7-day storage stabilities at 21 degrees C of the emulsions were monitored. The emulsifying activity index (EAI) of beta-lgPEG was not significantly different from the EAI of beta-lg. The EAI of beta-lg was enhanced following sulfitolysis of beta-lactoglobulin. The emulsifying activity increased more when the oxidatively modified protein was conjugated to polyethylene glycol. Emulsions made with beta-lgSO(3)PEG were more stable than emulsions made with beta-lg, beta-lgPEG, or beta-lgSO(3) under accelerated stability study and for 7 days at 21 degrees C. The stability of o/w emulsions stabilized with beta-lgSO(3)PEG increased because individual droplets were better protected, against protein bridging or coalescence, by the thick adsorbed protein-PEG layer.     

Genetic variation in pea seed globulin composition

A quantitative characterization of seeds from 59 pea (Pisum sativum L.) lines and relative taxa with various external characteristics and wide geographical origin was performed to explore the genetic variation of pea concerning its starch and protein contents and globulin composition. Pea lines, which produce round, wrinkled, flat, and round-dimpled seeds, have starch as the major reserve, with an average content of 46%. Protein content varied from 13.7 to 30.7% of the seed dry matter, with an overall average of 22.3%. Densitometric quantification of the individual globulins (legumin, vicilin, convicilin, and globulin-related proteins) based on SDS-PAGE gels showed no lines lacking any particular globulin. Among the lines tested, variation was shown in both their total globulins content and their globulin composition. The total globulin content ranged from 49.2 to 81.8% of the total pea protein extract (TPPE). Legumin content varied between 5.9 and 24.5% of the TPPE. Vicilin was the most abundant protein of pea, and its content varied between 26.3 and 52.0% of the TPPE. Both processed and nonprocessed vicilins occurred. The processed vicilin was the predominant one, with values between 17.8 and 40.8%, whereas the nonprocessed ones constituted between 3.1 and 13.5% of the TPPE. Convicilin was the least abundant globulin, and its content ranged from 3.9 to 8.3%. Finally, the globulin-related proteins were present in amounts ranging from 2.8 to 17.3%. They were less abundant in comparison with legumin and vicilin, but they showed the largest relative variation of the four globulin classes. Correlations between the different external characteristics and globulin composition were determined. Comparison with soybean showed that pea lines show more variety in the abundance of globulin proteins, enabling a wider range of food application.     

适宜物理-酶联合改性提高酸性条件下大豆分离蛋白乳化性

为提高酸性条件下大豆分离蛋白（soy protein isolates,SPI）的乳化性能,该文研究了物理-酶联合改性对SPI（pH值为4）的乳化性能影响,通过对比确定了物理-酶联合改性,即超声波-酶复合改性和挤压膨化-酶复合改性两种改性方法在酸性条件下的乳化性能效果最好;并通过对改性后 SPI（pH 值为4）进行溶解性、游离巯基、二硫键、粒径、扫描电镜（scanning electron microscope,SEM）和激光共焦扫描显微镜（confocal laser scanning microscopy,CLSM）分析,从蛋白结构变化上进一步揭示了乳化性能提高现象的原因。结果表明：超声波联合植酸酶-酸性蛋白酶改性的 SPI （Uphy-aci-SPI）的乳化活性（emulsifying activity index,EAI）为0.53 m2/g,比未改性SPI（0.18 m2/g）显著提高了196%（P<0.05）,乳化稳定性（emulsifying stability index,ESI）为17 min,比未改性SPI（13.5 min）显著提高了25.9%（P<0.05）;挤压膨化联合菠萝蛋白酶改性的SPI（Ebro-SPI）的EAI为0.46 m2/g,比未改性SPI显著增加了155%（P<0.05）,ESI为17 min,比未改性SPI显著增加了25.9%（P<0.05）。在pH值为4的条件下对物理-酶联合改性的SPI的性质分析发现,物理-酶联合改性的SPI与未改性SPI相比,物理-酶联合改性的SPI的溶解性显著增加（P<0.05）;物理-酶联合改性的SPI的乳状液平均粒径减小,CLSM观察乳状液中油与蛋白溶液稳定共融,改善了油滴之间的空间排斥力。物理-酶联合改性的SPI游离巯基的含量显著增加（P<0.05）,二硫键含量显著降低（P<0.05）。SEM观察物理-酶联合改性的SPI为结构松散、破碎均一的微观结构。由此可见,乳化性能的提高是通过深层改变蛋白的结构来实现的。该研究可为探索提高酸性条件下SPI的乳化性能的方法提供理论依据。     

Batch Chromium(VI), Cadmium(II) and Lead(II) Removal from Aqueous Solutions by Horticultural Peat

The selectivity and uptake capacity of horticultural peat available in Romania was evaluated with respect to the removal of Cd(II), Cr(VI) and Pb(II) ions from aqueous solution. The kinetics, sorption capacities, selectivity and pH dependence of sorption were determined. The influence of metal concentration in solution is discussed in the terms of Langmuir and Freundlich isotherm and constants. Sorption capacities increased with increasing metal concentration in solution. For solutions containing 300 mg/l of metal, the observed uptake capacities were 20 mg Cd(II)/g peat, 15 mg Cr(VI)/g peat and 30 mg Pb(II)/g peat. The study proved that horticultural peat is a suitable material for the removal of the studied heavy metal ions from aqueous solutions, achieving removal efficiencies higher than 90%, and could be considered as a potential material for treating effluent polluted with Cd(II), Cr(VI) and Pb(II) ions.     

Characterization of a cowpea (Vigna unguiculata) rhizobiophage and its effect on cowpea nodulation and growth

A cowpea rhizobiophage (JRW 3 phage) from Jamaican soil was isolated and characterized. The phage has a polyhedral head and a non-contractile tail; maximum adsorption of the phage to the host occurred after 5 min. A one-step growth experiment revealed that the latent period, rise period and burst size of JRW3 phage were 12 h, 16 h, and 28 plaque-forming units/cell, respectively. The JRW 3 phage was highly sensitive to heat, but survived well between pH 5 and 8. The phage was stable in EDTA, though completely inactivated in sodium citrate. Host range analysis showed that 7 of the 40Rhizobium andBradyrhizobium strains tested were sensitive to phage infection. The phage significantly reduced nodule numbers and shoot dry weight of cowpea plants when inoculated with rhizobia in combination with the phage.