A 16S rRNA amplicon approach to the structural and functional diversity of bacterial communities associated with horse gram crop for drought mitigation and sustainable productivity.

AIM: In this study, 16S rRNA amplicon sequencing analyses were performed to determine the diversity of the bacterial community present in the soil, rhizosphere region, root nodules and seeds of the horse gram plant. METHODS AND RESULTS: We observed the dominance of Proteobacteria, Actinobacteria, Firmicutes, Acidobacteria, Bacteroidetes, Planctomycetes and Gemmatimonadetes across all four domains of the horse gram plant. For community analyses, the significance of the alpha diversity was estimated using the Shannon index, Simpson index and Chao1 index, which revealed no significant difference among the samples. However, the estimation of the beta diversity indicated a significant difference among the samples, with p < 0.001 and R2  = 1. A strong positive correlation was found between the rhizosphere and root nodule samples. Comparative genomics of the 16S rRNA gene showed that ammonium-oxidizing metabolism (amoA), nitrite-reducing metabolism (nirK) and nitrogen-fixing metabolism (nifH) were prominent mechanisms in all samples. The genes involved in the biosynthesis of amino acids, purine metabolism and nitrogen metabolism were identified as the key genes associated with the functional traits of microbial domains in horse gram. CONCLUSION: The culturable microbes associated with horse gram can be used as a substitute for synthetic fertilizers to maintain soil fertility and ecological health in agricultural practices. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the survival strategies of bacterial communities that positively respond to multiple gate selection helps in understanding the structural diversity and functional traits primarily focused on the development of beneficial microbial consortium for promoting plant growth.

Influence of processing on bioactive compounds, Type-II diabetes related enzyme regulation potential and antiurolithiatic potential of underutilized legume Macrotyloma uniflorum.

The hydro-alcoholic extract of raw and processed Macrotyloma uniflorum seeds, an underutilized food legume was analysed for its bioactive compounds, Type-II diabetes enzyme regulation and antiurolithiatic potential. The study aimed to establish and promote the introduction of these new grains and enlarge the market of novel functional foods. The seed extract had phenolic content of 35.6 and 30.4 mg GAE/g dm, for PAIYUR-2 and GPM-6 respectively. Chlorogenic, coumaric, vanillic and ellagic were the major and, sinapic and syringic were limiting phenolic acids. The raw seeds extract exhibited ferric ion reducing potential (1125 and 1236 mmol Fe II/mg extract dm), free radical inhibition (EC50, 3.58 and 3.78 g dm/g DPPH), hydroxyl ion inhibition (46.71 and 45.44%) and superoxide ion inhibition (36.93 and 33.37%) for PAIYUR-2 and GPM-6 respectively. Further, considerable α-amylase (49.34 & 45.89%) and α-glucosidase (62.72 & 60.33%) inhibition potentials were observed along with antiurolithiatic activity of 48.12 and 46.31% in PAIYUR-2 and GPM-6 respectively. Although, processing had significant (p ≤ 0.05) impact on grain quality but still the significant (p ≤ 0.05) functional properties were retained. This proves the grain utility as a functional food in maintaining human health.

Morphological traits of drought tolerant horse gram germplasm: classification through machine learning.

BACKGROUND: Horse gram (Macrotyloma uniflorum (Lam.) Verdc.) is an underutilized pulse crop with good drought resistance traits. It is a rich source of protein. Conventional breeding methods for high yielding and abiotic stress tolerant germplasm are hampered by the scarcity of morphological data sets. Thus, horse gram cultivars considered for this study is classified based on prevailing growth factors showing homogenous genotype in various agro ecological zones. Nowadays, several machine learning (ML) methods are used in the field of plant phenotyping. RESULTS: We adopted unsupervised learning techniques from the K-means clustering algorithm to analyze important morphological traits: plant shoot length, total plant height, flowering percentage, number of pods per plant, pod length, number of seeds per plant, and seed length variants between germplasm. Unsupervised clustering revealed that 20 germplasm accessions were grouped in four clusters in which high-yielding traits were predominantly observed in cluster 2. CONCLUSION: These findings could guide ML-based classification to characterize suitable germplasms on the basis of high-yielding varieties for different agro-ecological zones. © 2020 Society of Chemical Industry.

Engineering properties of horse gram (Macrotyloma uniflorum) varieties as a function of moisture content and structure of grain.

The study was conducted to determine the engineering properties of horse gram varieties namely GPM-6, PAYIUR-2, and BHK as a function of moisture content in range of 10-30%. The average length, width, thickness, geometric mean diameter, arithmetic mean diameter, thousand kernel weight, sphericity, porosity and angle of repose ranged from 5.43 to 6.53 mm, 3.96 to 4.48 mm, 2.21 to 2.99 mm, 3.62 to 4.41 mm, 3.86 to 4.64 mm, 30.32 to 49.11 g, 63.56 to 72.66%, 35.20 to 38.76% and 22.72° to 29.86° respectively as the moisture content of the grain increased from 10.08 to 29.98%. The bulk density and true density of the grain decreased from initial range of 810-901 to 734-801 kg m-3 and 1250-1426 to 1168-1308 kg m-3. The volume, porosity and terminal velocity of the grain increased linearly with increase in moisture content. The coefficient of friction also showed positive correlation for all surface materials, the highest increase was found for plywood in all varieties of horse gram seeds. Dehulling properties of the grain found to be significantly affected by the change in moisture content. The overall dehulling ranged from 53.44 to 61.21% for GPM-6 and 55.58 to 61.06% for PAYIUR-2 variety of horse gram. Textural properties of the grains were also found to be significantly affected by the change in moisture content from 10 to 30%. The data generated in this study will be highly useful in optimization of post-harvest processing operations as well as to design and develop related processing equipment for horse gram.

Influence of processing methodology on phenolic acid content, fatty acid profile and oxidative stability of Macrotyloma uniflorum.

Study was conducted to determine the influence of different cooking methods namely pressure, flaking, infra-red, microwave and extrusion processing on rate of oxidative stability, fatty acid profile and functional properties of horse gram. The rate of auto-oxidation was significantly (p < 0.05) affected during storage and found maximum at 0.00 aw and minimum at 0.33 aw for both varieties namely GPM-6 and PAIYUR-2. The extrusion processed grains were more susceptible to auto-oxidation. The iron content of grain increased significantly (p < 0.05) during extrusion (1.22 ± 0.50 to 1.65 ± 0.15 mg 100 g-1 for PAIYUR-2 and 1.19 ± 0.45 to 1.59 ± 0.12 mg 100 g-1 for GPM-6). Whereas, tocopherol content decreased during extrusion (8.05 ± 0.15 to 2.28 ± 0.23 mg 100 g-1 for PAIYUR-2 and 6.48 ± 0.46 to 1.68 ± 0.15 mg 100 g-1 for GPM-6). Ellagic (12.36 ± 0.35 and 10.71 ± 0.29 mg 100 g-1), vanillic (15.20 ± 0.23 and 12.48 ± 0.18 mg 100 g-1), and coumaric acid (14.68 ± 0.71 and 8.97 ± 0.66 mg 100 g-1) were the major phenolic acids whereas, linoleic (35.53 ± 0.30 and 35.46 ± 0.19%), palmitic (26.08 ± 0.26 and 25.97 ± 0.33%), and linolenic acid (13.44 ± 0.18 and 10.13 ± 0.21%) were the major fatty acids present in raw grain for PAIYUR-2 and GPM-6 respectively. Phenolic and fatty acids were significantly (p < 0.05) affected during processing. The oxidative stability of microwave processed grains was maximum whereas, extrusion processed grains showed minimum oxidative stability during storage. Study explicitly describes that native grains and the grains which did not undergo destruction of naturally present cellular structure, were less prone to oxidation. The oxidation rate of grains was found dependent on composition, processing and storage environment of grains. Hence, all these factors need to be considered to ensure the stability of processed food during storage.

Global De Novo Protein-Protein Interactome Elucidates Interactions of Drought-Responsive Proteins in Horse Gram (Macrotyloma uniflorum).

Inspired by the availability of de novo transcriptome of horse gram (Macrotyloma uniflorum) and recent developments in systems biology studies, the first ever global protein-protein interactome (PPI) map was constructed for this highly drought-tolerant legume. Large-scale studies of PPIs and the constructed database would provide rationale behind the interplay at cascading translational levels for drought stress-adaptive mechanisms in horse gram. Using a bidirectional approach (interolog and domain-based), a high-confidence interactome map and database for horse gram was constructed. Available transcriptomic information for shoot and root tissues of a sensitive (M-191; genotype 1) and a drought-tolerant (M-249; genotype 2) genotype of horse gram was utilized to draw comparative PPI subnetworks under drought stress. High-confidence 6804 interactions were predicted among 1812 proteins covering about one-fourth of the horse gram proteome. The highest number of interactions (33.86%) in horse gram interactome matched with Arabidopsis PPI data. The top five hub nodes mostly included ubiquitin and heat-shock-related proteins. Higher numbers of PPIs were found to be responsive in shoot tissue (416) and root tissue (2228) of genotype 2 compared with shoot tissue (136) and root tissue (579) of genotype 1. Characterization of PPIs using gene ontology analysis revealed that kinase and transferase activities involved in signal transduction, cellular processes, nucleocytoplasmic transport, protein ubiquitination, and localization of molecules were most responsive to drought stress. Hence, these could be framed in stress adaptive mechanisms of horse gram. Being the first legume global PPI map, it would provide new insights into gene and protein regulatory networks for drought stress tolerance mechanisms in horse gram. Information compiled in the form of database (MauPIR) will provide the much needed high-confidence systems biology information for horse gram genes, proteins, and involved processes. This information would ease the effort and increase the efficacy for similar studies on other legumes. Public access is available at http://14.139.59.221/MauPIR/ .

Kaulath, a new fungal fermented food from horse gram.

Horse gram (Macrotyloma uniflorum) is used in the traditional method for treatmentof several health complications. It is also known that fermentation of such substrates yields a number of compounds that enhance the overall activities against several disease states. Solid state fermentation of horse gram using Penicillium camemberti showed an inhibition of pancreatic lipase and alpha glucosidase activities. The fermented material, termed Kaulath, showed 60 % increase in fat content. A reduction in sodium and increased levels of potassium and calcium was observed in Kaulath. In addition, a higher free radical scavenging activity was noted in this product compared to unfermented horse gram. Anti-nutritional factors, such as phytic acid and trypsin inhibitors showed a reduction in Kaulath. Furthermore, Kaulath, upto 1 g per kg body weight, did not exhibit any mortality or toxic effects in experimental rats after 14 days of administration. The hematological and clinical parameters were within safe limits between the groups, supported by the histopathology of liver and kidney. These results indicate potential food use of Kaulath in diets and as functional ingredients in formulated foods.

Effect of process and machine parameters on physical properties of extrudate during extrusion cooking of sorghum, horse gram and defatted soy flour blends.

Extrusion cooking of sorghum (Sorghum vulgaris), horse gram (Dolichos biflorus) and defatted soy (Glycine max) flour blends was done to prepare snacks by using a Brabender single-screw laboratory extruder. The combined effect of moisture content, blend ratio of feed, barrel temperature and screw speed of extruder on physical parameters of extrudate was studied. It was observed that 15% moisture content of feed, 80:10:10 (sorghum flour: horse gram flour: defatted soy flour) of blend ratio, 130 °C barrel temperature and 130 rpm of screw speed gave the highest sectional expansion index and longitudinal expansion index of extrudate, while 12% moisture content, 75:15:10 of blend ratio of feed, 135 °C of barrel temperature and 135 rpm of screw speed gave lowest bulk density of extrudate. A central composite rotable design (CCRD) of response surface methodology was used to develop prediction model. Second order quadratic regression model fitted adequately in the variation. The significance was established at p ≤ 0.05. It was also observed that increasing feed moisture content results in a higher density and lower expansion of extrudate. Increasing barrel temperature and screw speed reduced density but increased expansion of extrudate.

Comparative Metabolomic Profiling of Horse Gram (Macrotyloma uniflorum (Lam.) Verdc.) Genotypes for Horse Gram Yellow Mosaic Virus Resistance.

Horse gram (Macrotyloma uniflorum (Lam.) Verdc.) is an under-utilized legume grown in India. It is a good source of protein, carbohydrates, dietary fiber, minerals, and vitamins. We screened 252 horse gram germplasm accessions for horse gram yellow mosaic virus resistance using the percent disease index and scaling techniques. The percentage values of highly resistant, moderately resistant, moderately susceptible, susceptible, and highly susceptible were 0.34, 13.89, 38.89, 46.43, and 0.34, respectively. Repetitive trials confirmed the host-plant resistance levels, and yield loss was assessed. The present disease index ranged from 1.2 to 72.0 and 1.2 to 73.0 during the kharif and rabi seasons of 2018, respectively. The maximum percent yield loss was noticed in the HS (75.0 -89.4), while HR possessed the minimum (1.2-2.0). The methanolic leaf extracts of highly resistant and highly susceptible genotypes with essential controls were subjected to gas chromatography-mass spectrometry analysis. Differential accumulation of metabolites was noticed, and a total of 81 metabolites representing 26 functional groups were identified. Both highly resistant and susceptible genotypes harbored eight unique classes, while ten biomolecules were common. The hierarchical cluster analysis indicated a distinct metabolite profile. Fold change in the common metabolites revealed an enhanced accumulation of sugars, alkanes, and carboxylic acids in the highly resistant genotype. The principal component analysis plots explained 93.7% of the variation. The metabolite profile showed a significant accumulation of three anti-viral (octadecanoic acid, diphenyl sulfone, and 2-Aminooxazole), one insecticidal (9,10-Secocholesta-5,7,10(19)-triene-3,24,25-triol), one antifeedant (cucurbitacin B), and six metabolites with unknown biological function in the highly resistant genotype.

Studies on phytochemical, mineral content, in vitro anti-urolithiatic and anti-diabetic activities of horse gram flour extracts and its biosynthesized Ag nanoparticles.

The current study aims to examine the use of horse gram flour and its extracts as a low-cost source of mineral content with anti-urolithiatic, anti-diabetic properties and to compare the biological activities with its biosynthesized AgNPs. The secondary metabolites and the macro and micronutrients present in the selected herbal product, reinforces the utility of the horse gram as a promising herbal. Present investigation reveals that the biomass chosen for the study as a cheap natural source with valuable mineral content of calcium (43.06 mg/L), followed by potassium (13.78 mg/L) and sodium (6.94 mg/L). The grain's water extracts found to contain carbohydrate as dominating component with the value of (65.10 ± 0.95 mg g-1 equivalent). Whereas both water and ethanol extract contain TPC (phenol) viz; 60.13 ± 2.45 mg g-1, 68.24 ± 1.33 mg g-1, TTC (terpenoids)53.21 ± 1.4 mg g-1,51.27 ± 2.65 mg g-1, followed by TSC (sterol)as 45.58 ± 1.7 mg g-1, 57.27 ± 1.65 mg g-1 in moderate amounts respectively. The aqueous extract of Horse gram was used for the synthesis the AgNPs through a straightforward green approach and characterized by FESEM, TEM, zeta potential, X-ray diffraction, UV spectroscopy and particle size measurement. These studies demonstrate the production of AgNPs with an average particle size of 30 nm-60 nm. Investigation on anti-urolithiatic property with aqueous extract, (HGW), ethanol extract (HGE) and its biosynthesized Ag nanoparticles (HG-Ag) reveal that, among the three samples chosen, the biosynthesized Ag nanoparticles possess the best inhibiting activity. The disintegration of crystals in gel medium further validated the extracts and Ag nanoparticles crystal growth inhibitory activity, at concentrations of 2% for extracts and 200 µg/ml for AgNPs. Further, it is also found that the HG-Ag nanoparticle exhibit good anti-diabetic activity (75.36%) than the other two extracts (HGW Extract-67.18% & HGE Extract-44.29%). Thus, the seed flour extracts and its AgNps demonstrated to be a promising natural herbal product with potential mineral content, antidiabetic and anti-urolithiatic activities which could be a used as a value-added product in the dietary formulations.

In vitro exposed magnesium oxide nanoparticles enhanced the growth of legume Macrotyloma uniflorum.

Nanoparticles interact with plants to induce a positive, negative, or neutral effect on their growth and development. In this study, we document the positive influence of magnesium oxide (MgO) nanoparticles (NPs) on the morpho-biochemical parameters of Macrotyloma uniflorum (horse gram). Horse gram is a protein and polyphenol-rich legume crop. It is an important part of the human diet and nutrition. When exposed to MgO-NPs, a significant increment in the shoot-root length, fresh biomass, and chlorophyll content of horse gram was evident. Furthermore, there was a 4-20 and 18-127% increase in the accumulation of carbohydrate and protein content on MgO-NP exposure. The antioxidant potential was enhanced by 5-19% on NP treatment as a result of the increase in the accumulation of total polyphenolics. Total phenols and flavonoids were enhanced by 7-20 and 50-84% in the presence of MgO-NPs. The enzyme activity of SOD, CAT, and APX was also enhanced in MgO-NP-exposed horse gram. The observed alterations were also justified by the Pearson correlation. Overall, the MgO-NP-induced morpho-biochemical alterations in horse gram indicated their probable role as a nano-fertilizer. However, it further warrants the need to extensively investigate the responses of various other plant types to MgO-NPs before industry scale application.

A simple and efficient Agrobacterium-mediated in planta transformation protocol for horse gram (Macrotyloma uniflorum Lam. Verdc.).

BACKGROUND: Recalcitrant nature is a major constraint for the in vitro regeneration and genetic transformation of leguminous species members. Therefore, an improved genetic transformation in horse gram has been developed via in planta method, in which Agrobacterium strain harboring binary vector pCAMBIA2301 was used for the transformation. Several factors affecting in planta transformations were put forth viz. Agrobacterium cell density, co-cultivation, and sonication combined with vacuum infiltration duration which were optimized. RESULTS: Germinated seeds were sonicated and vacuum infiltrated with different densities of Agrobacterium culture and co-cultivated in half-strength MS medium with 100 µM of acetosyringone for 48 h. Seedlings were washed with cefotaxime and sowed in vermiculite soil for maturation. T1 plants were subjected to histochemical and molecular analysis to ensure transformation efficiency. Among various combinations analyzed, maximum transformation efficiency (20.8%) was attained with seeds of 5 min sonication combined with vacuum infiltration with 0.6 optical density of Agrobacterium culture. CONCLUSIONS: It concludes that a different Agrobacterium cell density with sonication combined with vacuum infiltration has improved transgenic efficiency in horse gram plants. This simple and efficient method is feasible for the stable expression of foreign genes that could be beneficial for future food security.

Effect of enzymatic hydrolysis on the functional, antioxidant, and angiotensin I-converting enzyme (ACE) inhibitory properties of whole horse gram flour.

Horse gram hydrolysate (HGH) with different degree of hydrolysis (DH) (20, 25, 35, 40, and 45%) was prepared from whole horse gram flour using alcalase. The amino acid composition of HGH showed the presence of essential amino acids. The alcalase hydrolysis (DH ≥ 20%) increased protein solubility with a notable difference in the pH range of 3-5 (p < 0.05). The emulsifying activity and stability of HGH improved with increase in pH, especially at DH ≥ 25% (p < 0.05). With increase in DH, the foaming properties reduced while the antioxidant and angiotensin I-converting enzyme inhibitory activities increased. Sensory evaluation showed no significant difference (p > 0.05) in preference between control soup and soup mixed with HGH. Thus, these results suggest the possibility of HGH to be used as an appropriate functional ingredient with different food applications including in management of oxidative stress as well as in controlling hypertension .

Nutritional implications and flour functionality of popped/expanded horse gram.

Utilization of horse gram and its flour in legume composite flours and products is limited due to the presence of antinutritional components, poor functional and expansion properties. Enzymatic treatment was used to improve the expansion and functional properties of horse gram to facilitate its use as an ingredient in food processing. Xylanase-mediated depolymerization of cell wall polysaccharides of horse gram lead to the development of a new expanded/popped horse gram. Expansion process of enzyme treated horse gram resulted in increased length (5.3-6.8mm) and higher yield of expanded grains (63-98%). The expanded horse gram had lower bulk density, higher protein digestibility and more resistant starch compared to the control raw grains. Dietary fibre content of raw and processed horse gram was in the range of 14.57-16.14%. High temperature short time (HTST) conditions used during expansion process lowered the levels of phytic acid, tannins and protease inhibitors by 46%, 61% and 92%, respectively. The flour obtained from xylanase treated and expanded horse gram had higher water (204.3g/100g) and oil absorption capacities (98.4g/100g) than unprocessed flour, which had 135.8g/100g and 74.6g/100g, respectively at ambient conditions. There was a decrease in foaming capacity and foam stability in expanded gram flour. However, emulsion stability increased significantly in the processed samples. Thus, the study indicated that nutritional value and flour functionality of horse gram could be improved by processing it into a new expanded product that can be used as an ingredient in food processing.

Comparative effect of horse gram and black gram on inflammatory mediators and antioxidant status.

A balanced diet is important for the overall wellbeing of an individual. Pulses are an important part of a nutritive diet. Pulses have been consumed for at least 10,000 years and are among the most extensively used foods in the world. They are a rich source of protein and fiber, as well as a significant source of vitamins and minerals, such as iron, zinc, and magnesium. The purpose of this study was to compare the effect of two pulses, horse gram and black gram, on inflammatory mediators and the antioxidant enzymes. Two sets of experiments were conducted in rats which were fed with boiled and unboiled horse gram and black gram, at a dose of 100 mg/100 g body weight, for 21 days and 60 days. The results showed that horse gram supplementation for 21 days and 60 days significantly increased the activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase and showed no significant changes in the activities of the inflammatory mediators such as cyclooxygenase, lipoxygenase, myeloperoxidase, nitric oxide synthase, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-α), interleukin-1-beta (IL-1ß), etc. However, the black gram (with skin and without skin) supplementation significantly increased activities of the inflammatory mediators and showed a significant decrease in the antioxidant enzymes in both the 21-day and 60-day experiments. Thus, these preliminary results demonstrate the anti-inflammatory and antioxidant potential of horse gram and the proinflammatory effects of black gram in rats. This is in accordance with the dietary regime advised by Ayurveda practitioners, where horse gram is to be included and black gram is to be excluded from the diet for conditions such as rheumatoid arthritis. Further studies are to be conducted to validate the same.

Quality evaluation of physical properties, antinutritional factors, and antioxidant activity of bread fortified with germinated horse gram (Dolichus uniflorus) flour.

Horse gram was germinated at 90% RH at 25°C for 72 h after 24 h soaking and then grinded to pass through 150 µm mesh size screens. The germination of horse gram result in increased protein, fiber, total polyphenol content, and antioxidant activity of horse gram flour whereas fat, ash, carbohydrate, iron, calcium, tannin, phytate, and oxalate were reduced due to germination. Bread was prepared by the incorporation of (2%, 4%, 6%, and 8%) germinated horse gram flour (GHF) by a straight dough method. The loaf volume and specific volume decreased with an increased use of percentage of GHF. The sensory evaluation revealed that the incorporation of GHF up to 6% was acceptable. The protein (% db), fiber (% db), ash (% db), iron (mg/100 g), calcium (mg/100 g), tannin (mg/g), phytate (mg/g), oxalate (mg/g), total polyphenol content (GAE/g), and antioxidant activity (DPPH % inhibition) was found to be 9.08 ± 0.01, 1.23 ± 0.15, 1.36 ± 0.11, 4.07 ± 0.03, 128 ± 0.26, 2.06 ± 0.15, 2.46 ± 0.15, 0.7 ± 0.1, 12.44 ± 0.40, and 31.13 ± 0.25, respectively, in 6% GHF incorporated bread. The research concludes that 6% GHF incorporation in bread enhance the polyphenol content and antioxidant properties.