Physicochemical properties and in vitro digestibility of flour and starch from pea (Pisum sativum L.) cultivars

Flours and isolated starches from three different cultivars (1544-8, 1658-11 and 1760-8) of pea grown under identical environmental conditions were evaluated for their physicochemical properties and in vitro digestibility. The protein content, total starch content and apparent amylose content of pea flour ranged from 24.4 to 26.3%, 48.8 to 50.2%, and 13.9 to 16.7%, respectively. In pea starches, the 1760-8 showed higher apparent amylose content and total starch content than the other cultivars. Pea starch granules were irregularly shaped, ranging from oval to round with a smooth surface. All pea starches showed C-type X-ray diffraction pattern with relative crystallinity ranging between 23.7 and 24.7%. Pea starch had only a single endothermic transition (12.1-14.2 J/g) in the DSC thermogram, whereas pea flour showed two separate endothermic transitions corresponding to starch gelatinization (4.54-4.71 J/g) and disruption of the amylose-lipid complex (0.36-0.78 J/g). In pea cultivars, the 1760-8 had significantly higher setback and final viscosity than the other cultivars in both pea flour (672 and 1170 cP, respectively) and isolated starch (2901 and 4811 cP). The average branch chain length of pea starches ranged from 20.1 to 20.3. The 1760-8 displayed a larger proportion of short branch chains, DP (degree of polymerization) 6-12 (21.1%), and a smaller proportion of long branch chains, DP ≥ 37 (8.4%). The RDS, SDS and RS contents of pea flour ranged from 23.7 to 24.1%, 11.3 to 12.8%, and 13.2 to 14.8%, respectively. In pea starches, the 1760-8 showed a lower RDS content but higher SDS and RS contents. The expected glycemic index (eGI), based on the hydrolysis index, ranged from 36.9 to 37.7 and 69.8 to 70.7 for pea flour and isolated pea starch, respectively.     

Extraction and chemical characterization of starch from S. lycocarpum fruits

In this study the pulp from Solanum lycocarpum fruits was used as raw material for extraction of starch, resulting in a yield of 51%. The starch granules were heterogeneous in size, presenting a conical appearance, very similar to a high-amylose cassava starch. The elemental analysis (CHNS) revealed 64.33% carbon, 7.16% hydrogen and 0.80% nitrogen. FT-IR spectroscopy showed characteristic peaks of polysaccharides and NMR analysis confirmed the presence of the α-anomer of d-glucose. The S. lycocarpum starch was characterized by high value of intrinsic viscosity (3515 mPa s) and estimated molecular weight around 645.69 kDa. Furthermore, this starch was classified as a B-type and high amylose content starch, presenting 34.66% of amylose and 38% crystallinity. Endothermic transition temperatures (T_o = 61.25 °C, T_p = 64.5 °C, T_c = 67.5 °C), gelatinization temperature (ΔT = 6.3 °C) ranges and enthalpy changes (ΔH = 13.21 J g⁻¹) were accessed by DCS analysis. These results make the S. lycocarpum fruit a very promising source of starch for biotechnological applications.     

In vitro and in vivo digestion of octenyl succinic starch

This study aimed to understand effects of octenyl succinic anhydride (OSA) modification of normal corn (NCS) and high-amylose corn (HA7) starch on their enzymatic hydrolysis rates. After modification with 3% and 10% OSA, resistant starch (RS) contents of the cooked OS-NCS increased from 0.8% of the control starch to 6.8% and 13.2% (Englyst Method), respectively, whereas that of the cooked OS-HA7 decreased from 24.1% to 23.7% and 20.9%, respectively. When the cooked NCS, HA7 and OS (10%)-HA7 were used to prepare diets for rats at 55% (w/w) starch, RS contents of the diets were 1.1%, 13.2% and 14.6%, respectively. After feeding to the rats, 20.2–31.1% of the starch in the OS (10%)-HA7-diet was not utilized in vivo and was found in rat feces, which was substantially larger than that of the HA7-diet (≤4.9%) and NCS-diet (≤0.2%). The body weights of the rats, however, remained similar between different groups.     

Multi-objective optimization of bioethanol production during cold enzyme starch hydrolysis in very high gravity cassava mash

Cold enzymatic hydrolysis conditions for bioethanol production were optimized using multi-objective optimization. Response surface methodology was used to optimize the effects of α-amylase, glucoamylase, liquefaction temperature and liquefaction time on S. cerevisiae biomass, ethanol concentration and starch utilization ratio. The optimum hydrolysis conditions were: 224 IU/g_starch α-amylase, 694 IU/g_starch glucoamylase, 77 °C and 104 min for biomass; 264 IU/g_starch α-amylase, 392 IU/g_starch glucoamylase, 60 °C and 85 min for ethanol concentration; 214 IU/g_starch α-amylase, 398 IU/g_starch glucoamylase, 79 °C and 117 min for starch utilization ratio. The hydrolysis conditions were subsequently evaluated by multi-objectives optimization utilizing the weighted coefficient methods. The Pareto solutions for biomass (3.655-4.380 × 10⁸ cells/ml), ethanol concentration (15.96-18.25 wt.%) and starch utilization ratio (92.50-94.64%) were obtained. The optimized conditions were shown to be feasible and reliable through verification tests. This kind of multi-objective optimization is of potential importance in industrial bioethanol production.     

Interplanting Annual Ryegrass,Wheat, Oat,and Corn to Mitigate Iron Deficiency in Dry Beans

This study evaluated whether grass intercropping can be used to alleviate Fe deficiency chlorosis in dry beans (Phaseolus vulgaris L.) grown in high pH, calcareous soils with low organic matter. Field studies were conducted at the University of Wyoming Sustainable Agriculture Research and Extension Center in 2009 and 2010. Black- and navy beans were grown alone or intercropped with annual ryegrass (Lolium multiflorum Lam.), oat (Avena sativa L.), corn (Zea mays L.), or spring wheat (Triticum aestivum L.) in a two-factor factorial strip-plot randomized complete block design. All four grass species increased chlorophyll intensity in dry beans. However, grass species did not increase iron (Fe) concentration in dry bean tissues suggesting inefficient utilization of Fe present in the dry bean tissues. In 2009, nitrate-nitrogen (NO₃-N) and manganese (Mn) concentration in bean tissue were greater in bean monoculture than in grass intercropped beans. Bean monoculture also had greater soil NO₃-N concentrations than grass intercropped treatments. In 2009, grass intercrops reduced dry bean yield >25% compared to bean monoculture. Annual ryegrass was the least competitive of the four annual grass species. This suggests that competition from grasses for nutrients, water, or light may have outweighed benefits accruing from grass intercropping. Additional studies are required to determine the appropriate grass and dry bean densities, as well as the optimum time of grass removal.     

Plasma glucose response and glycemic indices in pigs fed diets differing in in vitro hydrolysis indices

Different dietary starch sources can have a great impact in determining starch digestion potential, thus influencing the postprandial blood glucose response. Our objectives were to define: (i) the incremental plasma glucose response in pigs fed diets containing various sources of starch differing in in vitro digestion patterns, (ii) the in vivo glycemic index (GI) values for the same diets, (iii) the possible relationship between in vitro and in vivo data. Diets, formulated with 70% of starch from five heterogeneous sources, were characterized in depth by using two distinct in vitro evaluations. The first one was based on the Englyst-assay for nutritional classification of starch fractions, whereas the second one was based on a time-course multi-enzymatic assay up to 180 min from which the hydrolysis indices (HIs) were calculated and used as a link between the physicochemical properties of starch from diets and the in vivo responses. For the in vivo study, five jugular-catheterized pigs (35.3 ± 1.1 kg body weight) were fed one of the five diets for 6-day periods in a 5 × 5 Latin square design. On day 5, blood was collected for 8 h postprandially for evaluating glucose appearance. On day 6, blood was collected for 3 h postprandially for the estimation of the GI. Starchy diets differed for rapidly digestible starch (from 8.6% to 79.8% of total starch (TS)) and resistant starch contents (from 72.5% to 4.5% of TS). Wide between-diets variations were recorded for all the kinetic parameters and for the HI calculated from the in vitro digestion curves (P < 0.05). On the basis of the obtained HI, diets contained starch with a very low to a very high in vitro digestion potential (ranging from 26.7% to 100.0%; P < 0.05). The glucose response differed among diets (P < 0.05), with marked differences between 15 and 120 min postprandial. Overall, the ranking of incremental glucose appearance among diets agreed with their in vitro HI classification: high HI diets increased plasma glucose response more (P < 0.05) than low HI diets. Lastly, different in vivo GIs were measured (ranging from 30.9% to 100.0%; P < 0.05). The relationship between HI and GI showed a high coefficient of determination (R² = 0.95; root mean square error (RMSE) = 15.8; P < 0.05). In conclusion, diets formulated with starches with a wide range in HI potential can strongly affect the postprandial glucose response in pigs.     

Quantification of the In Vitro Radiosensitivity of Mung Bean Sprout Elongation to 6MV X-Ray: A Revised Target Model Study

In this study, a revised target model for quantifying the in vitro radiosensitivity of mung bean sprout elongation to 6-MV X-rays was developed. The revised target model, which incorporated the Poisson prediction for a low probability of success, provided theoretical estimates that were highly consistent with the actual data measured in this study. The revised target model correlated different in vitro radiosensitivities to various effective target volumes and was successfully confirmed by exposing mung beans in various elongation states to various doses of 6-MV X-rays. For the experiment, 5,000 fresh mung beans were randomly distributed into 100 petri dishes, which were randomly divided into ten groups. Each group received an initial watering at a different time point prior to X-ray exposure, resulting in different effective target volumes. The bean sprouts were measured 70 hr after X-ray exposure, and the average length of the bean sprouts in each group was recorded as an index of the mung bean in vitro radiosensitivity. Mung beans that received an initial watering either six or sixteen hours before X-ray exposure had the shortest sprout length, indicating that the maximum effective target volume was formed within that specific time period. The revised target model could be also expanded to interpret the “two-hit” model of target theory, although the experimental data supported the “one-hit” model. If the “two-hit” model was sustained, theoretically, the target size would be 2.14 times larger than its original size to produce the same results.     

Analysis of starch amylolysis using plots for first-order kinetics

Investigators often study product release from starches during prolonged incubations with α-amylase in vitro. The reaction time courses usually fit to a linear form of a first order rate equation, i.e., ln[(C_∞ − C_t)/C_∞] = −kt. This equation calls for an accurate estimate of C_∞, i.e., the concentration of product at the end of the reaction. Estimates of C_∞ from digestibility curves can be unreliable. The Guggenheim method does not require prior knowledge of C_∞ but seems not to have been applied to starch hydrolysis data. An alternative method is also available in which the logarithm of the slope (LOS) of a digestibility curve at various time points is plotted against time. This allows estimations of both k and C_∞ and can also reveal whether changes occur in digestion rate from rapid to slow as digestion proceeds. We describe the Guggenheim and LOS methods and provide examples of their application to starch digestibility data.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Effect of MEM vitamins and forskolin on embryo development and vitrification tolerance of in vitro-produced pig embryos

The aims of this study were (1) to determine the effect of in vitro maturation (IVM) medium supplementation with MEM vitamins on in vitro embryo development and sensitivity to vitrification of Day 6 blastocysts and (2) to evaluate whether the addition of forskolin to in vitro culture (IVC) medium enhances blastocyst survival following Super Open Pulled Straw (SOPS) vitrification. Cumulus–oocyte complexes (COCs; n = 4000) were matured with 0.0% or 0.05% (v/v) MEM vitamins. After 44 h of IVM, the oocytes were in vitro fertilized, and presumptive zygotes were cultured. At Day 5 of IVC, embryos from both experimental groups were cultured for 24 h with 0 or 10 μM forskolin, achieving a 2 × 2 factorial design. The blastocyst formation rate was assessed on Day 6, and subsets of samples from the four experimental groups were vitrified (n = 469) or kept fresh (n = 546). Fresh and vitrified-warmed blastocysts were cultured for 24 h prior to embryo survival and total blastocyst cell number assessment. The MEM vitamins increased (P < 0.001) the blastocyst formation rate at Day 6, but they did not affect embryo survival after vitrification. In contrast, the addition of forskolin to the culture medium enhanced (P < 0.05) the blastocyst vitrification tolerance. The total blastocyst cell number was similar among the groups. In conclusion, supplementation with 0.05% MEM vitamins improved the blastocyst formation rate, and the addition of 10 μM forskolin to the culture medium increased survival in Day 6 in vitro-produced blastocysts after SOPS vitrification.     

Nutritional characterization of Mucuna pruriens: 1. Effect of maturity on the nutritional quality of botanical fractions and the whole plant

This study was conducted to determine the stage of maturity at which the dry matter (DM) yield and nutritive value of velvet bean (Mucuna pruriens) is optimized. Mucuna was harvested at 77, 110 and 123 days after planting (DAP) from quadruplicate 5 m × 1 m plots within each of 6 blocks. At each DAP, DM yield, chemical composition, botanical composition, in vitro rumen fluid-pepsin DM digestibility (IVDMD) and concentrations of total polyphenols, l-dopa and tannins were determined on the whole plant and botanical fractions. Whole-plant Mucuna DM yield increased (P<0.01) linearly with maturity; proportions of leaves and stems decreased linearly (P<0.01), whereas proportion of pods increased (P<0.01). Concentrations of neutral-detergent fiber (aNDF) in whole plant, leaf, and stem increased (P<0.05), or tended (P<0.10) to increase linearly with maturity, as did the acid-detergent fiber concentration of leaves and stems. Maturity decreased (P<0.05) ether extract concentrations of leaves linearly, and stems quadratically, but increased (P<0.05) whole-plant and pod starch concentrations. Pods contained relatively high concentrations of lysine, histidine, phenylalanine, aspartate, glutamate, leucine, isoleucine, and valine, but low concentrations of methionine and cystine. The essential amino acid index did not vary with maturity. Most minerals in Mucuna are concentrated in the leaves and the whole plant contains sufficient Ca, P, K, Mg, Fe, Cu, Na, Mo, Mn, and Zn for growing sheep, although their bioavailability of these minerals is unknown. Total polyphenol concentration quadratically (P<0.01) increased with maturity in the whole plant, tended to increase (P<0.10) in pods, linearly (P<0.01) decreased in stems and fluctuated in leaves. Maturity quadratically increased l-dopa concentration of the whole plant (P<0.05) and stems (P<0.01), but did not affect those of leaves and pods. Maturity quadratically increased (P<0.05) total tannin concentration in the whole plant, but decreased (P<0.10) that of pods. The l-dopa was concentrated in the seeds and pods of mature (110–123 DAP) plants, but tannins were concentrated in leaves and stems. Whole-plant IVDMD was not affected by maturity, but digestible DM yield linearly (P<0.01) increased with increasing DM yield. There was a 2-week harvest window (110–123 DAP) during which whole-plant crude protein and IVDMD remained unchanged. Nevertheless, harvesting at 123 DAP gave the best combination of biomass yield and nutritive value.     

Isolation and characterization of lectins from kidney beans (Phaseolus vulgaris)

The bioactive properties of lectins obtained from raw and canned red kidney bean (Phaseolus vulgaris) were studied to determine the changes in their bioactivity during the canning process. Phytohaemagglutinin (PHA) was extracted using Affi-gel Blue gel and thyroglobulin-Sepharose and had a molecular weight of 32 kDa. Both the raw and the canned kidney beans possessed the ability to agglutinate red blood cells and inhibit α-glucosidase. The activity found in the canned beans was similar to that from the in the raw kidney beans. However, the amount of lectin that could be extracted from thyroglobulin-Sepharose was much less in the canned samples than in the raw kidney bean samples. The extracted lectin from the raw kidney beans was also subjected to a heating and cooling treatment using a differential scanning calorimeter. The lectin had a nonset denaturation temperature of 77.76 °C and it did not renature upon cooling. In this study, we demonstrated that extracts from raw red kidney bean and canned red kidney bean contain bioactive compounds capable of inhibiting HIV-1 RT in vitro.     

Purification and characterization of a thermostable α-amylase produced by the fungus Paecilomyces variotii

An α-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The α-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pI value of 4.5. Temperature and pH optima were 60 °C and 4.0, respectively. The enzyme was stable for 1 h at 55 °C, showing a t₅₀ of 53 min at 60 °C. Starch protected the enzyme against thermal inactivation. The α-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K_m of α-amylase on Reagen® and Sigma® starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to α-amylases from Bacillus sp. These results confirmed that the studied enzyme was an α-amylase ((1→4)-α-glucan glucanohydrolase).     

A titration approach to identify the capacity for starch digestion in milk-fed calves

Calf milk replacers (MR) commonly contain 40% to 50% lactose. For economic reasons, starch is of interest as a lactose replacer. Compared with lactose, starch digestion is generally low in calves. It is, however, unknown which enzyme limits the rate of starch digestion. The objectives were to determine which enzyme limits starch digestion and to assess the maximum capacity for starch digestion in milk-fed calves. A within-animal titration study was performed, where lactose was exchanged stepwise for one of four starch products (SP). The four corn-based SP differed in size and branching, therefore requiring different ratios of starch-degrading enzymes for their complete hydrolysis to glucose: gelatinised starch (α-amylase and (iso)maltase); maltodextrin ((iso)maltase and α-amylase); maltodextrin with α-1,6-branching (isomaltase, maltase and α-amylase) and maltose (maltase). When exceeding the animal’s capacity to enzymatically hydrolyse starch, fermentation occurs, leading to a reduced faecal dry matter (DM) content and pH. Forty calves (13 weeks of age) were assigned to either a lactose control diet or one of four titration strategies (n=8 per treatment), each testing the stepwise exchange of lactose for one SP. Dietary inclusion of each SP was increased weekly by 3% at the expense of lactose and faecal samples were collected from the rectum weekly to determine DM content and pH. The increase in SP inclusion was stopped when faecal DM content dropped below 10.6% (i.e. 75% of the average initial faecal DM content) for 3 consecutive weeks. For control calves, faecal DM content and pH did not change over time. For 87% of the SP-fed calves, faecal DM and pH decreased already at low inclusion levels, and linear regression provided a better fit of the data (faecal DM content or pH v. time) than non-linear regression. For all SP treatments, faecal DM content and pH decreased in time (P<0.001) and slopes for faecal DM content and pH in time differed from CON; P<0.001 for all SP), but did not differ between SP treatments. Faecal DM content of SP-fed calves decreased by 0.57% and faecal pH by 0.32 per week. In conclusion, faecal DM content and pH sensitively respond to incremental inclusion of SP in calf MR, independently of SP characteristics. All SP require maltase to achieve complete hydrolysis to glucose. We therefore suggest that maltase activity limits starch digestion and that fermentation may contribute substantially to total tract starch disappearance in milk-fed calves.     

Effect of corn preparation methods on dry-grind ethanol production by granular starch hydrolysis and partitioning of spent beer solids

Two corn preparation methods, rollermill flaking and hammermill grinding, were compared for efficient processing of corn into ethanol by granular starch hydrolysis and simultaneous fermentation by yeast Saccharomyces cerevisiae. Corn was either ground in a hammermill with different size screens or crushed in a smooth-surfaced rollermill at different roller gap settings. The partitioning of beer solids and size distribution of solids in the thin stillage were compared. The mean particle diameter d₅₀ for preparations varied with set-ups and ranged between 210 and 340 μm for ground corn, and 1180-1267 μm for flaked corn. The ethanol concentrations in beer were similar (18-19% v/v) for ground and flaked preparations, however, ethanol productivity increased with reduced particle size. Roller versus hammermilling of corn reduced solids in thin stillage by 28%, and doubled the volume percent of fines (d₅₀ ∼ 7 μm)in thin stillage and decreased coarse (d₅₀ ∼ 122 μm) by half compared to hammermilling.     

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

Barley limit dextrinase [Hordeum vulgare limit dextrinase (HvLD)] catalyzes the hydrolysis of α-1,6 glucosidic linkages in limit dextrins. This activity plays a role in starch degradation during germination and presumably in starch biosynthesis during grain filling. The crystal structures of HvLD in complex with the competitive inhibitors α-cyclodextrin (CD) and β-CD are solved and refined to 2.5 Å and 2.1 Å, respectively, and are the first structures of a limit dextrinase. HvLD belongs to glycoside hydrolase 13 family and is composed of four domains: an immunoglobulin-like N-terminal eight-stranded β-sandwich domain, a six-stranded β-sandwich domain belonging to the carbohydrate binding module 48 family, a catalytic (β/α)₈-like barrel domain that lacks α-helix 5, and a C-terminal eight-stranded β-sandwich domain of unknown function. The CDs are bound at the active site occupying carbohydrate binding subsites + 1 and + 2. A glycerol and three water molecules mimic a glucose residue at subsite − 1, thereby identifying residues involved in catalysis. The bulky Met440, a unique residue at its position among α-1,6 acting enzymes, obstructs subsite − 4. The steric hindrance observed is proposed to affect substrate specificity and to cause a low activity of HvLD towards amylopectin. An extended loop (Asp513-Asn520) between β5 and β6 of the catalytic domain also seems to influence substrate specificity and to give HvLD a higher affinity for α-CD than pullulanases. The crystal structures additionally provide new insight into cation sites and the concerted action of the battery of hydrolytic enzymes in starch degradation.     

Localization of the Fe(III)-Zn(II) Purple Acid Phosphatase in Dry Kidney Beans Using Immunofluorescence and Immunogold Electron Microscopy

Localization of purple acid phosphatase (PAP) from the seedsof kidney beans,Phaseolus vulgaris(L.), was performed usinglight and transmission electron microscopy. After rehydrationand aqueous fixation, cryo-sections of bean cotyledon tissueshowed a bright immunofluorescent signal in the cytoplasm ofcells whereas cell walls and reserve materials (starch, proteinbodies) remained unstained. In ultrathin sections of dry cotyledontissue anhydrously fixed in acrolein vapour and embedded inLowicryl resin, PAP mapped exclusively to ribosome-rich areasof the cytoplasm. In view of these results, we propose thatkidney bean PAP might possibly be engaged in mechanisms involvedin the triggering of seed dormancy.Copyright 1998 Annals ofBotany Company Phaseolus vulgaris(L.), kidney bean, purple acid phosphatase, immunofluorescence microscopy, immunogold electron microscopy, anhydrous vapour fixation, acrolein.