An economic approach for L-(+) lactic acid fermentation by Lactobacillus amylophilus GV6 using inexpensive carbon and nitrogen sources

AIMS: Development of cost-effective production medium by applying statistical designs for single-step fermentation of starch (corn flour - CF) to L-(+) lactic acid, using inexpensive nitrogen sources as substitutes for peptone and yeast extract in MRS medium by amylolytic Lactobacillus amylophilus GV6. METHODS AND RESULTS: A two-level Plackett-Burman design was employed for screening various available crude starches (flours) for L-(+) lactic acid production by Lact. amylophilus GV6 using red lentil flour (RL) and bakers yeast cells (YC) as substitutes for commercial peptone and yeast extract in MRS medium in anaerobic submerged fermentation. Of all the tested flours, CF was found to be the most significant. Central composite rotatable design was employed to determine maximum production of L-(+) lactic acid at optimum values of process variables, CF, RL, YC, CaCO(3) and incubation period (IP). minitab analyses showed that lactic acid production was significantly affected by the linear terms CF, RL, CaCO(3) and IP. The interactions of CF-RL, CF-YC, CF-CaCO(3), RL-YC and RL-CaCO(3) and the square terms CF and IP were significant. The maximum lactic acid production of 29 g/37 g of starch present in 50 g of CF was obtained at optimized concentrations of CF 5%, RL 0.7%, YC 0.8%, CaCO(3) 0.8% and IP 2.9 days. CONCLUSIONS: Successful application of Plackett-Burman design helped in identifying CF as the best carbon source among the tested flours for L-(+) lactic acid production using inexpensive nitrogen sources. Further optimization of the process variables by response surface methods (RSMs) led to maximum production of lactic acid (29 g lactic acid from 37 g of starch present in 50 g of flour). SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus amylophilus GV6 showed 78.4% lactic acid production efficiency (g lactic acid produced/g starch taken) and 96% lactic acid yield efficiency (g lactic acid produced/g starch utilized). Information from the present studies provides a better understanding on production of L-(+) lactic acid on fermentation of CF using inexpensive nitrogen sources and on changes in the production as a response from interaction of factors. Use of inexpensive nitrogen sources and starch as substrate in MRS medium for single-step fermentation of lactic acid can become an efficient, economic and viable process. This report is on optimization of inexpensive nitrogen sources completely replacing peptone and yeast extract in single-step submerged fermentation of starch (present in CF) to lactic acid with high production efficiency.     

The effects of reaction conditions on the production of γ-cyclodextrin from tapioca starch by using a novel recombinant engineered CGTase

A novel mutant enzyme namely H43T CGTase can produce up to 39% γ-cyclodextrin (γ-CD) compared to the native enzyme which produces only 10% γ-CD. The effect of the reaction conditions on γ-CD production was studied using this mutant CGTase. The effects of substrate–buffer combination, starch pretreatment and concentration, pH, additives and finally the use of a debranching enzyme improved the γ-CD ratio further. The tapioca–acetate pair gave the highest conversion (16% conversion) among four types of starch and four buffer system combinations. Gelatinized starch was preferred compared to raw tapioca starch in producing a high percentage of γ-CD and conversion rate. Higher pH especially pH 8–9 led to a higher proportion of γ-CD, and was relatively more apparent when the concentration of starch was increased. Forty-six percent γ-CD was produced using 2.5% gelatinized tapioca starch at pH 8. Pullulanase enzyme was found to be useful in reducing the viscosity of tapioca starch paste thus increasing the efficiency of utilization of starch by CGTase by at least 20- to 30-fold. Up to 48% γ-CD can be produced when 4% pullulanase-pretreated tapioca starch was reacted with the CGTase mutant. It was also found that the supplementation of the reaction mixture with glucose, toluene, or cyclododecanone improved the γ-CD yield by 42.2, 46.4, 43.4, and 43.4%, respectively. All the parameters involved have been shown to affect the product specificity of the mutant H43T CGTase transglycosylation mechanism.     

A novel multiplexed immunoassay identifies CEA,IL-8 and prolactin as prospective markers for Dukes’ stages A-D colorectal cancers

Background

Current methods widely deployed for colorectal cancers (CRC) screening lack the necessary sensitivity and specificity required for population-based early disease detection. Cancer-specific protein biomarkers are thought to be produced either by the tumor itself or other tissues in response to the presence of cancers or associated conditions. Equally, known examples of cancer protein biomarkers (e.g., PSA, CA125, CA19-9, CEA, AFP) are frequently found in plasma at very low concentration (pg/mL-ng/mL). New sensitive and specific assays are therefore urgently required to detect the disease at an early stage when prognosis is good following surgical resection. This study was designed to meet the longstanding unmet clinical need for earlier CRC detection by measuring plasma candidate biomarkers of cancer onset and progression in a clinical stage-specific manner. EDTA plasma samples (1 μL) obtained from 75 patients with Dukes’ staged CRC or unaffected controls (age and sex matched with stringent inclusion/exclusion criteria) were assayed for expression of 92 human proteins employing the Proseek® Multiplex Oncology I proximity extension assay. An identical set of plasma samples were analyzed utilizing the Bio-Plex Pro™ human cytokine 27-plex immunoassay.

Results

Similar quantitative expression patterns for 13 plasma antigens common to both platforms endorsed the potential efficacy of Proseek as an immune-based multiplex assay for proteomic biomarker research. Proseek found that expression of Carcinoembryonic Antigen (CEA), IL-8 and prolactin are significantly correlated with CRC stage.

Conclusions

CEA, IL-8 and prolactin expression were found to identify between control (unaffected), non-malignant (Dukes’ A + B) and malignant (Dukes’ C + D) stages.

Electronic supplementary material

The online version of this article (doi:10.1186/s12014-015-9081-x) contains supplementary material, which is available to authorized users.     

Effectiveness of Antegrade Access in Bladder Tumors With Inaccessible Urethra

Inaccessible urethra with no retrograde endoscopic access due to multiple/diffuse strictures or multiple urethrocutaneous fistulas with acute urinary retention due to posturethral instrumentation (transurethral resection of bladder tumor [TURBT], or TURBT with transurethral resection of the prostate [TURP]), is a rare entity. Management of such a case with a bladder tumor for TURBT/surveillance cystoscopy poses a great challenge. The authors present 12 cases of bladder tumor with inaccessible urethra, 10 cases due to multiple strictures (post-TURBT and/or TURP), and 2 cases due to urethrocutaneous fistulas (post-TURBT), who presented to our emergency department with acute urinary retention. Emergent suprapubic catheterization was used as a temporary treatment method.Key words: Suprapubic cystostomy, Inaccessible urethra, Bladder tumors, Tract seedlingBladder tumors are the most common neoplasm of the lower urinary tract, comprising 6% of all malignancies in men and 2% of those in women.¹ A majority of patients present with gross painless hematuria, usually as the sole presenting symptom.² Bladder carcinoma is unique among human neoplasms in that many of its etiologic factors are known; the urologist should be aware of the possible occupational exposures to urothelial carcinogens.³ Initial symptoms of urothelial carcinoma of the bladder (UCB) include microhematuria, painless macrohematuria, and/or irritative voiding symptoms, and require further investigation. Carcinoma in situ of the bladder causes irritative lower urinary tract symptoms (LUTS) more often than does papillary UCB. Histopathologic evaluation is necessary to assess stage and grade with sufficient certainty after the appearance of bladder tumors.⁴ Bladder tumors spread by implantation in abdominal wounds, denuded epithelium, resected prostatic fossa, or traumatized urethra⁵; implantation occurs most often with high-grade tumors.     

Discovery of Predictive Biomarkers for Litter Size in Boar Spermatozoa

Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that l-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5′-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility.Prognosis and diagnosis of male fertility is a major concern in both animals and humans worldwide. In humans, about half of the fertility problems arise because of male factors. In addition, 50% of breeding system failures that are contributed by the sire lead to huge economic drawbacks in the animal industry (1 –5). Therefore, the development of new methods is needed to ensure more accurate prognosis and diagnosis of male fertility.Worldwide, artificial insemination (AI)¹ has been extensively performed in animal industries. Recent data revealed that more than 90% of the sows in Europe and the United States have been bred using AI during last three decades (6). The AI system sufficiently contributes to the achievement of high performance swine production through the selection of quality semen. Moreover, AI has been implemented extensively in swine industries for genetic up-grading (7, 8). However, the selection of high quality semen still depends on conventional sperm analyses such as the analysis of sperm morphology (9), motility (10), and sperm penetration assays (11, 12). Although these tests are commonly used to evaluate the male factor of fertility/infertility, the clinical value is still debated (13). Therefore, to evaluate the limits of conventional sperm analyses, the development of new methods to assess sperm function and fertility should be seriously considered for animal species of economic importance as well as for humans. Additionally, it is important to note that the optimization of sperm production will be possible when the methods to choose superior sires with greater efficiency become available. In this regard, the identification of global protein biomarkers using comprehensive proteomic tools represents a new method on the horizon that may facilitate the prediction of superior sires.Recently, several studies have reported that proteomics is an effective tool that has the potential to transform our understanding of spermatozoa (14 –16) by acquiring new biomarkers of male infertility and/or fertility. In addition, the development of mass spectrometry (MS) allows the potential identification of sperm proteins (17, 18). In fact, increased knowledge of the sperm proteome allows us to identify new molecular markers.In this study, we used high- and low-litter size boar spermatozoa to develop suitable biomarkers. First, sperm motility (%), motion characteristics, and capacitation status were evaluated by computer-assisted sperm analysis (CASA) and combined Hoechst 33258/chlortetracycline fluorescence assessment. Second, to find deferentially expressed proteins (>threefold) between high- and low-litter size boar spermatozoa, a 2-DE proteomic approach was applied following the identification of proteins by ESI-MS/MS and a MASCOT search. The 2-DE results were confirmed by a Western blot analysis that was performed with five commercially available antibodies. Third, to validate discovered markers for male fertility prediction, the expression levels of five proteins from 20 randomly selected boar spermatozoa with broad fertility ranges (i.e. litter size) were analyzed by enzyme-linked immunosorbent assay (ELISA), and the relationship between protein expression and male fertility was determined. Moreover, to represent the entire proteomic event, biological functions and interactions of the deferentially expressed proteins were schematized by a signaling pathway.     

Foraging and growth responses of cotton armyworm Spodoptera litura to the biophysical characteristics of five cotton varieties

This study investigated the morphological and biochemical characteristics of the CB1, CB3, CB5, CB8 and CB12 cotton varieties and evaluated their effects on third instar larval movement, and body weight of the cotton armyworm Spodoptera litura at different developmental stages. The cotton varieties differed in their plant architecture, branching, stem color and hairiness, leaf color and hairiness, leaf trichome density, flower color, numbers of leaves and bolls per plant, concentrations of protein and starch, and boll length, width and weight. The CB1 and CB3 varieties possessed significantly higher trichome densities, while CB8 produced larger and heavier bolls. Boll bearing was found to be highest in CB1 and lowest in CB8. Biochemical analyses indicated the highest percentage of protein in CB5, and of starch in CB8; concentrations of both were lowest in CB12. Cotton varieties did not affect larval foraging, but their abundance on leaflet, mature and square differed significantly. Analysis of the growth response parameters of S. litura as a result of feeding on the tested varieties revealed that larval instars, pupae and adult moths attained the highest body weight on CB8, followed by CB5, and the lowest weight on CB12. Collectively, the results of this study show that the CB5 and CB8 varieties are favorable host‐plants for cotton armyworms; therefore, these varieties are the least suitable for cultivation.     

Bioconcentration of some macrominerals in soil,forage and buffalo hair continuum: A case study on pasture irrigated with sewage water

The present study aimed to evaluate the bioaccumulation of some macrominerals in grazing buffaloes fed forage irrigated with sewage water or canal water. In particular, the transfer of sodium (Na), magnesium (Mg), potassium (K) and calcium (Ca) from soil to plant and in turn to animals was evaluated under sub-tropical environmental conditions. Samples of soil, forage and buffalo hair were collected and digested by wet method. Sodium and K concentrations were significantly higher in the soil but lower in the forages; however, Mg and Ca concentrations in both soil and forages were higher. The correlation between soil, forage and hair showed an imbalanced flow of Na, Mg and K and a balanced flow of Ca from soil to forage and then to animals. Based on the findings, the highest rates of transfer of minerals were found for sewage water treatment, whereas lowest rates were found for canal water treatment, except for Na. As the transfer of minerals depends on their bioavailability, the highest values may be due to the high rates of mineral uptake by plants. Thus, the high transfer rate of some elements by plants could become toxic in future causing detrimental effect to grazing livestock.     

Studies on the role of goat heart galectin-1 as an erythrocyte membrane perturbing agent

Galectins are β-galactoside binding lectins with a potential hemolytic role on erythrocyte membrane integrity and permeability. In the present study, goat heart galectin-1 (GHG-1) was purified and investigated for its hemolytic actions on erythrocyte membrane. When exposed to various saccharides, lactose and sucrose provided maximum protection against hemolysis, while glucose and galactose provided lesser protection against hemolysis. GHG-1 agglutinated erythrocytes were found to be significantly hemolyzed in comparison with unagglutinated erythrocytes. A concentration dependent rise in the hemolysis of trypsinized rabbit erythrocytes was observed in the presence of GHG-1. Similarly, a temperature dependent gradual increase in percent hemolysis was observed in GHG-1 agglutinated erythrocytes as compared to negligible hemolysis in unagglutinated cells. The hemolysis of GHG-1 treated erythrocytes showed a sharp rise with the increasing pH up to 7.5 which became constant till pH 9.5. The extent of erythrocyte hemolysis increased with the increase in the incubation period, with maximum hemolysis after 5 h of incubation. The results of this study establish the ability of galectins as a potential hemolytic agent of erythrocyte membrane, which in turn opens an interesting avenue in the field of proteomics and glycobiology.