Engineering soya bean seeds as a scalable platform to produce cyanovirin‐N,a non‐ARV microbicide against HIV

There is an urgent need to provide effective anti‐HIV microbicides to resource‐poor areas worldwide. Some of the most promising microbicide candidates are biotherapeutics targeting viral entry. To provide biotherapeutics to poorer areas, it is vital to reduce the cost. Here, we report the production of biologically active recombinant cyanovirin‐N (rCV‐N), an antiviral protein, in genetically engineered soya bean seeds. Pure, biologically active rCV‐N was isolated with a yield of 350 μg/g of dry seed weight. The observed amino acid sequence of rCV‐N matched the expected sequence of native CV‐N, as did the mass of rCV‐N (11 009 Da). Purified rCV‐N from soya is active in anti‐HIV assays with an EC₅₀ of 0.82–2.7 nM (compared to 0.45–1.8 nM for E. coli‐produced CV‐N). Standard industrial processing of soya bean seeds to harvest soya bean oil does not diminish the antiviral activity of recovered rCV‐N, allowing the use of industrial soya bean processing to generate both soya bean oil and a recombinant protein for anti‐HIV microbicide development.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Towards the development of a sustainable soya bean‐based feedstock for aquaculture

Soya bean (Glycine max (L.) Merr.) is sought after for both its oil and protein components. Genetic approaches to add value to either component are ongoing efforts in soya bean breeding and molecular biology programmes. The former is the primary vegetable oil consumed in the world. Hence, its primary usage is in direct human consumption. As a means to increase its utility in feed applications, thereby expanding the market of soya bean coproducts, we investigated the simultaneous displacement of marine ingredients in aquafeeds with soya bean‐based protein and a high Omega‐3 fatty acid soya bean oil, enriched with alpha‐linolenic and stearidonic acids, in both steelhead trout (Oncorhynchus mykiss) and Kampachi (Seriola rivoliana). Communicated herein are aquafeed formulations with major reduction in marine ingredients that translates to more total Omega‐3 fatty acids in harvested flesh. Building off of these findings, subsequent efforts were directed towards a genetic strategy that would translate to a prototype design of an optimal identity‐preserved soya bean‐based feedstock for aquaculture, whereby a multigene stack approach for the targeted synthesis of two value‐added output traits, eicosapentaenoic acid and the ketocarotenoid, astaxanthin, were introduced into the crop. To this end, the systematic introduction of seven transgenic cassettes into soya bean, and the molecular and phenotypic evaluation of the derived novel events are described.     

Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.     

Isolation and characterization of lipid-degrading Bacillus thermoleovorans IHI-91 from an icelandic hot spring

An efficient lipid-degrading thermophilic aerobic bacterium was isolated from an icelandic hot spring and classified as Bacillus thermoleovorans IHI-91. The aerobic bacterium grows optimally at 65°C and pH 6.0 and secretes a high level of lipase (300 U l⁻¹). The newly isolated strain utilizes several lipids such as palmitic acid, stearic acid, lanolin, olive oil, sunflower seed oil, soya oil, and fish oil as sole carbon and energy source without an additional supply of growth factors. The degradation of about 93% of triolein, which is present in olive oil, was observed after only 7 h of fermentation at a maximal growth rate of 1.0 h⁻¹. During growth at optimal conditions on yeast extract, the doubling time was only 15 min. Based on 16S rDNA studies, DNA–DNA hybridization and morphological and physiological properties, the isolate IHI-91 was identified as Bacillus thermoleovorans IHI-91 sp. nov. Because of its production of high concentrations of thermoactive lipases and esterases and the capability of degrading a wide range of lipids at high temperatures, the isolated strain is an ideal candidate for application in various biotechnological processes such as wastewater treatment. Received: August 25, 2000 / Accepted: September 26, 2000     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Evaluation of alpha-galactosidase biosynthesis by Streptomyces griseoloalbus in solid-state fermentation using response surface methodology

Aim: The present study was aimed at evaluating the effects of the three crucial factors, galactose concentration, inoculum size and moisture content, on α‐galactosidase production by the filamentous actinobacterium Streptomyces griseoloalbus in solid‐state fermentation. Methods and Results: Central Composite design was adopted to derive a statistical model for the optimization of fermentation conditions. Maximum α‐galactosidase yield (117 U g^–1 of dry fermented substrate) was obtained when soya bean flour supplemented with 1·5% galactose and with initial moisture content of 40% was inoculated with 1·9 × 10⁶ CFU g^?1 initial dry substrate. Conclusions: The model was valid and could result in considerably enhanced enzyme yield. Significance and Impact of the Study: The results indicated a cost effective method for the production of α‐galactosidase using soya bean flour. This is the first report on exploitation of the potential of filamentous bacterium for the production of α‐galactosidase, an enzyme having versatile applications.     

Fermentation characteristics of cell-wall sugars from soya bean meal,and from separated endosperm and hulls of soya beans

Two experiments were conducted to investigate the degradation of cell-wall sugars from soya bean meal (in situ), and soya bean endosperm and hulls (in vitro). Soya bean meal, soya bean endosperm, and soya bean hulls were extracted with different chemicals to obtain the cell-wall fraction. Soya bean meal cell walls were incubated in the rumen of a fistulated cow. The individual cell-wall sugars were degraded at different rates: galactose (13.6% h⁻¹), arabinose (7.8% h⁻¹), uronic acids (5.1% h⁻¹), xylose (3.5% h⁻¹) and glucose (3.2% h⁻¹). Microscopic evaluation of the cell walls and degraded material revealed the presence of two cell wall types, with distinctly different degradation characteristics: one originating from the hull (thick, slowly degraded) and one from the endosperm (thin, rapidly degraded). Furthermore, the cell-wall sugar composition of endosperm and hull cell walls was different, most markedly for galactose (281 vs. 12 g kg⁻¹) and glucose (132 vs. 508 g kg⁻¹). The degradation of endosperm and hull cell walls was measured in vitro by use of in vitro cumulative gas production. Degradation rates of the individual cell-wall sugars for hull cell walls were similar (ranging from 2.4% to 4.6% h⁻¹). For endosperm cell walls, the degradation rates of the individual sugars were different but with the same ranking as in the in situ experiment (ranging from 20.9% to 7.0% h⁻¹). It was concluded that for soya bean meal cell walls, the cell-wall sugar degradation pattern is influenced by the presence of two cell-wall types (hull and endosperm cell-wall), which differ in their rate of degradation and sugar composition. The difference in cell-wall sugar degradation pattern between hull and endosperm cell walls is likely to be caused by a combined effect of particle size and cell-wall thickness.     

A Cocktail ELISA Semi‐nested RT‐PCR Assay to Detect Bean pod mottle virus in Soya bean Seeds

Bean pod mottle virus (BPMV) has been identified as an important pathogen for plant quarantine in China because large quantities of soya bean seeds (approximately 7 × 10⁷ tons) are imported annually. To develop a practical detection programme for BPMV, a cocktail enzyme‐linked immunosorbent assay (ELISA) nested RT‐PCR using a combination of serological and molecular methods was designed for soya bean seeds. The single‐vessel detection assay was performed in a 96‐well ELISA plate, which served as a carrier for the subsequent nested RT‐PCR assay. Assay specificity was demonstrated by the production of the expected 330‐ and 296‐bp bands using the external and internal primers, respectively. This method was 10⁴‐fold more sensitive than immunocapture‐RT‐PCR (IC‐RT‐PCR). In particular, it is important to note that this assay resulted in successful micro‐extraction from soya bean seeds and combined the advantages of each individual technique. The cocktail ELISA nested RT‐PCR is a specific, sensitive, rapid and economical procedure to rapidly identify and characterize BPMV and could be suitable for both primary‐level platforms and laboratories.     

Black soldier fly as dietary protein source for broiler quails: apparent digestibility,excreta microbial load,feed choice,performance, carcass and meat traits

In order to expand with validated scientific data the limited knowledge regarding the potential application of insects as innovative feed ingredients for poultry, the present study tested a partial substitution of soya bean meal and soya bean oil with defatted black soldier fly (Hermetia illucens) larvae meal (H) in the diet for growing broiler quails (Coturnix coturnix japonica) on growth performance, mortality, nutrients apparent digestibility, microbiological composition of excreta, feed choice, carcass and meat traits. With this purpose, a total of 450 10-day-old birds were allocated to 15 cages (30 birds/cage) and received three dietary treatments: a Control diet (C) and two diets (H1 and H2) corresponding to 10% and 15% H inclusion levels, respectively (H substituted 28.4% soya bean oil and 16.1% soya bean meal for H1, and 100% soya bean oil and 24.8% soya bean meal for H2, respectively). At 28 days of age, quails were slaughtered, carcasses were weighed, breast muscles were then excised from 50 quails/treatment, weighed, and ultimate pH (pHu) and L*, a*, b* colour values were measured. Breast muscles were then cooked to assess cooking loss and meat toughness. For the digestibility trial, a total of 15 28-day-old quails were assigned to the three feeding groups. The excreta samples were subjected to chemical and microbiological analysis. The same 15 quails were then simultaneously provided with C and H2 diets for a 10-day feed choice trial. Productive performance, mortality and carcass traits were in line with commercial standards and similar in all experimental groups. With the exception of ether extract digestibility, which was lower in H1 group compared with C and H2 (P=0.0001), apparent digestibility of dry matter, CP, starch and energy did not differ among treatments. Microbial composition of excreta was also comparable among the three groups. Feed choice trial showed that quails did not express a preference toward C or H2 diets. Breast meat weight and yield did not differ among C, H1 and H2 quails. Differently, the inclusion of H meal reduced meat pHu compared with C. In conclusion, this study demonstrated that H. illucens larvae meal can partially replace conventional soya bean meal and soya bean oil in the diet for growing broiler quails, thus confirming to be a promising insect protein source for the feed industry. Further research to assess the impact of H meal on intestinal morphology as well as on meat quality and sensory profile would be of utmost importance.     

Prediction of the amino acid digestibility of legume seeds in growing pigs: a meta-analysis approach

On pig farms, a high proportion of the cost of production comes from feed costs. However, the use of alternative ingredients such as legume seeds may help to reduce this cost. In fact, legume seeds are an important source of essential amino acids (EAA) and can therefore be an alternative to oilseed meals. However, the accurate use of these legume seeds requires a precise knowledge of the standardized ileal digestibility (SID) of EAA, which may vary depending on its botanical variety. A meta-analysis was performed on a database compiling data from 41 studies published between 1981 and 2013 and 178 dietary treatments. Models of prediction of the SID of EAA as well as the dietary concentration of digestible standardized EAA (dEAA) were obtained, based on the chemical composition of ingredients reported in the publications. The effect of the type of legume seeds (faba bean, lupin, pea and soya bean), surgical procedures (T-cannula, re-entrant cannulas, post valve T-cannulas and ileo-rectal anastomosis), and BW of pigs (BW⩽25 kg BW>25 kg) were also tested in each model. Results showed that dietary CP and crude fibre (CF) were, respectively, the best predictors of each EAA SID for faba bean, lupin and pea (R²=0.42 to 0.89) and soya bean (R²=0.32 to 0.77). For the dEAA content, the best prediction models included dietary CP and ADF for faba bean, lupin and pea and soya bean, respectively, with R² ranging from 0.66 to 0.98. Models developed in this study allow predicting the digestibility of EAA in these alternatives feedstuffs.     

Use of unextracted sunflower seeds as a protein source

Substitution of unextracted sunflower seeds for either 0, 25, 50 or 100% of the soya bean meal in pig diets produced no significant differences in digestible energy or apparent nitrogen retention. However, all diets containing unextracted sunflower seeds had significantly higher digestible nitrogen than the diets containing soya bean meal as the protein source. Replacement of 25% of the soya bean meal with unextracted sunflower seeds produced the greatest increase in digestibility. Rate and efficiency of gain in rats were used to evaluate the effects of autoclaving unextracted sunflower seeds at 115°C with 1.05 kg/cm² pressure for 0, 5 or 10 minutes. Rats fed on the basal maize-soya bean meal diet gained significantly faster and more efficiently than the rats fed on the diets containing the sunflower seeds. An increase in heating time of the sunflower seeds produced a significant reduction in rate and efficiency of rat gain.     

Turnover of Storage Protein in Seeds of Soya Bean and Pea

We were interested in determining whether the low protein contentof pea seeds (Pisum sativum L.) as compared to soya bean seeds(Glycine max L. Merrill) might be due to faster degradationof the pea storage proteins during development of the seed.Pea and soya bean cotyledons were subjected to a ‘pulse-chase’experiment using [³H]glycine in in-vitro cultures. In peas,legumin had a half-life of 146 days, while vicilin had a half-lifeof 39 days. There was no measureable degradation of soya beanstorage proteins. Even with the pea storage proteins, the half-liveswere so much longer than the maturation time of seeds that degradationof storage proteins could not account for the lower proteincontent of peas as compared to soya beans. The validity of theseresults was indicated by the finding that non-storage proteinshad much shorter half-lives and that omission of a carbon ora nitrogen source greatly accelerated degradation. Labelledglycine was found to be a good probe for protein turnover studiesbecause it was very rapidly metabolized. Glycine max L. Merrill, soya bean, Pisum sativum, L. pea, protein turnover, storage proteins, legumin, vicilin     

Effect of culture media on protease and oxytetracycline production with mycelium and protoplasts of Streptomyces rimosus

For the simultaneous production of protease and oxytetracycline, mycelium and protoplasts of Streptomyces rimosus TM-55 were cultivated in basal medium containing soluble starch, corn steep liquid, ammonium sulphate, calcium carbonate, sodium chloride and soybean oil. Protease and oxytetracycline production increased with decreasing in ratio of culture broth to vessel volume from 1:2 to 1:5. Each ml of broth with 0.286 mg fresh mycelia yielded 168–204 units of protease and 785–972 g of oxytetracycline after replacement of corn steep liquor, sodium chloride and soybean oil with beef extract and sunflower oil, while each ml of broth with 7.5 × 10⁷ protoplasts produced 141–153 units of protease and 504–615 g of oxytetracycline. Protease and oxytetracycline production were low when the pH was 5.1 or 9.0. Soluble starch and ammonium sulphate were the best carbon and nitrogen sources, respectively. Supplementation with calcium carbonate enhanced protease and oxytetracycline production. The productivity of protoplasts decreased sharply when the incubation temperature increased from 28 to 34 °C, while the productivity of mycelium was almost unchanged.     

Improvement of Photorhabdus temperata bioinsecticides production in low‐cost media through adequate fermentation technology

To develop a cost effective process for bioinsecticides production by Photorhabdus temperata, dissolved oxygen (DO) requirements were investigated in both the complex and the optimized media using diluted seawater as a source of micronutrients. By varying DO concentrations, tolerance to hydrogen peroxide was shown to be medium dependant. Indeed, P. temperata cells grown in the complex medium, exhibited higher tolerance than cells grown in the optimized medium (OM). Tolerance to H₂O₂ was shown to be related to intracellular reactive oxygen species (ROS) accumulation during soya bean meal or glucose assimilation, as shown by flow cytometry analysis. To avoid oxidative stress damages in P. temperata cells cultured in the OM, DO concentration should be constant 50% saturation throughout the fermentation. However, a DO‐shift control strategy was demonstrated to be beneficial for P. temperata bioinsecticide production in the complex medium. By using such a strategy biomass, culturability, and oral toxicity reached 16.5 × 10⁸, 1.15 × 10⁸ cells/mL and 64.2%, respectively, thus was 16.19, 26.37, and 12.2% more than in the cultures carried out at a constant 50% saturation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effects of amendments on biodegradation of crude petroleum by sediment bacteria from Bonny River Estuary

The biodegradation of Bonny light crude petroleum by bacteria in batch culture was enhanced by the addition to culture media, of 0.2 mg of urea and soya bean lecithin per 100 ml of crude oil, sediment and water mixture. Biodegradation was found to be purely an aerobic process. There was a direct relationship between hydrocarbon content and proportion (%) of total heterotrophic count that was capable of growing on crude petroleum as sole carbon and energy source.     

Enhanced mannanase production by submerged culture of Aspergillus niger NCH-189 using defatted copra based media

Copra and other mannans including locust bean gum, guar gum, konjac flour, copra and defatted copra were used to produce extracellular mannanase by shaken flask cultures of Aspergillus niger NCH-189 in this study. The best carbon source for mannanase production was defatted copra, which provided more nitrogen source and mannan content. The peak mannanase activity at 28 U ml⁻¹ was obtained on the day 3 at 30 °C, which was four times of those obtained from other carbon sources. Presence of oil in copra depressed the mannanase production of the fungus and the amount should be less than 3% (w/w). The copra suspension could be sequentially treated by boiling and refrigeration, followed by using n-hexane to remove copra oil.     

CRISPR/Cas9‐mediated targeted mutagenesis of GmFT2a delays flowering time in soya bean

Flowering is an indication of the transition from vegetative growth to reproductive growth and has considerable effects on the life cycle of soya bean (Glycine max). In this study, we employed the CRISPR/Cas9 system to specifically induce targeted mutagenesis of GmFT2a, an integrator in the photoperiod flowering pathway in soya bean. The soya bean cultivar Jack was transformed with three sgRNA/Cas9 vectors targeting different sites of endogenous GmFT2a via Agrobacterium tumefaciens‐mediated transformation. Site‐directed mutations were observed at all targeted sites by DNA sequencing analysis. T1‐generation soya bean plants homozygous for null alleles of GmFT2a frameshift mutated by a 1‐bp insertion or short deletion exhibited late flowering under natural conditions (summer) in Beijing, China (N39°58′, E116°20′). We also found that the targeted mutagenesis was stably heritable in the following T2 generation, and the homozygous GmFT2a mutants exhibited late flowering under both long‐day and short‐day conditions. We identified some ‘transgene‐clean’ soya bean plants that were homozygous for null alleles of endogenous GmFT2a and without any transgenic element from the T1 and T2 generations. These ‘transgene‐clean’ mutants of GmFT2a may provide materials for more in‐depth research of GmFT2a functions and the molecular mechanism of photoperiod responses in soya bean. They will also contribute to soya bean breeding and regional introduction.     

Overcoming the production limitations of Photorhabdus temperata ssp. temperata strain K122 bioinsecticides in low-cost medium

For low-cost production of Photorhabdus temperata ssp. temperata strain K122 bioinsecticide, a cheap complex medium was optimized. Diluted seawater was used as the source of micronutrients, especially sodium chloride, involved in the improvement of cell density, culturability and oral toxicity of the bacterium P. temperata against Ephestia kuehniella larvae. Thus, the new formulated medium was composed only of 10 g/l of soya bean meal, used as the carbon and nitrogen main source, mixed in sevenfold diluted seawater. At such conditions, several limitations of P. temperata bioinsecticide productions were shown to be overcome. The appearance of variants small colony polymorphism was completely avoided. Thus, the strain K122 was maintained at the primary form even after prolonged incubation. Moreover, the viable but nonculturable state was partially overcome, since the ability of P. temperata cells to form colonies on the solid medium was prolonged until 78 h of incubation. In addition, when cultured in the complex medium, P. temperata cells were produced at high cell density of 12 × 10⁸ cells/ml and exhibited 81.48% improvement of oral toxicity compared to those produced in the optimized medium. With such medium, the large-scale bioinsecticides production into 3-l fully controlled fermenter improved the total cell counts, CFU counts and oral toxicity by 20, 5.81 and 16.73%, respectively. This should contribute to a significant reduction of production cost of highly potent P. temperata strain K122 cells, useful as a bioinsecticide.