Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.     

Effect of Variations in the Fatty Acid Chain of Oligofructose Fatty Acid Esters on Their Foaming Functionality

In this article the effect of variations in the fatty acid chain of oligofructose fatty acid esters (OFAE) on foamability and foam stability is described. First, oligofructose (OF) mono-esters containing saturated fatty acid chains ranging between C4 and C18 were studied. Additionally, a mono-ester containing a C16 mono-unsaturated fatty acid chain and a C12 di-ester were studied. Finally, to investigate the influence of the size of the hydrophilic group, commercially available sucrose esters were studied. The surface tension and surface rheological properties of air/water interfaces stabilized by the esters were determined, as well as the foaming properties of the esters, at a bulk concentration of 0.2 % (w/v). OF mono-esters with intermediate fatty acid chain lengths (C10-C16) were able to migrate quickly to the interface producing foams with small bubbles (0.4 mm), a relatively narrow bubble size distribution, and a high stability. For oligofructose mono-esters containing fatty acids C4 and C8, the bulk concentration of 0.2 % (w/v) was below the CMC, resulting in insufficient surface coverage, and low foamability and foam stability. The OF C18 mono-ester and the OF C12 di-ester were slow to migrate to the interface resulting in low foamability. Despite similar surface tension values, the foam half-life time of OFAE was higher than of the corresponding sucrose esters. OFAE gave higher surface dilatational moduli compared to sucrose esters. Based on the frequency dependence of the modulus and analysis of Lissajous plots, we propose that OFAE may be forming a soft glass at the interface.     

Carbohydrate Storage in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana was grown in 1% (wt/vol) gelatin-liquid media singly supplemented with a monosaccharide (glucose or fructose), a disaccharide (maltose or trehalose), a polyol (glycerol, mannitol, or sorbitol), or the amino sugar N-acetyl-d-glucosamine. The relative contributions of the carbohydrate, protein, and water contents in the fungal biomass were determined. Carbohydrates composed 18 to 42% of the mycelial dry weight, and this value was lowest in unsupplemented medium and highest in medium supplemented with glucose, glycerol, or trehalose. Biomass production was highest in liquid cultures supplemented with trehalose. When liquid cultures were grown in medium supplemented with 0 to 1% (wt/vol) glucose, trehalose, or N-acetyl-d-glucosamine, there was an increase in the biomass production and the contribution of carbohydrate to mycelial dry weight. Regardless of the glucose concentration in the culture, water content of the mycelia remained about 77.5% (wt/wt). Mycelial storage carbohydrates were determined by capillary gas chromatography. In gelatin-liquid medium supplemented with 1% (wt/vol) glucose, B. bassiana stored glycogen (12.0%, wt/dry wt) and the polyols mannitol (2.2%), erythritol (1.6%), glycerol (0.4%), and arabitol (0.1%). Without glucose, B. bassiana stored glycogen (5.4%), mannitol (0.8%), glycerol (0.6%), and erythritol (0.6%) but not arabitol. To our knowledge, this is the first report of carbohydrate storage in an entomopathogenic fungus, and the results are discussed in relation to other fungi and the potential implications to commercial formulation and insect-fungus interactions.     

Removal of peroxides in polyethylene glycols by vacuum drying: Implications in the stability of biotech and pharmaceutical formulations

The purpose of this study was to investigate the utility of vacuum drying for removing peroxides from polyethylene glycols (PEGs). PEG solutions (PEG 1450 and PEG 20000) containing varying levels of peroxides were prepared by storing under different light and temperature conditions. PEGs containing low and high levels of peroxides were vacuum dried from dilute and concentrated solutions (2.5%, 7.5%, 15%, and 50% wt/vol of PEG 1450 and 2.5%, 7.5%, 15%, and 25% wt/vol of PEG 20000). Ferrous ion oxidation in presence of ferric ion indicator xylenol orange (FOX) colorimetric assay was used to determine the concentration of peroxides. Peroxide content in PEGs increased upon storage. The increase was more pronounced when PEGs were stored at higher temperatures and exposed to light. Vacuum drying at 0.1 mm Hg for 48 hours at 25°C resulted in greater than 90% decrease in the level of peroxides in all cases except when high peroxide containing 25% wt/vol solution of PEG 20000 or 50% wt/vol solution of PEG 1450 were dried. The reduction in the level of peroxides for PEGs dried from high peroxide containing 25% wt/vol solution of PEG 2000 and 50% wt/vol solution of PEG 1450 was found to be 88% and 52%, respectively. Oxidation of methionine in Met-Leu-Phe peptide was significantly reduced when vacuum-dried PEGs were used. Vacuum drying PEG solutions at low pressures is an effective method for the removal of the residual peroxides present in commercially available PEGs. Published: July 28, 2006     

Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25°C/60% RH climate chamber, before placing all vials in climate chambers (25°C/60% RH and 40°C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40°C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.     

Recrystallization of Ice Crystals in Trehalose Solution at Isothermal Condition

An isothermal ice recrystallization behavior in trehalose solution was investigated. The isothermal recrystallization rate constants of ice crystals in trehalose solution were obtained at ?5 °C, ?7 °C, and ?10 °C. Then the results were compared to those of a sucrose solution used as a control sample. Simultaneous estimation of water mobility in the freeze-concentrated matrix was conducted by ¹H spin–spin relaxation time T₂ to investigate mechanisms causing the different ice crystal recrystallization behaviors of sucrose and trehalose. At lower temperatures, lower recrystallization rates were obtained for both trehalose and sucrose solutions. The ice crystallization rate constants in trahalose solution tended to be smaller than those in sucrose solution at the same temperature. Although different ice contents (less than 3.6%) were observed between trehalose and sucrose solutions at the same temperature, the recrystallization behaviors of ice crystals were not markedly different. The ¹H spin–spin relaxation time T₂ of water components in a freeze-concentrated matrix for trehalose solution was shorter than in a sucrose solution at the same temperature. Results show that the water mobility of trehalose solutions in freeze-concentrated matrix was less than that of sucrose solutions, which was suggested as the reason for retarded ice crystal growth in a trehalose solution. Results of this study suggest that the replacement of sucrose with trehalose will not negatively affect deterioration caused by ice crystal recrystallization in frozen foods and cryobiological materials.     

The composition of fresh and stored oyster mushrooms (Pleurotus ostreatus)

Soluble carbohydrate, protein, polysaccharide and cell wall composition were assayed in freshly harvested Pleurotus ostreatus sporophores and those stored for 4 days at 2° or 18°. Mannitol and trehalose were present at 1.8 and 6.5% dry wt respectively in fresh sporophores, and at reduced levels in those stored at 18°. In sporophores stored at 2°, trehalose levels increased by up to 122%. Soluble polysaccharide appeared to be composed of glycogen-like material, which was susceptible to post-harvest breakdown, and components containing mannose and other sugars. The total protein content was 42% dry wt; no protein degradation was seen in sporophores stored at 2°, but about 25% of the protein disappeared during storage at 18°. Cell wall polysaccharide was utilised during storage. Respiration rate was about 8–10 ml CO₂/g dry wt/hr at harvest and declined to about 5 ml/g dry wt/hr after 40 hr storage at 18°.     

Effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drops through excised goat, sheep, and buffalo corneas

The purpose of this investigation was to evaluate the effect of formulation factors on in vitro permeation of moxifloxacin from aqueous drop through freshly excised goat, sheep, and buffalo corneas. Aqueous isotonic ophthalmic solutions of moxifloxacin hydrochloride of different concentrations (pH 7.2) or 0.5% (wt/vol) solutions of different pH or 0.5% solutions (pH 7.2) containing different preservatives were made. Permeation characteristics of drug were evaluated by putting 1 mL formulation on freshly excised cornea (0.50 cm²) fixed between donor and receptor compartments of an all-glass modified Franz diffusion cell and measuring the drug permeated in the receptor (containing 10 mL bicarbonate ringer at 37°C under stirring) by spectrophotometry at 291 nm, after 120 minutes. Statistical analysis was done by one-way analysis of variance (ANOVA) followed by Dunnett’s test. Increase in drug concentration in the formulation resulted in an increase in the quantity permeated but a decrease in percentage permeation. Increase in pH of the solution from 5.5 to 7.2 increased drug permeation, indicating pH-dependent transport. Compared with control formulation, moxifloxacin 0.5% (wt/vol) solution (pH 7.2) containing disodium edetate (EDTA) (0.01% wt/vol) produced significantly (P<.05) higher permeation with all the corneas. Formulation with benzyl alcohol significantly (P<.05) increased permeation with buffalo cornea compared with its control. Presence of benzalkonium chloride (BAK) (0.01% wt/vol) and EDTA (0.01% wt/vol) in the formulation increased permeation to the maximum with all the corneas. The results suggest that moxifloxacin 0.5% ophthalmic solution (pH 7.2) containing BAK (0.01%) and EDTA (0.01%) provides increased in vitro ocular availability through goat, sheep, and buffalo corneas. Published: February 10, 2006 Formerly College of Pharmacy, University of Delhi, Pushp Vihar, Sector III, New Delhi-110017, India     

Preparation of spray-dried wettable powder formulations of Bacillus thuringiensis-based biopesticides

Bacillus thuringiensis is the most widely used biopesticide among many methods available to control insects. To make a saleable product, B. thuringiensis must be substantially concentrated by removal of water and formulated to improve longevity, efficacy, and ease of transport of the product. B. thuringiensis subsp. aizawai culture broth as an active ingredient was mixed with various adjuvants and then spray dried. The optimum conditions for spray drying were found to be an outlet temperature of 60-85 degrees C and an inlet temperature of 120-180 degrees C. Various adjuvants had different effects on physical and biological properties of the dried product. Gelatinized tapioca starch and milk powder improved suspensibility but adversely affected wettability of the dried formulated product. Vegetable oil and Tween 20 enhanced wettability but resulted in poor suspensibility. Silica fume was used to enhance flowability because it reduced clumping and caking of the powder resulting from the addition of vegetable oil. Formulation containing 10% wt:wt B. thuringiensis, 10% wt:wt gelatinized tapioca starch, 10% wt:wt sucrose, 38% wt:wt tapioca starch, 20% wt:wt milk powder, 10% wt:wt silica fume, 2% wt:wt polyvinyl alcohol, 5% vol:vol Tween 20, 1% vol:vol refined rice bran oil, and 1% vol:vol antifoam solution was found to be optimum in terms of the physical and biological properties of the dried product. This formulation had 55% suspensibility, 24 s for wetting time, and 5.69 x 10(4) CFU/ml of LC50 value against Spodoptera exigua larvae.     

Thermal Destabilization of Stem Bromelain by Trehalose

Trehalose, a naturally occurring osmolyte, is considered as a universal protein stabilizer. We investigated the effect of the disaccharides, trehalose and sucrose, on the thermal stability and conformation of bromelain. To our surprise, bromelain in the presence of 1 M trehalose/sucrose was destabilized under thermal stress. The average Tm values as determined by UV spectroscopy and CD spectropolarimetry decreased by 5° and 7°C for bromelain in 1 M sucrose or trehalose solutions, respectively. The enzyme was also found to inactivate faster at 60°C in the presence of these osmolytes. The tertiary and secondary structure of bromelain undergoes small changes in the presence of sucrose/trehalose. Studies on the binding of these osmolytes with the native and the heat denatured enzyme revealed that sucrose/trehalose lead to preferential hydration of the denatured bromelain as compared to the native one, hence stabilizing more the denatured conformation. This is perhaps the first report on the destabilization of a protein by trehalose.     

去甲基万古霉素菌种低温冷冻、冷干保藏条件优化及10 年菌种保藏效果

【目的】针对去甲基万古霉素产生菌不耐保藏的问题,改进菌种保藏方法,对超低温液氮保藏、-80°C低温冷冻保藏、冷干保藏方法跟踪考察10年保藏稳定性,评价不同保藏方法对去甲基万古霉素产生菌的保藏适用性。【方法】采用甘油作基础保护剂进行超低温液氮保藏和-80°C低温冷冻保藏,采用脱脂牛奶作基础保护剂进行冷干保藏,针对超低温液氮保藏进行降温速率考察,研究非渗透性冷冻保护剂海藻糖、聚乙烯吡咯烷酮(PVP)等对3种保藏方法的冻存影响,对优选出的保藏方法进行10年跟踪考察。【结果】3种保藏方法冻后菌种存活率依次为:-80°C低温冷冻保藏超低温液氮保藏冷干保藏。液氮保藏最适降温速率为快速冷冻。优选出最佳保护剂配方:超低温液氮保藏为甘油8.0%,海藻糖3.5%;-80°C低温冷冻保藏为甘油6.0%,PVP 5.0%;冷干保藏为脱脂牛奶,6.0%海藻糖。采用优化保藏条件,液氮保藏10年存活率稳定在70.6%,菌种发酵水平为入藏水平的92.9%。【结论】在优化条件下,尤以超低温液氮保藏适合于去甲基万古霉素产生菌长期保藏。     

Adventitious shoot formation and plant regeneration from Pinus pinaster Sol. ex Aiton

Summary Pinus pinaster plants were regenerated from cotyledons excised from in vitro germinated seeds and axenically cultured on induction medium (GMD). 6-Benzyladenine (2.2 μM) induced the highest frequency of direct bud formation from cotyledons. An average of 13.1 ± 2.1 elongated shoots per cotyledon was obtained. Germination time influenced shoot induction, and the organogenic potential decreased with explant age. Cotyledons remained for 21 d on induction medium, and in order to promote adventitious shoot elongation, they were transferred to Gupta and Durzan’s DCR medium without growth regulators, containing 0.5% (wt/vol) activated charcoal and 3% (wt/vol) sucrose. Rooting was achieved by application of an indole-3-butyric acid, (396.6 μM) pulse (24 h at 4° C), followed by transfer to a sterile mixture of peat plus perlite (1:1 vol/vol). Ninety-eight to 100% of the regenerated plants were successfully acclimatized. All plants have survived after transfer to the field.     

Enhancing viability of two biocontrol yeasts in liquid formulation by applying sugar protectant combined with antioxidant

Survivals of Cryptococcus laurentii and Pichia membranaefaciens in liquid formulations with sugar protectants (trehalose and galactose) and L-ascorbic acid (Vc) were investigated during storage at 4°C and 25°C. When galactose or trehalose was used alone as protectant, C. laurentii maintained relatively high viability in potassium phosphate buffer. Addition of Vc to trehalose improved its protective effect. P. membranaefaciens maintained viability >60% after 90 days at 4°C when 5% galactose served as a protectant, and its combination with Vc was the most effective at maintaining viability. Moreover, liquid formulation kept higher viability of the two yeasts at 4°C than at 25°C. Biocontrol efficiency of the two yeasts was maintained after formulation and storage. The results indicate that trehalose is considered as a suitable protectant for liquid formulation of C. laurentii, while galactose is better for P. membranaefaciens. Combining Vc with the sugars improves the protective efficiency.     

Vitrification of dog spermatozoa: Effects of two cryoprotectants (sucrose or trehalose) and two warming procedures

Sperm vitrification is a low cost and simple technique that does not require special equipment and may represent an attractive alternative to the costly and time consuming conventional dog spermatozoa cryopreservation techniques. The objective of this study was to evaluate different cryoprotectants and warming temperatures on the vitrification of dog spermatozoa. Pooled semen samples from 10 beagle dogs were vitrified with four extenders, based on Tris, citric acid and glucose, 20% egg yolk (TCG-20% EY) and different combinations of sucrose and/or trehalose: 250 mM sucrose; 250 mM trehalose; 125 mM sucrose + 125 mM trehalose; 250 mM sucrose + 250 mM trehalose. Samples were vitrified by dropping 50 μL of sperm suspension directly into liquid nitrogen. After vitrification, warming was done either fast (at 65 °C for 2–5 s) or slow (at 37 °C for one minute). Motility was assayed using a computer-aided sperm analysis (CASA) system; membrane integrity and acrosomal status were analyzed by fluorescence microscopy. For comparison, samples were also conventionally frozen in liquid nitrogen vapor using a TCG-20% egg yolk extender plus 5% glycerol. Frozen straws were thawed in a water bath at 37 °C for 30 s. Poorer motility results (P < 0.05) but similar viability were obtained when vitrification was performed, compared to conventional freezing (P > 0.05). When vitrification was used, cryoprotectants containing either 250 mM sucrose or 250 mM trehalose and warmed at 37 °C returned the best sperm quality variables.     

GUSTATORY REACTION TIME AND TIME INTENSITY MEASUREMENTS OF TREHALOSE AND SUCROSE SOLUTIONS AND THEIR MIXTURES

Dynamic sweetness perception of commercial food grade trehalose, sucrose solutions and their mixtures were studied for a wide range of concentrations. For gustatory reaction time (GRT), concentrations ranged from 2.3 to 13.8% for sucrose and up to 23.0% for trehalose. For time intensity (T-I) sucrose or trehalose solutions (concentration range 2.3–36.8%) and their combinations (23.0 and 36.8% total solids) were analyzed. Trehalose had bigger GRT along the studied range. Both sugars presented similar values for persistence and times of plateau and to maximum intensity, while a significant difference was observed in intensity and GRT at equal concentrations. Trehalose had longer persistence than sucrose in equi -sweet solutions. Overall sweetness profile of some sucrose solutions (i.e., 29.9% sucrose solution and 0.6 sucrose/trehalose ratio mixture at 36.8% total solids) were perceived as similar to mixtures of sucrose/trehalose or single trehalose solutions, which suggests the possibility of sugar replacement without completely modifying sweetness perception.

PRACTICAL APPLICATIONS

It has been suggested that trehalose may be a potential substitute for sucrose and other sugars used in food formulation because, although its chemical structure is very similar to that of sucrose, it is more stable at low pH and high temperatures. It is not involved in caramelization and does not participate in Maillard reaction with amino acids/proteins. In order to fully establish the potential of trehalose as a functional replacement of sucrose we have determined the sweetness dynamic profile (gustatory reaction time and time-intensity curves) of trehalose solutions and sucrose/trehalose solutions; this aspect is needed for adequately replacing (partially or totally) sucrose in food systems.     

Isolation of indigenous enteroviruses from chemically treated and dewatered sludge samples.

Samples of wastewater sludge were examined for infectious enteroviruses before and after they had been chemically conditioned and dewatered. The least virus was recovered from the cake produced by filter pressing of sludge, which had a greatly increased solids content (39 to 45% [wt/vol]) relative to the untreated sludge (4.2 to 6.2% [wt/vol]) and in one plant was at pH 11 due to the lime conditioner used. Conditioning with a cationic polyelectrolyte before dewatering by centrifugation produced a watery sludge (2.7 to 5.3% [wt/vol]) from which high titers of infectious virus were recovered which were often greater than those isolated from the untreated sludge (0.6 to 1.4% [wt/vol]). This was thought to be due to saturation of virus and sludge floc adsorption sites by the polyelectrolyte, resulting in the liberation of virions from the sludge solids.     

Comparison of the effect of tromethamine and polyvinylpyrrolidone on dissolution properties and analgesic effect of nimesulide

The solubilizing and absorption enhancer properties towards nimesulide (ND) of tromethamine (Tris) and polyvinylpyrrolidone (PVP) have been investigated. Solid binary systems were prepared at various drug-polymer ratios by mixing or coprecipitation, characterized by differential scanning calorimetry, X-ray diffractometry, and Fourier transform infrared spectroscopy, and tested for dissolution behavior. Both carriers improved drug dissolution and their performance depended on concentration of the hydrophilic carrier in coprecipitates. Tris was more effective than PVP, despite the amorphizing power of PVP as revealed by solid state analyses. Complete drug amorphiztion was attained at 1∶3 (wt/wt) drug: PVP, 25% (wt/wt) ND in PVP. According to thermal behavior of ND and Tris, ND-Tris systems present a eutectic behavior. The eutectic composition was 30% ND-70% Tris at ∼129°C. Amorphous ND-PVP and eutectic ND-Tris mixtures showed an improvement of 5.55 and 6.6 times of drug dissolution efficiency, respectively. In vivo experiments in mice demonstrated that administration of 60 mg/kg of drug coprecipitated with PVP or Tris resulted, respectively, in a 50% and 94% reduction of acetic acid-induced writhings in comparison with pure drug, which, instead, was statistically ineffective as compared with the control group. Moreover, the eutectic mixture of ND-Tris demonstrated antiwrithing potency 1.88 times higher than amorphous ND-PVP coprecipitate. Thus, the solubilizing power, dissolution-enhancing effect, and analgesic effect enhancer ability toward the drug make Tris particularly suitable for developing a reduced-dose, fast-release solid oral dosage form of nimesulide. Published: August 10, 2007     

Isolation of indigenous enteroviruses from chemically treated and dewatered sludge samples

Samples of wastewater sludge were examined for infectious enteroviruses before and after they had been chemically conditioned and dewatered. The least virus was recovered from the cake produced by filter pressing of sludge, which had a greatly increased solids content (39 to 45% [wt/vol]) relative to the untreated sludge (4.2 to 6.2% [wt/vol]) and in one plant was at pH 11 due to the lime conditioner used. Conditioning with a cationic polyelectrolyte before dewatering by centrifugation produced a watery sludge (2.7 to 5.3% [wt/vol]) from which high titers of infectious virus were recovered which were often greater than those isolated from the untreated sludge (0.6 to 1.4% [wt/vol]). This was thought to be due to saturation of virus and sludge floc adsorption sites by the polyelectrolyte, resulting in the liberation of virions from the sludge solids.     

Cryopreservation of Luciola praeusta Kiesenwetter (Coleoptera: Lampyridae) embryos by vitrification

There is an urgent need to preserve the ever-decreasing number of different species of fireflies all over the world. We sought to develop a vitrification procedure to cryopreserve the firefly embryos. The late stages of Luciola praeusta Kiesenwetter embryos were collected. Several impermeable and permeable protectants with various concentrations in different mediums (TNM-FH insect medium, Grace's medium, Dulbecco's Modification of Eagle's Medium (DMEM) and Dulbecco's Phosphate-Buffered Saline (DPBS)) were used. Embryos culturing in TNM-FH medium yielded the highest survival rate of 75.3 ± 3.6%. One-step, two-step and three-step methods were used in equilibrium procedure respectively. The highest survival rate (73.7% ±3.2%) occurred in embryos treated by three-step method ((1.5 M ethylene glycol (EG) + 2 M EG plus 8% polyvinylpyrrolidone (PVP) + 3 M EG, 8% PVP and 15% trehalose). Additionally, embryos exposed to 0.5 M trehalose presented a significantly higher survival rate (71.8 ± 2.7%) than embryos preserved in 0.5 M sucrose.     

Screening of different adjuvants for wastewater/wastewater sludge-based Bacillus thuringiensis formulations

Screening of different adjuvants, namely, suspending agents, phagostimulants, stickers, antimicrobial agents, and UV screens to develop aqueous biopesticidal suspensions of Bacillus thuringiensis (Bt) variety kurstaki HD-1 fermented broths, specifically, nonhydrolyzed sludge, hydrolyzed sludge, starch industry wastewater, and soya (commercial medium), were investigated. The selected suspending agents [20% (wt:vol)] included sorbitol, sodium monophosphate, and sodium metabisulfite with corresponding suspendibility of 74-92, 69-85, and 71-82%, respectively. Molasses [0.2% (wt:vol)] increased adherence by 84-90% for all fermented broths. The optimal phagostimulants [0.5% (wt:vol)], namely, soya and molasses, caused entomotoxicity increase of 3-13 and 7-13%, respectively. Sorbic and propionic acids showed high antimicrobial action [0.5% (wt:vol)], irrespective of fermentation medium. Sodium lignosulfonate, molasses, and Congo red, when used as UV screens [0.2% (wt:vol)], showed percent corresponding entomotoxicity losses of 3-5, 0.5-5 and 2-16, respectively. The Bt formulations, when exposed to UV radiation, showed higher half-lives (with and without UV screens) than the fermented broths or semisynthetic soya medium and commercial Bt formulation. UV screen-amended nonhydrolyzed, hydrolyzed, and starch industry wastewater formulations showed 1.3-1.5-fold higher half-lives than commercial Bt formulation. Thus, the recommended formulation comprises sorbitol, sodium monophosphate, sodium metabisulfite (suspending agents); molasses, soya flour (phagostimulants); molasses and skimmed milk powder (rainfasteners); sorbic and propionic acids (antimicrobial agents) and sodium lignosulfate; and molasses and Congo red (UV screens). These waste-based Bt formulations offer better UV resistance in comparison with commercial formulation.