Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Preparation of Komagataeibacter xylinus inoculum for bacterial cellulose biosynthesis using magnetically assisted external-loop airlift bioreactor

The aim of this study was to demonstrate the applicability of a novel magnetically-assisted external-loop airlift bioreactor (EL-ALB), equipped with RMF generators for the preparation of Komagataeibacter xylinus inoculum during three-cycle repeated fed-batch cultures, further used for bacterial cellulose (BC) production. The fermentation carried out in the RMF-assisted EL-ALB allowed to obtain an inoculum of more than 200x higher cellular density compared to classical methods of inoculum preparation. The inoculum obtained in the RMF-assisted EL-ALB was characterized by a high and stable metabolic activity during repeated batch fermentation process. The application of the RMF-assisted EL-ALB for K. xylinus inoculum production did not induce the formation of cellulose-deficient mutants. It was also confirmed that the ability of K. xylinus to produce BC was at the same level (7.26 g/L of dry mass), regardless of inoculum age. Additionally, the BC obtained from the inoculum produced in the RMF-assisted EL-ALB was characterized by reproducible mechanical strength, nanostructure and total crystallinity index. The results obtained in this study may find multiple applications in any biotechnological processes requiring a high-quality bacterial inoculum.

Effects of Water Temperature, Inoculum Concentration and Age, and Sanitizer Presence on Infection of Ceratocystis fimbriata, Causal Agent of Black Rot in Sweetpotato

Black rot, caused by Ceratocystis fimbriata, is a devastating postharvest disease of sweetpotato that recently re-emerged in 2014. Although the disease is known to develop in storage and during export to overseas markets, little is known as to how pathogen dispersal occurs. This study was designed to investigate dump tank water as a means of dispersal through four different types of water treatments: inoculum concentration (0, 5, 5 × 101, 5 × 102, and 5 × 103 spores/ml), inoculum age (0, 24, 48, 96, and 144 h), water temperature (10, 23, 35, and 45˚C), and presence of a water sanitizer (DryTec, Sanidate, FruitGard, and Selectrocide). Wounded and non-wounded sweetpotato storage roots were soaked in each water treatment for 20-min, stored at 29˚C for a 14-day period, and rated for disease incidence every other day. Disease was observed in sweetpotato storage roots in all water treatments tested, except in the negative controls. Disease incidence decreased with both inoculum concentration and inoculum age, yet values of 16.26% and up to 50% were observed for roots exposed to 5 spores/ml and 144 h water treatments, respectively. Sanitizer products that contained a form of chlorine as the active ingredient significantly reduced disease incidence in storage roots when compared to control roots and roots exposed to a hydrogen-peroxide based product. Finally, no significant differences in final incidence were detected in wounded sweetpotato storage roots exposed to water treatments of any temperature, but a significant reduction in disease progression was observed in the 45˚C treatment. These findings indicate that if packing line dump tanks are improperly managed, they can aid C. fimbriata dispersal through the build-up of inoculum as infected roots are unknowingly washed after storage. Chlorine-based sanitizers can reduce infection when applied after root washing and not in the presence of high organic matter typically found in dump tanks.

Optimization of Fungal Dextranase Production and Its Antibiofilm Activity, Encapsulation and Stability in Toothpaste

Dextranase catalyzes the degradation of the substrate dextran, which is a component of plaque biofilm. This enzyme is involved in antiplaque accumulation, which can prevent dental caries. The activity of crude dextranase from Penicillium roquefortii TISTR 3511 was assessed, and the maximum value (7.61 unit/g) was obtained at 37 °C and pH 6. The Plackett–Burman design was used to obtain significant factors for enhancing fungal dextranase production, and three influencing factors were found: Dextran, yeast extract concentration and inoculum age. Subsequently, the significant factors were optimized with the Box–Behnken design, and the most suitable condition for dextranase activity at 30.24 unit/g was achieved with 80 g/L dextran, 30 g/L yeast extract and five day- old inoculum. The use of 0.85% alginate beads for encapsulation exhibited maximum dextranase activity at 25.18 unit/g beads, and this activity was stable in toothpaste for three months of testing. This study explored the potential production of fungal dextranase under optimal conditions and its encapsulation using alginate for the possibility of applying encapsulated dextranase as an additive in toothpaste products for preventing dental caries.

Inoculum Size and Age Studies on Single and Mixed Strain Fermentation of Grape Juice

Single and mixed strain fermentation were compared to check the effect on properties of wine. Two strains of Saccharomyces cerevisiae (MTCC 11815 & MTCC 170) were used to study the effect of inoculum age and inoculum size on fermentation of grape juice. The inoculum sizes used were 2%, 5%, 10% and 15%, while inoculum age effect was studied using 24 h, 48 h and 60 h old inoculum. Fermentation efficiency of 77.2% was achieved in mixed strain culture using 15% inoculum, 17% initial sugars giving ethanol concentration of 6.70% (w/v) after 48 hrs. Fermentation efficiency of 84.65% was achieved with MTCC170 using 15% inoculum and 17% initial sugars giving ethanol concentration of 7.34% (w/v) in 48 hrs. Strain MTCC11815 produced 8.5% (w/v) ethanol from 17% initial sugars giving 98% efficiency using 2 and 5% inoculum. Concentration of phenolics increased with inoculum concentration while nitrogen and phosphates did not show any regular trend. The nitrogen and phosphate concentration was affected by type of strain rather than other factors.

ASPERGILLUS TERREUS KMBF1501 A POTENTIAL PIGMENT PRODUCER UNDER SUBMERGED FERMENTATION

Objective: The present study was aimed to identify the fungal isolate from soil and to understand the different optimized parameters better to facilitate the pigment production that has high yield and stability.Methods: Aspergillus sp. was isolated from Western Ghats soil by the conventional serial dilution technique and assessed as a potential pigment producer. Different broth medium such as potato dextrose broth (PDB), czapek-dox broth (CDB), malt extract broth (MEB), rose bengal broth (RBB), sabouraud dextrose broth (SDB), yeast malt extract broth (YEMB), pH (3-9), temperature (24, 27, 30, 33, 37 and 40 °C), carbon (lactose,glucose,sucrose, maltose, galactose and fructose) and nitrogen source (peptone, yeast extract, urea and inorganic nitrogen sources like potassium nitrate, ammonium chloride and sodium nitrate), mineral salts such as sodium dihydrogen phosphate (Na2H2Po4), magnesium sulphate (Mg2So4), calcium chloride (CaCl2), copper sulphate (Cu2So4), potassium dihydrogen phosphate (KH2Po4) and manganese sulphate (Mn2So4) and inoculum age (2-7 d) of the medium related to high pigment production were analysed.Results: Aspergillus terreus KMBF1501 was identified by ribosomal DNA sequencing showing 99% similarity with other Aspergillus terreus and the Accession number (KX113516) was assigned. The optimum culture conditions for pigment production by Aspergillus terreus KMBF1501 was achieved at pH 5 (0.563±0.012 nm), temperature of 27 °C (0.382±0.001 nm) with glucose (0.501±0.002 nm) as carbon source, peptone (2.147±0.004 nm) as nitrogen source, Mg2SO4 (0.401±0.001 nm)as mineral salt and 4 d (0.324±0.001 nm) of inoculum age in PDB (0.761±0.006 nm).Conclusion: Aspergillus terreus KMBF1501 produced maximum pigment when cultured in modified PDB than in common PDB medium. The high concentration of the pigment can be used for various industrial purposes.

Effect of inoculum age and physical parameters on in vitro culture of the entomopathogenic nematode Steinernema feltiae

Entomopathogenic nematodes (EPNs) of the families Steinernematidae and Heterorhabditidae have a symbiotic association with bacteria which makes them virulent against insects. EPNs have been mass produced using in vivo and in vitro methods, including both solid and liquid fermentation. This study assessed the effect of nematode inoculum age on the production of Steinernema feltiae in liquid, solid and biphasic processes. Several physical parameters were also assessed: the effect of medium viscosity, flask size and aeration speed on the recovery and yield of infective juveniles (IJs). Inoculum age treatments included inoculum liquid cultures that were 7, 14, 21 and 28 days old. Nematodes from the same inoculum were added to one liquid medium (liquid culture), one solid medium with bacteria previously grown in sponge (solid culture) and a variation of the solid medium (a biphasic culture), in which the bacteria were first grown in liquid and, then, soaked into the sponges, with the purpose of providing a more homogeneous bacterial culture before nematode inoculation. Experiments were conducted in Erlenmeyer flasks. Eight treatments were established involving combinations of three variables: two media (with and without 0.2% agar), two flask sizes (250 and 150 ml) and two agitation speeds (180 and 280 rpm). The study showed increases in nematode yield for liquid cultures, but not for solid or biphasic cultures, with the advance of the inoculum age up to 28 days of growth. Furthermore, the addition of 0.2% agar to the liquid medium and increasing the aeration rate by using larger flasks with higher agitation speed may increase nematode recovery and final yield. The experiments were conducted using shake flasks but the results may also be applicable for bioreactors.

Zn(II) and Cu(II) removal byNostoc muscorum: a cyanobacterium isolated from a coal mining pit in Chiehruphi, Meghalaya, India

Nostoc muscorum was isolated from a coal mining pit in Chiehruphi, Meghalaya, India, and its potential to remove Zn(II) and Cu(II) from media and the various biochemical alterations it undergoes during metal stress were studied. Metal uptake measured as a function of the ions removed by N. muscorum from media supplemented independently with 20 μmol/L ZnSO4and CuSO4established the ability of this cyanobacterium to remove 66% of Zn2+and 71% of Cu2+within 24 h of contact time. Metal binding on the cell surface was found to be the primary mode of uptake, followed by internalization. Within 7 days of contact, Zn2+and Cu2+mediated dissimilar effects on the organism. For instance, although chlorophyll a synthesis was increased by 12% in Zn2+-treated cells, it was reduced by 26% in Cu2+-treated cells. Total protein content remained unaltered in Zn2+-supplemented medium; however, a 15% reduction was noticed upon Cu2+exposure. Copper enhanced both photosynthesis and respiration by 15% and 19%, respectively; in contrast, photosynthesis was unchanged and respiration dropped by 11% upon Zn2+treatment. Inoculum age also influenced metal removal ability. Experiments in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (a photosynthetic inhibitor), carbonyl cyanide m-chlorophenyl hydrazone (an uncoupler), and exogenous ATP established that metal uptake was energy dependent, and photosynthesis contributed significantly towards the energy pool required to mediate metal removals.

Optimization of Culture Conditions for Production of the Anti-Leukemic Glutaminase Free L-Asparaginase by Newly IsolatedStreptomyces olivaceusNEAE-119 Using Response Surface Methodology

Among the antitumor drugs, bacterial enzyme L-asparaginase has been employed as the most effective chemotherapeutic agent in pediatric oncotherapy especially for acute lymphoblastic leukemia. Glutaminase free L-asparaginase producing actinomycetes were isolated from soil samples collected from Egypt. Among them, a potential culture, strain NEAE-119, was selected and identified on the basis of morphological, cultural, physiological, and biochemical properties together with 16S rRNA sequence asStreptomyces olivaceusNEAE-119 and sequencing product (1509 bp) was deposited in the GenBank database under accession number KJ200342. The optimization of different process parameters for L-asparaginase production byStreptomyces olivaceusNEAE-119 using Plackett-Burman experimental design and response surface methodology was carried out. Fifteen variables (temperature, pH, incubation time, inoculum size, inoculum age, agitation speed, dextrose, starch, L-asparagine, KNO3, yeast extract, K2HPO4, MgSO4·7H2O, NaCl, and FeSO4·7H2O) were screened using Plackett-Burman experimental design. The most positive significant independent variables affecting enzyme production (temperature, inoculum age, and agitation speed) were further optimized by the face-centered central composite design-response surface methodology.