Enhanced high ß-carotene yeast cell production by Rhodotorula paludigena CM33 and in vitro digestibility in aquatic animals.

This study assessed Rhodotorula paludigena CM33's growth and ß-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and ß-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a ß-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Qx) of 0.91 g/L.h and remarkable ß-carotene production rates (Qp) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale ß-carotene production for aquatic animal nutrition.

ß-sitosterol isolated from the leaves of Trema orientalis (Cannabaceae) promotes viability and proliferation of BF-2 cells.

Trema orientalis is a pioneer species in the cannabis family (Cannabaceae) that is widely distributed in Thai community forests and forest edges. The mature leaves are predominantly used as an anti-parasite treatment and feed for local freshwater fish, inspiring investigation of their phytochemical composition and bioactivity. The purpose of this work was to investigate the bioactive compounds in T. orientalis leaf extract and their cytotoxicity in the BF-2 fish cell line (ATCC CCL-91). Flash column chromatography was used to produce 25 mL fractions with a mixture solvent system comprised of hexane, diethyl ether, methanol, and acetone. All fractions were profiled with HPLC-DAD (mobile phase methanol:aqueous buffer, 60:40 v/v) and UV detection (wavelengths 256 and 365 nm). After drying, a yellowish powder was isolated from lipophilic leaf extract with a yield of 280 µg/g dry weight. Structure elucidation by nuclear magnetic resonance (NMR) indicated it to consist of pure ß-sitosterol. The lipophilic extract and pure compound were evaluated for cytotoxicity using BF-2 cells. MTT assays showed both leaf extract and pure compound at 1 µg/mL to increase cell viability after 24 h treatment. The respective half maximal inhibitory concentration (IC50) values of leaf extract and ß-sitosterol were 7,027.13 and 86.42 µg/ml, indicating a lack of toxicity in the BF-2 cell line. Hence, T. orientalis can serve as a source of non-toxic natural lipophilic compounds that can be useful as bioactive ingredients in supplement feed development.

Potential of Pm11 antimicrobial peptide against bovine mastitis pathogens.

OBJECTIVE: To investigate an alternative treatment for bovine mastitis by using Pm11 antimicrobial peptide. SAMPLE: 5 bovine mastitis pathogens that were previously isolated from cows affected by either clinical or subclinical mastitis. PROCEDURES: The current study introduces Pm11 antimicrobial peptide as an alternative treatment for bovine mastitis. The antibacterial activity of Pm11 was tested against Escherichia coli strain SCM1249, Klebsiella spp strain SCM1282, Staphylococcus aureus strain CM967, Streptococcus agalactiae strain SCM1084, and Streptococcus uberis strain SCM1310 using minimum bactericidal concentrations (MBCs) and time-kill kinetics. The pathogens' morphological changes were demonstrated using a scanning electron microscope (SEM). The cytotoxicity of Pm11 was assessed using the minimum hemolytic concentration assay. RESULTS: MBCs ranged from 2.5 to 10 µM and IC50 ranged from 0.32 to 2.07 µM. Time-kill kinetics at MBC demonstrated that Pm11 reduced viable cell counts of S agalactiae strain SCM1084 and S uberis strain SCM1310 from 105 to 0 CFU/mL within 1 h. E coli strain SCM1249 and S aureus strain CM967 were reduced from 105 to 0 CFU/mL within 4 h. The average Pm11-induced hemolytic activity was < 10% for all Pm11 concentrations tested except at the maximum concentration tested (160 µM: 10.19 ± 2.29%). Based on SEM, Pm11 induced morphological and cellular changes in S aureus and E coli. CLINICAL RELEVANCE: Pm11 antimicrobial peptide demonstrated in vitro antibacterial activity against the common bovine mastitis pathogens E coli, S aureus, S agalactiae, and S uberis, except Klebsiella spp, and should be further investigated in vivo.

Shrimp protected from a virus by feed containing yeast with a surface-displayed viral binding protein.

Recombinant Pichia pastoris biomass surface-expressing the viral binding protein PmRab7 (YSD-PmRab7) was prepared by fed-batch, aerobic fermentation with methanol induction for 48 h. By cell based ELISA assay, immunofluorescence and flow cytometry, 45% of the YSD-PmRab7 cells were positive for PmRab7. Freeze dried YSD-PmRab7 cells were added to formulated shrimp feed pellets at 0.25 g and 0.5 g per g feed and fed to 2 shrimp groups for 7 days prior to challenge with white spot syndrome virus (WSSV). Controls consisted of 1 shrimp group fed normal pellets and one fed pellets containing P. pastoris carrying an empty gene cassette. At 10 days post challenge, survival in the two control groups was 6.7 ± 6.6%, while it was 26.7 ± 6.6% in the 0.25 g YSD-PmRab7 group and significantly higher (p < 0.05) in the 0.5 g YSD-PmRab7 group at 46.7 ± 10.1%. Nested PCR assays and histopathological analysis revealed significantly lower WSSV replication levels in the 0.5 g YSD-PmRab7 group. The results indicated potential for development of YSD-PmRab7 cells as an oral prophylactic against WSSV in shrimp.

Cationic cassava starch and its composite as flocculants for microalgal biomass separation.

Commercial- and laboratory modified- cationic cassava starches and their composites with magnetic particles were examined for characteristics and separation efficiency. Scanning electron micrographs showed that cationic starch with an increasing degree of substitution (DS) value (0.0180 to 0.91) showed greater clumped polyhedral granules and became markedly enlarged with disintegrated boundaries. Zeta potential analysis revealed that the increase in the DS value in cationic starches resulted in an increase in positive charge. The maximum harvesting efficiency of 92.86 ± 0.46% was achieved when commercial cationic starch with DS 0.040 at 1.0 g L-1 was added to the Chlorella sp. solution. The maximum recovery capacity (10.20 ± 0.16 g DCW g starch-1) was recorded by using commercial cationic starch with DS 0.040 at a lower dosage of 0.1 g L-1. Their composites showed lower separation efficiency than the commercial cationic starches. The results suggest that the commercial cationic cassava starch with 0.040 DS shows great potential as a flocculant for algal separation. This first report of using commercial cationic cassava starch as a flocculant provides a low cost and convenient process to separate algal cells from the culture medium. Moreover, uncontaminated magnetic particle biomass allows for wide range of algal utilization in food and pharmaceutical biotechnologies.

Repeated cultures of Saccharomyces cerevisiae SC90 to tolerate inhibitors generated during cassava processing waste hydrolysis for bioethanol production.

Large amount of cassava pulp is produced as by-product of industrial tapioca production. The value-added process of this low-cost waste is to use it as a substrate for bioethanol production. However, during the pulp pretreatment by acidification combined with steam explosion, many yeast inhibitors including acetic acid, formic acid, levulinic acid, furfural and 5-hydroxymethylfurfural are generated and these compounds have negative effects on the subsequent fermentation step. Therefore, the objective of this study was to investigate whether the repeated cultures of Saccharomyces cerevisiae SC90 could alleviate this problem. To obtain the inhibitor tolerable cells, the repeated culture was performed by growing yeast cells to a specific growth rate (µ) of 0.22 h-1 or higher (80% of the µ in control) and then transferring them to progressively higher concentrations of hydrolysate ranging from 20 to 100% (v/v). The results showed a tendency of longer lag phase as well as time to reach maximum cell number (t maxc) with an increase in hydrolysate concentration. However, the repeated culture at the same hydrolysate concentration could shorten both lag period and t maxc. Interestingly, the growth and fermentation efficiency of adapted cells in 100% hydrolysate were significantly higher (p ≤ 0.05) than those of non-adapted cells by 38% and 27%, respectively.

High Cell Density Process for Constitutive Production of a Recombinant Phytase in Thermotolerant Methylotrophic Yeast Ogataea thermomethanolica Using Table Sugar as Carbon Source.

The yeast Ogataea thermomethanolica has recently emerged as a potential host for heterologous protein expression at elevated temperature. To evaluate the feasibility of O. thermomethanolica as heterologous host in large-scale fermentation, constitutive production of fungal phytase was investigated in fed-batch fermentation. The effect of different temperatures, substrate feeding strategies, and carbon sources on phytase production was investigated. It was found that O. thermomethanolica can grow in the temperature up to 40 °C and optimal at 34 °C. However, the maximum phytase production was observed at 30 °C and slightly decreased at 34 °C. The DOT stat control was the most efficient feeding strategy to obtain high cell density and avoid by-product formation. The table sugar can be used as an alternative substrate for phytase production in O. thermomethanolica. The highest phytase activity (134 U/mL) was obtained from table sugar at 34 °C which was 20-fold higher than batch culture (5.7 U/mL). At a higher cultivation temperature of 38 °C, table sugar can be used as a low-cost substrate for the production of phytase which was expressed with an acceptable yield (85 U/mL). Lastly, the results from this study reveal the industrial favorable benefits of employing O. thermomethanolica as a host for heterologous protein production.

Mutational analysis in the glycone binding pocket of Dalbergia cochinchinensis ß-glucosidase to increase catalytic efficiency toward mannosides.

Dalcochinase and Abg are glycoside hydrolase family 1 ß-glucosidases from Dalbergia cochinchinensis Pierre and Agrobacterium sp., respectively, with 35% sequence identity. However, Abg shows much higher catalytic efficiencies toward a broad range of glycone substrates than dalcochinase does, possibly due to the difference in amino acid residues around their glycone binding pockets. Site-directed mutagenesis was used to replace the amino acid residues of dalcochinase with the corresponding residues of Abg, generating three single mutants, F196H, S251V, and M369E, as well as the corresponding three double mutants and one triple mutant. Among these, the F196H mutant showed increases in catalytic efficiency toward almost all glycoside substrates tested, with the most improved catalytic efficiency being a 3-fold increase for hydrolysis of p-nitrophenyl ß-D-mannoside, suggesting a preferred polar residue at this position and consistent with the presence of histidine at this position in two other GH1 glycosidases from barley and rice that prefer ß-mannosides. In addition, the M369E mutation resulted in a small increase in catalytic efficiency for cleavage of p-nitrophenyl ß-D-galactoside. By contrast, the multiple mutants were up to 8-fold less efficient than the recombinant wild-type dalcochinase, and displayed primarily antagonistic interactions between these residues. Thus, differences in catalytic efficiency between dalcochinase and Abg are therefore not primarily due to differences in the residues that directly contact the substrate, but derive largely from contributions from more remote residues and the overall architecture of the active site.

Improvement of recombinant endoglucanase produced in Pichia pastoris KM71 through the use of synthetic medium for inoculum and pH control of proteolysis.

The long lag time in basal salts medium (BSM) and an occurrence of proteolysis are major problems for recombinant protein production in Pichia pastoris KM71. In this study, optimal conditions were explored for fed-batch cultivation of recombinant fungal endoglucanase in P. pastoris KM71. It was found that lag and process times were much reduced when the synthetic FM22 medium was used for the inoculum compared with enriched buffered glycerol complex (BMGY) medium. The highest endoglucanase activity was obtained at 30°C which was more than 10 fold higher than that produced from shake flask. At 30°C, the specific endoglucanase activity was dependent on culture pH and a higher specific activity was observed at pH 5.0 than at pH 6.0. The higher activity was likely due to lower rate of proteolysis, since a truncated protein species was apparent at pH 6.0, but not pH 5.0. Thus, production of endoglucanase at 30°C and pH 5.0 is the optimal condition suitable for economical production in large scale. The combination of using synthetic FM22 medium for inoculum and proteolysis control by growth at lower pH could be applied for production of other recombinant proteins in P. pastoris.

Process technology for production and recovery of heterologous proteins with Pichia pastoris.

Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion.

Interfacing Pichia pastoris cultivation with expanded bed adsorption.

For improved interfacing of the Pichia pastoris fed-batch cultivation process with expanded bed adsorption (EBA) technique, a modified cultivation technique was developed. The modification included the reduction of the medium salt concentration, which was then kept constant by regulating the medium conductivity at low value (about 8 mS/cm) by salt feeding. Before loading, the low conductivity culture broth was diluted only to reduce viscosity, caused by high cell density. The concept was applied to a one-step recovery and purification procedure for a fusion protein composed of a cellulose-binding module (CBM) from Neocallimastix patriciarum cellulase 6A fused to lipase B from Candida antarctica (CALB). The modified cultivation technique resulted in lower cell death and consequently lower concentration of proteases and other contaminating proteins in the culture broth. Flow cytometry analysis showed 1% dead (propidium-stained) cells compared to 3.5% in the reference process. During the whole process of cultivation and recovery, no proteolysis was detected and in the end of the cultivation, the product constituted 87% of the total supernatant protein. The lipase activity in the culture supernatant increased at an almost constant rate up to a value corresponding to 2.2 g/L of CBM-CALB. In the EBA process, no cell-adsorbent interaction was detected but the cell density had to be reduced by a two-times dilution to keep a proper bed expansion. At flow velocity of 400 cm/h, the breakthrough capacity was 12.4 g/L, the product yield 98%, the concentration factor 3.6 times, the purity about 90%, and the productivity 2.1 g/L x h.

Recovery of recombinant beta-glucosidase by expanded bed adsorption from Pichia pastoris high-cell-density culture broth.

Methanol limited fed-batch cultivation was applied for production of a plant derived beta-glucosidase by Pichia pastoris. The beta-glucosidase was recovered by expanded bed adsorption chromatography applied to the whole culture broth. The new Streamline Direct HST1 adsorbent was compared with Streamline SP. Higher bead density made it possible to operate at two times higher feedstock concentration and at two times higher flow velocity. The higher binding capacity in the conductivity range 0-48 mS cm(-1) of Streamline Direct HST1 might be caused by the more complex interaction of multi-modal ligand in Streamline Direct HST1 compared to the single sulphonyl group in Streamline SP. Harsher elution condition had to be applied for dissociation of beta-glucosidase from Streamline Direct HST1 due to stronger binding interaction. The 5% dynamic binding capacity was 160 times higher for Streamline Direct HST1 compared to Streamline SP. The yield of beta-glucosidase on Streamline Direct HST1 (74%) was significantly higher than on Streamline SP (48%). Furthermore, beta-glucosidase was purified with a factor of 4.1 and concentrated with a factor of 17 on Streamline Direct HST1 while corresponding parameters were half of these values for Streamline SP. Thus, for all investigated parameters Streamline Direct HST1 was a more suitable adsorbent for recovery of recombinant beta-glucosidase from unclarified P. pastoris high-cell-density cultivation broth.

Oxygen-limited fed-batch process: an alternative control for Pichia pastoris recombinant protein processes.

An oxygen-limited fed-batch technique (OLFB) was compared to traditional methanol-limited fed-batch technique (MLFB) for the production of recombinant Thai Rosewood beta-glucosidase with Pichia pastoris. The degree of energy limitation, expressed as the relative rate of respiration (q(O)/q(O,max)), was kept similar in both the types of processes. Due to the higher driving force for oxygen transfer in the OLFB, the oxygen and methanol consumption rates were about 40% higher in the OLFB. The obligate aerobe P. pastoris responded to the severe oxygen limitation mainly by increased maintenance demand, measured as increased carbon dioxide production per methanol, but still somewhat higher cell density (5%) and higher product concentrations (16%) were obtained. The viability was similar, about 90-95%, in both process types, but the amount of total proteins released in the medium was much less in the OLFB processes resulting in substantially higher (64%) specific enzyme purity for input to the downstream processing.