Development of a modified serum-free medium for Vero cell cultures: effects of protein hydrolysates, l-glutamine and SITE liquid media supplement on cell growth.

Vero cells have been widely used in the viral vaccine production due to the recommendation of the World Health Organization regarding its safety and non-tumorigenicity. The aim of this study was to describe the development a modified serum-free medium for Vero cell cultures. Two protein hydrolysates (Bacto™ soytone and Bacto™ yeast extract), vitamin C, vitamin B12, SITE liquid media supplement, and recombinant human epidermal growth factor (rEGF) were investigated as serum substitutes. A sequential experiment of fractional factorial and central composite design was applied. A modified serum-free medium obtained (named as SFM01-M) was verified. Contrary to P0, the cell yields obtained at P1, P2, and P3 decreased continuously during the verification experiments indicating that Vero cells could not adapt to SFM01-M as expected according to the empirical mathematical model. To improve cell growth after P0, protein hydrolysates, l-glutamine, and SITE liquid media supplement were further investigated. The results showed that cell yields gradually decreased from P1 to P3 when a fixed concentration of Bacto™ yeast extract (7.0 g/L) combined with various concentrations of Bacto™ soytone (0.1-7.0 g/L) in SFM01-M were used. Similarly, cell yields also gradually decreased from P1 to P3 when a fixed concentration of Bacto™ soytone (7.0 g/L) combined with various concentrations of Bacto™ yeast extract (0.1-7.0 g/L) in SFM01-M were used. However, the combination of Bacto™ soytone at 0.1 g/L and Bacto™ yeast extract at 7.0 g/L or Bacto™ soytone at 7.0 g/L and Bacto™ yeast extract at 0.1 g/L in SFM01-M could give the maximum cell yield at P3 when compared with other combinations. In addition, the addition of SITE liquid media supplement (0.1-2.0% v/v) in SFM01-M in which the concentrations of Bacto™ soytone, Bacto™ yeast extract, and l-glutamine were fixed at 0.1 g/L, 0.1 g/L, and 4.0 mM, respectively, the results showed that the cell yields obtained at P3 were not significantly different. From this study, the optimum concentrations of SFM01-M components were as follows: Bacto™ soytone (0.1 g/L), Bacto™ yeast extract (0.1 g/L), vitamin C (9.719 mg/L), vitamin B12 (0.1725 mg/L), SITE liquid media supplement (0.1-2.0% v/v), rEGF (0.05756 mg/L), l-glutamine (4.0 mM), MEM non-essential amino acids (1.0% v/v), sodium pyruvate (1.0 mM), MEM (9.4 g/L), and sodium hydrogen carbonate (2.2 g/L). However, to evaluate SFM01-M in the long-term subculture of Vero cells, the efficiency of SFM01-M will be further investigated.

Impact of Crystalline Structural Differences Between α- and ß-Chitosan on Their Nanoparticle Formation Via Ionic Gelation and Superoxide Radical Scavenging Activities.

α- and ß-Chitosan nanoparticles were obtained from shrimp shell and squid pen chitosan with different set of deacetylation degree (%DD) and molecular weight (MW) combinations. After nanoparticle formation via ionic gelation with sodium tripolyphosphate (TPP), the % crystallinity index (%CI) of the α- and ß-chitosan nanoparticles were reduced to approximately 33% and 43% of the initial %CI of the corresponding α- and ßchitosan raw samples, respectively. Both forms of chitosan and chitosan nanoparticles scavenged superoxide radicals in a dose-dependent manner. The %CI of α- and ß-chitosan and chitosan nanoparticles was significantly negatively correlated with superoxide radical scavenging abilities over the range of concentration (0.5, 1, 2 and 3 mg/mL) studied. High %DD, and low MW ß-chitosan exhibited the highest superoxide radical scavenging activity (p < 0.05). α- and ß-Chitosan nanoparticles prepared from high %DD and low MW chitosan demonstrated the highest abilities to scavenge superoxide radicals at 2.0-3.0 mg/mL (p < 0.05), whereas α-chitosan nanoparticles, with the lowest %CI, and smallest particle size (p < 0.05), prepared from medium %DD, and medium MW chitosan showed the highest abilities to scavenge superoxide radicals at 0.5-1.0 mg/mL (p < 0.05). It could be concluded that α- and ß-chitosan nanoparticles had superior superoxide radical scavenging abilities than raw chitosan samples.

Fuzzy logic control of rotating drum bioreactor for improved production of amylase and protease enzymes by Aspergillus oryzae in solid-state fermentation.

In this study, we compared the performance of two control systems, fuzzy logic control (FLC) and conventional control (CC). The control systems were applied for controlling temperature and substrate moisture content in a solidstate fermentation for the biosynthesis of amylase and protease enzymes by Aspergillus oryzae. The fermentation process was achieved in a 200 L rotating drum bioreactor. Three factors affecting temperature and moisture content in the solid-state fermentation were considered. They were inlet air velocity, speed of the rotating drum bioreactor, and spray water addition. The fuzzy logic control system was designed using four input variables: air velocity, substrate temperature, fermentation time, and rotation speed. The temperature was controlled by two variables, inlet air velocity and rotational speed of bioreactor, while the moisture content was controlled by spray water. Experimental results confirmed that the FLC system could effectively control the temperature and moisture content of substrate better than the CC system, resulting in an increased enzyme production by A. oryzae. Thus, the fuzzy logic control is a promising control system that can be applied for enhanced production of enzymes in solidstate fermentation.

Statistical optimization for Monacolin K and yellow pigment production and citrinin reduction by Monascus purpureus in solid-state fermentation.

Monacolin K and yellow pigment, produced by Monascus sp., have each been proven to be beneficial compounds as antihypercholesterolemic and anti-inflammation agents, respectively. However, citrinin, a human toxic substance, was also synthesized in this fungus. In this research, solidstate fermentation of M. purpureus TISTR 3541 was optimized by statistical methodology to obtain a high production of monacolin K and yellow pigment along with a low level of citrinin. Fractional factorial design was applied in this study to identify the significant factors. Among the 13 variables, five parameters (i.e., glycerol, methionine, sodium nitrate, cultivation time, and temperature) influencing monacolin K, yellow pigment, and citrinin production were identified. A central composite design was further employed to investigate the optimum level of these five factors. The maximum production of monacolin K and yellow pigment of 5,900 mg/kg and 1,700 units/g, respectively, and the minimum citrinin concentration of 0.26 mg/kg were achieved in the medium containing 2% glycerol, 0.14% methionine, and 0.01% sodium nitrate at 25°C for 16 days of cultivation. The yields of monacolin K and yellow pigment were about 3 and 1.5 times higher than the basal medium, respectively, whereas citrinin was dramatically reduced by 36 times.

Fermentation process development of recombinant Hansenula polymorpha for gamma-linolenic acid production.

Development in the strain and the fermentation process of Hansenula polymorpha was implemented for the production of gamma-linolenic acid (GLA, C18:3 delta 6,9,12), which is an n-6 polyunsaturated fatty acid (PUFA) and has been reported to possess a number of health benefits. The mutated delta 6-desaturase (S213A) gene of Mucor rouxii was expressed in H. polymorpha under the control of the methanol oxidase (MOX) promoter. Without utilization of methanol a high cell-density culture of the yeast recombinant carrying the delta 6-desaturase gene was achieved by fed-batch fermentation using glycerol-limited conditions. The delta 6-desaturated products, octadecadienoic acid (C18:2 delta6,9), GLA and stearidonic acid (C18:4 delta6,9,12,15), accumulated at high levels under the derepression condition. The GLA production was also optimized by adjusting specific growth rates. The results show that the specific growth rate affected both lipid content and fatty acid composition of the GLA-producing recombinant. Among the various specific growth rates studied, the highest GLA concentration, which was at of 697 mg/l, was obtained in the culture with the specific growth rate of 0.08 /h. Interestingly, the fatty acid profile of the yeast recombinant bearing the Mucor delta 6-desaturase gene was similar to that of blackcurrant oil with both containing similar proportions of n-3 and n-6 essential fatty acids.