Enhanced production of periplasmic interferon alpha-2b by Escherichia coli using ion-exchange resin for in situ removal of acetate in the culture

The possibility of using in situ addition of anion-exchange resin for the removal of acetate in the culture aimed at improving growth of E. coli and expression of periplasmic human interferon-α2b (PrIFN-α2b) was studied in shake flask culture and stirred tank bioreactor. Different types of anion-exchange resin were evaluated and the concentration of anion-exchange resin was optimized using response surface methodology. The addition of anion-exchange resins reduced acetate accumulation in the culture, which in turn, improved growth of E. coli and enhanced PrIFN-α2b expression. The presence of anion-exchange resins did not influence the physiology of the cells. The weak base anion-exchange resins, which have higher affinity towards acetate, yielded higher PrIFN-α2b expression as compared to strong anion-exchange resins. High concentrations of anion-exchange resin showed inhibitory effect towards growth of E. coli as well as the expression of PrIFN-α2b. The maximum yield of PrIFN-α2b in shake flask culture (501.8 μg/L) and stirred tank bioreactor (578.8 μg/L) was obtained at ion exchange resin (WA 30) concentration of 12.2 g/L. The production of PrIFN-α2b in stirred tank bioreactor with the addition of ion exchange resin was about 1.8-fold higher than that obtained in fermentation without ion exchange resin (318.4 μg/L)

Primary recovery of miraculin from miracle fruit, Synsepalum dulcificum by AOT reverse micellar system

Miracle fruit, Synsepalum dulcificum, contains a glycoprotein known as miraculin. After consuming this glycoprotein, sour foods taste sweet and the effect lasts for up to 4 h. With increasing demand for natural and “low-calorie” sweeteners, the use of miraculin as an additive is increasing enormously in the food, medicine and cosmetic industries. In this study, we used reverse micelles formed from a sodium di (2-ethylhexyl) sulfosuccinate (AOT)/isooctane system to purify miraculin from S. dulcificum. We studied factors affecting purification performance, such as surfactant (AOT) concentration and the pH of the crude during forward extraction. During backward extraction, we examined the effects of NaCl concentration, the pH of the aqueous phase and addition of isopropanol. We found that 0.1 mol/L AOT/isooctane solution mixed with crude extract at pH 8 during the forward extraction stage and 0.5 mol/L NaCl solution at pH 11 during the backward stripping stage were optimal purification conditions, from which 22% miraculin was recovered with a purity of 94.8%

Enhancement of BLIS production by Pediococcus acidilactici kp10 in optimized fermentation conditions using an artificial neural network

The present study was aimed at enhancing the production of bacteriocin-like inhibitory substance (BLIS) produced by Pediococcus acidilactici Kp10 through optimizing the fermentation parameters. M17 was chosen in preliminary study as a culture medium because BLIS production was nine times higher (1427.7 AU mL−1) compared to that produced by MRS (160 AU mL−1). The fermentation parameters such as temperature, inoculum size, buffer strength, concentration of tween 80 and agitation speed were screened using two level half-factorial design. BLIS production is influenced by three most significant factors identified as temperature, inoculum size and agitation speed, which were further optimized using an artificial neural network (ANN). ANN predicted that a maximum activity of 5262.64 AU mL−1 would be obtained at optimum conditions of 120 rpm, 3% and 28.5 °C. The observed BLIS activity at the predicted optimum levels of the tested variables in ANN was 5118.5 AU mL−1, which was close to the predicted BLIS activity. Increased BLIS activity in the final solution, which resulted from the optimized process, would reduce downstream steps such as concentrating the product during purification

Optimization of conditions for the single step IMAC purification of miraculin from Synsepalum dulcificum

In this study, the methods for extraction and purification of miraculin from Synsepalum dulcificum were investigated. For extraction, the effect of different extraction buffers (phosphate buffer saline, Tris-HCl and NaCl) on the extraction efficiency of total protein was evaluated. Immobilized metal ion affinity chromatography (IMAC) with nickel-NTA was used for the purification of the extracted protein, where the influence of binding buffer pH, crude extract pH and imidazole concentration in elution buffer upon the purification performance was explored. The total amount of protein extracted from miracle fruit was found to be 4 times higher using 0.5M NaCl as compared to Tris-HCl and phosphate buffer saline. On the other hand, the use of Tris-HCl as binding buffer gave higher purification performance than sodium phosphate and citrate-phosphate buffers in IMAC system. The optimum purification condition of miraculin using IMAC was achieved with crude extract at pH 7, Tris-HCl binding buffer at pH 7 and the use of 300 mM imidazole as elution buffer, which gave the overall yield of 80.3% and purity of 97.5%. IMAC with nickel-NTA was successfully used as a single step process for the purification of miraculin from crude extract of S. dulcificum

Optimization of an induction strategy for improving interferon-α2b production in the periplasm of Escherichia coli using response surface methodology

Induction strategies for the periplasmic production of recombinant human IFN-alpha2b (interferon-alpha2b) by recombinant Escherichia coli Rosetta-gami 2(DE3) were optimized in shake-flask cultures using response surface methodology based on the central composite design. The factors included in the present study were induction point, which related to the attenuance of the cell culture, IPTG (isopropyl beta-D-thiogalactoside) concentration and induction temperature. Second-order polynomial models were used to correlate the abovementioned factors to soluble periplasmic IFN-alpha2b formation and percentage of soluble IFN-alpha2b translocated to the periplasmic space of E. coli. The models were found to be significant and subsequently validated. The proposed induction strategies consisted of induction at an attenuance of 4 (measured as D600), IPTG concentration of 0.05 mM and temperature of 25 degrees C. The optimized induction strategy reduced inclusion-body formation as evidenced by electron microscopy and yielded 323.8 ng/ml of IFN-alpha2b in the periplasmic space with translocation of 74% of the total soluble product. In comparison with the non-optimized condition, soluble periplasmic production and the percentage of soluble IFN-alpha2b translocated to the periplasmic space obtained in optimized induction strategies were increased by approx. 20-fold and 1.4-fold respectively

Preliminary assessment of Polytrichum commune extract as an antimicrobial soap ingredient

Mosses have long been used in traditional Chinese medicine due to the presence of secondary metabolites which have shown high biological activities. In particular, these secondary metabolites have demonstrated effective antibacterial activity against pathogenic microorganisms. In this study, the influence of different extraction solvents on the antibacterial activities of the Polytrichum commune was carried out using the disc diffusion method. Results showed that both 12.5 mg/mL of methanol moss extract and 6.25 mg/mL of ethanol moss extract were the most effective concentrations against Bacillus cereus and Pseudomonas aeruginosa. Additionally, the P. commune extracts were included as an added ingredient in soap bases to produce antibacterial soap prototypes where the effectiveness of the soaps containing the extracts in removing microorganisms from actual test individuals was carried out. Results of the thumb impression test of test individuals showed that the growth of microbial reduced after washing hands with the usage of both liquid and solid soap with the addition of P. commune extracts. Moreover, the antibacterial soaps performed better in eliminating microorganisms in comparison to control soaps without P. commune extracts. Taken together, P. commune extract could be a good candidate as a value-added ingredient utilized to produce antibacterial soaps due to its antibacterial properties

Influence of culture conditions and medium compositions on the production of bacteriocin-like inhibitory substances by Lactococcus lactis Gh1

Antibacterial peptides or bacteriocins produced by many strains of lactic acid bacteria have been used as food preservatives for many years without any known adverse effects. Bacteriocin titres can be modified by altering the physiological and nutritional factors of the producing bacterium to improve the production in terms of yield and productivity. The effects of culture conditions (initial pH, inoculum age and inoculum size) and medium compositions (organic and inorganic nitrogen sources; carbon sources) were assessed for the production of bacteriocin-like inhibitory substances (BLIS) by Lactococcus lactis Gh1 in shake flask cultures. An inoculum of the mid-exponential phase culture at 1% (v/v) was the optimal age and size, while initial pH of culture media at alkaline and acidic state did not show a significant impact on BLIS secretion. Organic nitrogen sources were more favourable for BLIS production compared to inorganic sources. Production of BLIS by L. lactis Gh1 in soytone was 1.28-times higher as compared to that of organic nitrogen sources ((NH4)2SO4). The highest cell concentration (XmX = 0.69 ± 0.026 g·L−1) and specific growth rate (μmax = 0.14 h−1) were also observed in cultivation using soytone. By replacing carbon sources with fructose, BLIS production was increased up to 34.94% compared to BHI medium, which gave the biomass cell concentration and specific growth rate of 0.66 ± 0.002 g·L−1 and 0.11 h−1, respectively. It can be concluded that the fermentation factors have pronounced influences on the growth of L. lactis Gh1 and BLIS production. Results from this study could be used for subsequent application in process design and optimisation for improving BLIS production by L. lactis Gh1 at larger scale

Effects of dual impeller system of Rushton turbine, concave disk turbine and their combinations on the performance of kojic acid fermentation by Aspergillus flavus Link 44-1

In the present work, the effects of dual impeller configurations comprising of Rushton turbine (RT) and Concave-bladed disk turbine (CD-6) on the oxygen transfer profile and fermentation performance of kojic acid production by Aspergillus flavus Link 44-1 was investigated. Batch cultivations of A. flavus Link 44-1 were performed in 2 L stirred tank bioreactor. The fermentations were conducted using different dual impeller systems; (1) RT-RT, (2) CD6-CD6, (3) RT-CD6, and (4) CD6-RT (bottom-top impeller). It was perceived that dual CD-6 system was able to improve oxygen transfer rate by about 25–45% over the hybrids of RT and CD-6 and the typically configured dual RT system. While no substantial disparity could be seen on the fungal growth rate by the manipulation of the impeller, high concentration of kojic acid (44.93 g L−1) was attained with the use of dual CD-6 as the mixer. Efficient agitating system that can facilitate good gas dispersion capability is crucially required in order to counteract the problem of oxygen solubility limitation faced in such viscous fungal fermentation broth. The results from this work suggested the promising capability of dual CD-6 configuration in enhancing productivity of kojic acid fermentation in stirred tank bioreactor

Improved cultivation of gdhA derivative Pasteurella multocida B: 2 for high density of viable cells through in situ ammonium removal using cation-exchange resin for use as animal vaccine

Pasteurella multocida serotype B:2 is the causative agent of hemorrhagic septicemia, a fatal disease of cattle and buffaloes. A live attenuated vaccine, gdhA derivative P. multocida B:2 mutant, was created to defeat the disease. During the cultivation of P. multocida B:2 mutant, substantial amount of ammonium was accumulated in the culture, which greatly inhibited the growth of this bacterium. The feasibility of using integrated cultivation with in situ removal of ammonium by cation-exchange resin for the improvement of growth and viability of P. multocida cells was investigated. The ability of various cation-exchange resins, which include Amberlite IRC86, Amberlite IR120 H, and Dowex DRG8 H, to selectively adsorbed ammonium was first investigated using sorption isotherm experiments. Amberlite IRC86 has the highest ability for ammonium adsorption. The incorporation of 10 g/L of Amberlite IRC86 resin into the shake flask culture (100 mL) of P. multocida B:2 mutant, improved the final viable cell concentration (7.2 × 1010 cfu/mL) by about 13-fold compared to that obtained in cultivation without resin (5.5 × 109 cfu/mL). In cultivation with Amberlite IRC86 resin, approximately 41% of the ammonium accumulated in the culture was removed

Phytochemicals, nutritionals and antioxidant properties of miracle fruit Synsepalum dulcificum

Synsepalum dulcificum, also called the miracle fruit, which has the sweet-inducing activity can be used as additives in food, medicine and cosmetic industries. Some selected chemical properties of miracle fruit including percentage by weight, total anthocyanin, phenolic and antioxidant content of different parts of miracle fruit as well as physicochemical analysis of seed oil, nutritional elements of fruit juice were determined in this study. The results showed that miracle fruit contains a large amount of vitamin C (40.1 mg/100 g fresh fruit weight (FW)), phenolic content (625.57 mg GAE/100 g FW), high antioxidant capacity (457.3 μmol Trolox/100 g FW) and low total sugar content (5.6 g/100 g FW), suggesting that the fruit is healthy for human consumption. According to its fatty acid composition and Triacylglycerol (TAG) profile, miracle fruit seed oil is rich in oleic and palmitic acid