Survival of microencapsulated Lactobacillus casei (prepared by vibration technology) in fruit juice during cold storage

Background: Foods including probiotics are considered "functional foods." As an alternative to dairy products, we investigated the behavior of Lactobacillus casei when exposed to low-pH fruit juice. Juices of fruits such as pineapple, raspberry, and orange were assessed. Free and microencapsulated forms of L. casei were compared, and the viability of the probiotic was evaluated under storage at 4°C for 28 d. Microbiological analyses were carried out to ensure a safe and healthy product for consumers who look for foods with probiotics from sources other than dairy. Results: Low pH affected L. casei survival during storage depending on the type of fruit juice. In the case of pineapple juice, some microcapsules were broken, but microcapsules recovered at the end of the storage period had 100% viability (2.3 × 107 CFU/g spheres). In the case of orange juice, more than 91% viability (5.5 × 106 CFU/g spheres) was found. In raspberry juice, viability decreased rapidly, disappearing at the end of the storage period, which was caused by the absorption of high concentrations of anthocyanin inside microcapsules more than low pH. Conclusion: Low pH affected the survival of L. casei under refrigeration; even when they were microencapsulated, acidic conditions impacted their viability. Although pH affects viability, its value is very sensitive and will depend on the type of fruit juice and its composition. Some fruit juices contain compounds used as substrates for Lactobacillus and other compounds with antimicrobial effects.

Polyphenolic extracts of walnut (Juglans regia) green husk containing juglone inhibit the growth of HL-60 cells and induce apoptosis

BACKGROUND: Juglone is a naphthoquinone currently obtained by chemical synthesis with biological activities including antitumor activity. Additionally, juglone is present in the green husk of walnut, which suggests evaluating the effect of GH extracts on carcinogenic cell lines. RESULTS: Walnut green husk ethanolic extract was obtained as 169.1 mg juglone/100 g Green Husk and antioxidant activity (ORAC) of 44,920 µmol Trolox Equivalent/100 g DW Green Husk. At 1 µM juglone in HL-60 cell culture, green husk extract showed an antiproliferative effect, but pure juglone did not; under these conditions, normal fibroblast cells were not affected. A dose-dependent effect on mitochondrial membrane potential loss was observed. Apoptosis of HL-60 was detected at 10 µM juglone. Despite high ORAC values, neither purified juglone nor the extract showed protective effects on HL-60 cells under oxidative conditions. CONCLUSIONS: Green husk extract generates an antiproliferative effect in HL-60 cells, which is related to an induction of the early stages of apoptosis and a loss of mitochondrial membrane potential. The normal cells were not affected when juglone is present at concentrations of 1 µM, while at higher concentrations, there is loss of viability of both cancerous and healthy cells.

Impact of sodium butyrate and mild hypothermia on metabolic and physiological behaviour of CHO TF 70R cells

Background: To reduce costs associated with productivity of recombinant proteins in the biopharmaceutical industry, research has been focused on regulatory principals of growth and survival during the production phases of the cell culture. The main strategies involve the regulation of cell proliferation by the modulation of cell cycle control points (G1/S or G2/M) with mild hypothermia and the addition of sodium butyrate (NaBu). In this study, batch culture strategies were evaluated using CHO TF 70R cells producing the recombinant human tissue plasminogen activator (rh-tPA), to observe their individual and combined effect on the cellular physiological state and relevant kinetic parameters. Results: NaBu addition has a negative effect on the mitochondrial membrane potential (ΔΨm), the values of which are remarkably diminished in cultures exposed to this cytotoxic compound. This effect was not reflected in a loss of cell viability. NaBu and mild hypothermic conditions increased the doubling time in the cell cultures, suggesting that these strategies triggered a general slowing of each cell cycle phase in a different way. Finally, the individual and combined effect of NaBu and mild hypothermia produced an increase in the specific rh-tPA productivity in comparison to the control at 37°C without NaBu. Nevertheless, both strategies did not have a synergistic effect on the specific productivity. Conclusions: The combination of NaBu addition and mild hypothermic condition causes an impact on physiological and metabolic state of CHO TF 70R cells, decreasing cell growth rate and improving glucose consumption efficiency. These results therefore provide a promising strategy to increase specific productivity of rh-tPA.

Antiproliferative evaluation of tall-oil docosanol and tetracosanol over CHO-K1 and human melanoma cells

Background Polycosanols derived from plant species have traditionally been used in medicine as antiproliferative agents for treating various viruses (primarily the herpes simplex virus). However, few studies have studied their effects on hyperproliferative cell lines. In this work, the antiproliferative capacity of polycosanols from tall-oil pitch, obtained from black liquor soaps in the kraft pulping process of cellulose (specifically from Pinus radiata, Pinus taede, and Eucalyptus globulus), was evaluated on CHO-K1 and CRL-1974 human melanoma cell lines. Results The proliferative capacities and cell viabilities were measured for 72 and 140 h, respectively. Treatment with docosanol produced differential effects on the CHO-K1 and human melanoma cells and significantly affected their proliferation rates, but not their cell viabilities. Tetracosanol produced a significant negative effect on the proliferation of human melanoma cells, and this effect was less than that caused by docosanol. However, it had no effect on the proliferation of CHO-K1 cells and did not induce any significant effect on the viability of the studied cell lines. Conclusion Docosanol and tetracosanol induced antiproliferative effects on the studied cell lines and exhibited significantly greater effects on the oncogenic cell lines. Prior to this study, the capacity of these polycosanols has never been investigated. Future studies will be necessary to determine their mechanisms of action on these cell systems.

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.

Simultaneous environmental manipulations in semi-perfusion cultures of CHO cells producing rh-tPA

We evaluated the combined effect of decreasing the temperature to a mild hypothermia range (34 and 31ºC) and switching to a slowly metabolizable carbon source (glucose substituted by galactose) on the growth and production of a recombinant human tissue plasminogen activator (rh-tPA) by Chinese hamster ovary cells in batch and semi-perfusion cultures. In batch cultures using glucose as a carbon source, decreasing the temperature caused a reduction in cell growth and an increase in specific productivity of rh-tPA of 32 percent at 34ºC and 55 percent at 31ºC, compared to cultures at 37ºC. Similar behaviour was observed in cultures at 34ºC using galactose as a carbon source. Nonetheless, at 31ºC, the specific productivity of rh-tPA strongly decreased (about 58 percent) compared to the culture at 37ºC. In semi-perfusion culture, the highest rh-tPA specific productivity was obtained at 34ºC. Similarly, whether a decrease in the temperature is accompanied of the replacement of glucose by galactose, the rh-tPA specific productivity improved about 112 percent over that obtained in semi-perfusion culture carried out at 37ºC with glucose as the carbon source. A semi-perfusion culture strategy was implemented based on the combined effect of the chosen carbon source and low temperatures, which was a useful approach for enhance the specific productivity of the recombinant protein.

Specific nutrient supplementation of defined serum-free medium for the improvement of CHO cells growth and t-PA production

Recombinant CHO TF70R cells are able to grow and produce t-PA on serum-free medium BIOPRO1 (BioWhitaker Europe, Belgium). The purpose of the present study was to determine the effect of medium supplementation with vitamins, lipids, and specific amino acids on cell growth, t-PA production and biological functionality. Among vitamins, only biotin, folic acid, cobalamine and benzoic acid were required for improving growth and t-PA production. Lipid supplement allowed a significant increase cell concentration and t-PA specific activity and concentration, though its specific production rate decreased slightly. Medium supplementation with proline, serine and asparagine had also positive effects on cell growth. Besides, the addition of asparagine (even in the presence of glutamine) was essential for the production and biological quality of the t-PA. This systematic approach for media supplementation produced an increase in cell concentration around 100 percent and in t-PA production around 80 percent, with no detrimental effect on its biological activity. The effect of asparagine on t-PA production was unexpected and needs to be further studied. The above modifications of the production medium did not produce a significant effect on the metabolism of the main carbon and energy sources (glucose and glutamine) and the level of by-product formation (lactate and ammonia).