Pichia pastoris X-33 has probiotic properties with remarkable antibacterial activity against Salmonella Typhimurium.

Probiotics are live microorganisms which are beneficial for the host when ingested at high enough concentrations. The methylotrophic yeast Pichia pastoris is widely used as heterologous protein production platform. However, its use as probiotic is poorly studied. The objective of this study was to evaluate some probiotic properties of the P. pastoris strain X-33 wild type. The resistance to in vitro and in vivo gastrointestinal conditions, stability in feed, safety, and antibacterial activity against Salmonella Typhimurium were evaluated. The yeast remained viable and persisted at appropriate concentration in the diet for at least 2 months, survived the stresses of the gastrointestinal tract in vitro and in vivo, caused no behavioral changes or lesions when administered to mice, inhibited the growth of S. Typhimurium in culture media, and reduced adhesion of the bacteria to the intestinal cells HCT-116. In the challenge experiment with a LD50 of virulent S. Typhimurium strain, mice supplemented with the yeast had a higher survival rate (50 % when administered by gavage and 80 % via the diet, compared with 20 and 50 %, respectively, in the control group). In addition, the S. Typhimurium concentration in the intestine of the surviving mice was lower; the score of intestinal lesions, lower; and the pathogen, not detected in the liver, spleen, and feces when compared to the control group (p < 0.05). It was concluded that the yeast Pichia pastoris X-33 has probiotic properties with remarkable antibacterial activity against S. Typhimurium.

Development of a microencapsulated probiotic delivery system with whey, xanthan, and pectin.

Pediococcus pentosaceus is a lactic acid bacterium that has probiotic potential proven by studies. However, its viability can be affected by adverse conditions such as storage, heat stress, and even gastrointestinal passage. Thus, the aim of the present study was to microencapsulate and characterize microcapsules obtained by spray drying and produced only with whey powder (W) or whey powder combined with pectin (WP) or xanthan (WX) in the protection of P. pentosaceus P107. In the storage test at temperatures of - 20 °C and 4 °C, the most viable microcapsule was WP (whey powder and pectin), although WX (whey powder and xanthan) presented better stability at 25 °C. In addition, WX did not show stability to ensure probiotic potential (< 6 Log CFU mL-1) for 110 days and the microcapsule W (whey powder) maintained probiotic viability at the three temperatures (- 20 °C, 4 °C, and 25 °C) for 180 days. In the exposition to simulated gastrointestinal juice, the WX microcapsule showed the best results in all tested conditions, presenting high cellular viability. For the thermal resistance test, WP microcapsule was shown to be efficient in the protection of P. pentosaceus P107 cells. The Fourier transform infrared spectroscopy (FTIR) results showed that there was no chemical interaction between microcapsules of whey powder combined with xanthan or pectin. The three microcapsules produced were able to protect the cell viability of the microorganism, as well as the drying parameters were adequate for the microcapsules produced in this study.

Curcumin and Quercetin-Loaded Lipid Nanocarriers: Development of Omega-3 Mucoadhesive Nanoemulsions for Intranasal Administration.

Curcumin (CUR) and quercetin (QU) are potential compounds for treatment of brain diseases such as neurodegenerative diseases (ND) because of their anti-inflammatory and antioxidant properties. However, low water solubility and poor bioavailability hinder their clinical use. In this context, nanotechnology arises as a strategy to overcome biopharmaceutical issues. In this work, we develop, characterize, compare, and optimize three different omega-3 (ω-3) fatty acids nanoemulsions (NEs) loaded with CUR and QU (negative, cationic, gelling) prepared by two different methods for administration by intranasal route (IN). The results showed that formulations prepared with the two proposed methods exhibited good stability and were able to incorporate a similar amount of CUR and QU. On the other side, differences in size, zeta potential, in vitro release kinetics, and permeation/retention test were observed. Considering the two preparation methods tested, high-pressure homogenization (HPH) shows advantages, and the CQ NE- obtained demonstrated potential for sustained release. Toxicity studies demonstrated that the formulations were not toxic for Caenorhabditis elegans. The developed ω-3 fatty acid NEs have shown a range of interesting properties for the treatment of brain diseases, since they have the potential to increase the nose-to-brain permeation of CUR and QU, enabling enhanced treatments efficiency.

Optimization of Ralstonia solanacearum cell growth using a central composite rotational design for the P(3HB) production: Effect of agitation and aeration.

The intracellular accumulation of polyhydroxyalkanoates (PHAs) normally occurs after cell growth, during the second fermentation stage and under nutrient-limited conditions in the presence of a carbon excess. However, some microorganisms are able to accumulate PHAs as poly(3-hydroxybutyrate) [P(3HB)] during the first fermentation stage, the cell growth phase, without nutrient limitation, once they have been reported to utilize type II metabolism during the polymer accumulation phase. This study evaluated the effect of aeration and agitation on cell growth and P(3HB) accumulation in Ralstonia solanacearum RS, performed in a bioreactor for 24h at 32°C. A 22 central composite rotational design (CCRD) was used, with agitation (150 to 250 rpm) and aeration (0.3 to 1 vvm) as independent variables and optical density (OD600nm), dry cell weight (DCW), and P(3HB) yield as dependent variables. A significant polymer accumulation, until 70% of P(3HB), was observed, proving that R. solanacearum RS exhibited metabolism type II, regardless of the aeration process. The best results were obtained for 1 vvm and 250 rpm (+1, +1), with values of OD600nm (18.04) and DCW (4.82 g.L-1).

Electrospinning of native and anionic corn starch fibers with different amylose contents.

Through starch phosphorylation and solution aging treatments, the aim of this work was to produce electrospun fibers derived from native and anionic (modified with sodium tripolyphosphate) corn starches with amylose contents of <70% (w/w). The fibers of native and anionic corn starches (regular amylose and high amylose Hylon V/Hylon VII) were prepared by electrospinning of starch solutions dissolved in aqueous 75% formic acid (v/v) solvent. The effects of the aging (24, 48, and 72 h) on the rheology and electrical conductivity of the starch solutions, as well as the material properties (size distribution, morphology, and infrared spectrum) of the resulting electrospun fibers, were evaluated. Fibers produced from Hylon VII and Hylon V starches showed homogeneous morphologies, whereas the fibers from regular corn starches exhibited droplets and had heterogeneous morphologies, with diameter varied from 70 to 264 nm. Both native and anionic corn starches, with amylose contents of <70% (w/w), produced smooth continuous fibers. The electrospun corn starch fibers potentially can be used as carriers for the encapsulation of active components in food and packaging applications.

Saccharomyces boulardii improves humoral immune response to DNA vaccines against leptospirosis.

PURPOSE: Saccharomyces boulardii may improve the immune response by enhancing the production of anti-inflammatory cytokines, T-cell proliferation and dendritic cell activation. The immunomodulator effect of this probiotic has never been tested with DNA vaccines, which frequently induce low antibody titers. This study evaluated the capacity of Saccharomyces boulardii to improve the humoral and cellular immune responses using DNA vaccines coding for the leptospiral protein fragments LigAni and LigBrep. BALB/c mice were fed with rodent-specific feed containing 108 c.f.u. of Saccharomycesboulardii per gram. METHODOLOGY: Animals were immunized three times intramuscularly with 100 µg of pTARGET plasmids containing the coding sequences for the above mentioned proteins. Antibody titers were measured by indirect ELISA. Expression levels of IL-4, IL-10, IL-12, IL-17, IFN-γ and TGF-ß were determined by quantitative real-time PCR from RNA extracted from whole blood, after an intraperitoneal boost with 50 µg of the recombinant proteins.Results/Key findings. Antibody titers increased significantly after the second and third application when pTARGET/ligAni and pTARGET/ligBrep were used to vaccinate the animals in comparison with the control group (P<0.05). In addition, there was a significant increase in the expression of the IL-10 in mice immunized with pTARGET/ligBrep and fed with Saccharomyces boulardii. CONCLUSION: The results suggested that Saccharomyces boulardii has an immunomodulator effect in DNA vaccines, mainly by stimulating the humoral response, which is often limited in this kind of vaccine. Therefore, the use of Saccharomyces boulardii as immunomodulator represents a new alternative strategy for more efficient DNA vaccination.

Lectin I from Bauhinia variegata (BVL-I) expressed by Pichia pastoris inhibits initial adhesion of oral bacteria in vitro.

Lectins are non-immune proteins that reversibly bind to carbohydrates in a specific manner. Bauhinia variegata lectin I (BVL-I) is a Gal/GalNAc-specific, single-chain lectin isolated from Bauhinia variegata seeds that has been implicated in the inhibition of bacterial adhesion and the healing of damaged skin. Since the source of the native protein (nBVL) is limited, this study aimed to produce recombinant BVL-I in Pichia pastoris (rBVL-Ip). The coding sequence for BVL-I containing preferential codons for P. pastoris was cloned into the pPICZαB plasmid. A single expressing clone was selected and fermented, resulting in the secretion and glycosylation of the protein. Fed-batch fermentation in 7L-scale was performed, and the recombinant lectin was purified from culture supernatant, resulting in a yield of 1.5mg/L culture. Further, rBVL-Ip was compared to nBVL and its recombinant version expressed in Escherichia coli BL21 (DE3) (rBVL-Ie). Although it was expressed as a monomer, rBVL-Ip retained its biological activity since it was able to impair the initial adhesion of Streptococcus mutans and S. sanguinis in an in vitro model of biofilm formation and bacterial adhesion. In summary, rBVL-Ip produced in Pichia pastoris represents a viable alternative to large-scale production, encouraging further biological application studies with this lectin.

Functional, thermal and rheological properties of oat ß-glucan modified by acetylation.

Fibers of ß-glucan have been added to foods for their thickening properties, their ability to form gel at low concentrations, but mainly for their appeal in health promotion. Current analysis evaluates the influence of acetylation (4% and 6% acetic anhydride for 10 and 20 min) on the functional, thermal, morphological and rheological properties of the concentrate containing 31% of oat ß-glucan. The degree of substitution of the acetylated ß-glucans ranged from 0.03 to 0.12, suitable for use in foods. Acetylation increased the heterogeneity of molecule degradation and promoted a more compacted hole-less microstructure. Functional properties such as the swelling power and bile acid binding capacity were increased by acetylation. The ß-glucan gel showed a reduction in hardness and adhesiveness, which was confirmed by its rheological behavior similar to liquid. The above information is relevant to establish the industrial application of acetylated ß-glucan.