Simulated Gastric Fluid Treatment Research Articles

Membrane modification as a survival mechanism through gastric fluid in non-acid adapted enteropathogenic Escherichia coli (EPEC)

In bacterial cells, the cytoplasmic membrane forms a barrier between the environment and the cell's cytoplasm. This barrier regulates which substances (and the amount) that leave and enter the cell, to maintain homeostasis between the cytoplasm and the external environment. One of the mechanisms employed to maintain structure and functionality during exposure to environmental stress is adaptation of the membrane lipids. The aim of this study was to investigate membrane alteration as a possible survival method of non-acid adapted enteropathogenic Escherichia coli (E. coli) (EPEC) (as could be found in contaminated water or unprocessed food) through simulated gastric fluid (SGF). Enteropathogenic E. coli was grown in nutrient-rich media and then exposed to SGF of various pH (1.5, 2.5, 3.5, or 4.5) for 180 min. Flow cytometry was utilised to examine membrane integrity; and morphological changes were investigated using transmission electron microscopy (TEM). Gas chromatography-mass spectrometry (GC-MS) was used to assess the membrane lipid composition. The results of this study showed that SGF treatment caused membrane damage, as well as cell wall thickening and irregular plasma membranes. The morphological changes were accompanied by membrane lipid changes indicative of decreased membrane fluidity and increased rigidity. The findings suggest that non-acid adapted EPEC can perceive pH change in the environment and adapt accordingly.

Effect of gums coating materials on the survival of microencapsulated probiotics under simulated gastrointestinal conditions

Probiotic bacteria have been extensively merged into numerous dietary supplements and dairy commodities and have been sold as functional foods. Microencapsulation is one of those approaches used to increase probiotic survival and delivery. The aim of this work is to analyze the effect of three types of coating materials, xanthan, carrageenan and acacia gum on the viability of microencapsulated probiotics during simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) exposure. The probiotics, Lactobacillus casei Shirota (LcS) were microencapsulated into uncoated alginate beads and were coated with 3 different concentrations of xanthan, carrageenan and acacia gums (0.1%, 0.3% and 0.5%). Free cells, uncoated and coated beads were subjected to SGF (1.5 h and 3 h) and SIF (24 h) and their viability was assessed after incubation period. Results denoted that glazed alginate beads with gums increased the viability of cells after cultivation in SGF and SIF. After SGF treatment, the viability of most of the coated beads was significantly higher compared to free cells. Among the three coating materials used in this study, xanthan gum coating provided the best cell protection in both treatments. Increasing concentration of gums had an optimistic outcome on the existence of LcS in GIT's replicated severe environments. However, microencapsulation of LcS with alginate does not significantly improve probiotic survivals under simulated gastrointestinal conditions.

Hybrid coating of alginate microbeads based on protein-biopolymer multilayers for encapsulation of probiotics.

A hybrid coating based on multilayers of proteins and biopolymers was developed to enhance the protection performance of alginate microbeads against acidic conditions for delivery of probiotics (Lactobacillus rhamnosus GG). Zeta potential measurements and quartz crystal microbalance with dissipation confirmed layer-by-layer deposition of protein-polymer layers. The stability of protein-based coatings during simulated gastric fluid (SGF) treatment was monitored by microscopy. Protein-coated microbeads were partially dismantled, whereas polymer-coated microbeads were intact after a sequential treatment in simulated gastric and intestinal fluids. This suggests that hybrid formulation offers an advantage over the coatings based on biopolymer multilayers in terms of better release of bacteria. Uncoated alginate microbeads completely dissolved and could not protect bacteria after SGF treatment whereas microbeads with hybrid coating showed increased physical stability and a modest decrease of culturability of 3.8 log units. Therefore, this work provides a concept for future protein-based hybrid coatings for bacterial delivery systems.

Effects of thermal processing methods and simulated digestion on the phenolic content and antioxidant activity of lotus leaves

The release and antioxidant activity of the phenolics in lotus leaves were changed by various processing methods and in vitro simulated gastric fluid (SGF) and intestinal fluid (SIF) digestion. The total phenolic content (TPC), total flavonoid content (TFC), and antioxidant capacity of FRAP and ABTS were the highest in the sample processed by microwave vacuum drying. The SGF treatment significantly reduced the TFC in lotus leaves treated by various thermal processing methods but had no significant effect on the TPC. However, SIF digestion reduced the TPC in lotus leaves treated with different thermal processing methods but increased the TFC. The SGF digestion reduced the ABTS of lotus leaves, whereas SIF digestion had no significant effect on the ABTS antioxidant capacity. However, both SGF and SIF digestion increased the FRAP antioxidant capacity. Previous steam heated followed by microwave vacuum drying is the optimal method for drying lotus leaves, after considering all factors. PRACTICAL APPLICATIONS: Phenolics and flavonoid from lotus leaf have antioxidant capacity that brings health benefits. However, the antioxidant capacity is affected by various processing methods and digestion. Therefore, the development of processing methods is very important. Our results show that previous steam heated followed by microwave vacuum drying is the optimal method for drying lotus leaves, considering the antioxidant capacity and flavor.

Oral administration of coated PEDV-loaded microspheres elicited PEDV-specific immunity in weaned piglets

Porcine epidemic diarrhea virus (PEDV) infects pigs of all ages by invading villous epithelial cells of the small intestine causing severe diarrhea with high mortality rate in suckling piglets. Mucosal immunity is believed to play an important role in PEDV control and mucosal delivery of vaccines induces mucosal immunity more efficiently than parenteral vaccination. In this study, coated PEDV-loaded microspheres with the size range of 700–900 μm in diameter were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system to protect PEDV antigens against the complex gastrointestinal environment by detecting the live virus particles in microspheres after the simulated gastric fluid treatment and the PEDV RNA in fecal swabs collected from all weaned piglets (100%) orally inoculated with coated PEDV-loaded microspheres. Weaned piglets orally immunized with coated PEDV-loaded microspheres developed higher levels of PEDV-specific antibodies (IgG and IgA) in their sera and saliva than those negative control groups (p < 0.001 or p < 0.01). Furthermore, neutralization assays demonstrated that serum antibodies in coated PEDV-loaded microspheres groups could significantly inhibit virus infection in Vero cells, compared to PEDV only group (p < 0.05). Overall, our results indicate that the coated PEDV-loaded microspheres might serve as an effective way to induce PEDV-specific mucosal immunity in pigs against PEDV.

Effects of in vitro digestion on the composition of flavonoids and antioxidant activities of the lotus leaf at different growth stages

SummaryThe lotus leaf is rich in phenolic compounds and has good antioxidant activity. The effects of simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) digestions on the active substances and antioxidant activity of the lotus leaf in different growth stages (D25, D45 and D65) were investigated. The total phenolic content of D25 and D45 significantly increased by SGF digestion but not D65 compared to the DW treatment. However, SIF treatment saw markedly increased total phenolic content across all growth stages (D25, D45 and D65). Moreover, the total flavonoid content at all growth stages was increased by SIF digestion compared to SGF treatment. The pepsin and trypsin had different effects on the FRAP and ABTS (2, 2'‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulphonic acid)) antioxidant activities of the lotus leaf. The SGF digestion increased the FRAP antioxidant capacity of the lotus leaf, and SIF digestion increased the ABTS antioxidant capacity of the lotus leaf. The HPLC results showed that the main flavonoid compound in the lotus leaf was isoquercetin. This study provides a theoretical basis for the development and utilisation of the lotus leaf as an inexpensive natural and functional food ingredient.

Superporous Hydrogel Composites of Acrylamide Using Starch-silicone Dioxide Coprecipitate as Composite Agent

Aim: Present study includes the development and evaluation of superporous hydrogel composites of acrylamide using starch silicone dioxide coprecipitate (SSC) as composite agent. Methodology: Gas blowing method was used for synthesis of superporous hydrogels. Different ingredients were mixed in specified amount in test tube and foaming agent was added at last. Simultaneous foaming and gelation lead to formation of porous hydrogel networks. Effect of different treatments (acetone dehydration and simulated gastric fluid (SGF) treatment) and drying conditions on the porous structure was evaluated. The prepared superporous hydrogels were evaluated for density, porosity, equilibrium swelling ratio, equilibrium swelling time, mechanical strength, scanning electron microscopy studies. The pure drug (Verapamil Hydrochloride) mixed with Carbopol 934 (1:0.5 and 1:1 ratio) was loaded into the selected batches of SPHC using in situ method of drug loading Original Research Article British Journal of Pharmaceutical Research, 4(3): 338-351, 2014 339 (drug added directly into the reaction mixture). The in vitro drug release was carried out using USP II dissolution apparatus. Results: Acetone dehydrated superporous hydrogel composites were observed as better floating devices due to least density and maximum porosity values. Maximum swelling ratio was found in case of oven dried conventional superporous hydrogel and superporous hydrogel composites with equilibrium swelling time of more than 25 min. On the other hand, acetone dehydrated SPH showed less equilibrium swelling ratio with equilibrium swelling time of 5-10 min. Minimum swelling ratio was observed in case of SGF treated hydrogels with equilibrium swelling time of 15-19 min. Mechanical strength was improved by addition of starch silicone dioxide coprecipitate as composite agent. SGF treatment also leads to enhanced mechanical strengthbut compromised swelling characteristics. SEM images showed uniformity in pore structure in case of acetone dehydrated hydrogels. Acetone dehydrated SPHC showed sustained drug release behavior with 71.14% and 57.84% of verapamil released in 12 h in the batches with Drug: Carbopol 934ratio of (1:0.5 and 1:1) respectively. Conclusion: Acetone dehydrated SPHC were found to be promising formulations with sufficient swelling and mechanical properties for achieving sustained drug delivery.

Assessing the digestion of a genetically modified tomato (Solanum lycopersicum) R8 DNA in simulated gastric fluid using event-specific real-time PCR

There are numerous studies that have demonstrated the digestion of plant DNA in the gastrointestinal (GI) tracts of various animal species. In this study, we investigate the process of DNA degradation in the GI tract using simulated gastric fluid treatment and quantitative PCR approach. Event-specific SYBR Green and TaqMan real-time PCR methods were developed in the present study for measuring the degradation of GM tomato R8 DNA. The selectivity, sensitivity, accuracy, and precision of real-time PCR detection methods were feasible for detection and quantification purposes. The results of real-time PCR analysis revealed that approximately 99.98% of an endogenous gene was degraded and could not be amplified after 180-min simulated gastric fluid treatment. The rapid acid hydrolysis of tomato fruit DNA in the SGF suggests that only less than 1% of genetically modified tomato fruit DNA could survive through the human digestive system.

Evaluation of cell surface-displayed protein stability against simulated gastric fluid

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.

Microencapsulation of Lactobacillus plantarum spp in an alginate matrix coated with whey proteins

Whey proteins were used as a coating material to improve encapsulation of Lactobacillus plantarum strains in calcium alginate beads. L. plantarum 299v, L. plantarum 800 and L. plantarum CIP A159 were used in this study. Inactivation experiments were carried out in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Cross-sections of freeze-dried beads revealed the random distribution of bacteria throughout the alginate network. From an initial count of 10.04 ± 0.01 log 10 CFU g − 1 for L. plantarum 299v, 10.12 ± 0.04 for L. plantarum CIP A159 and 10.03 ± 0.01 for L. plantarum 800, bacteria in coated beads and incubated in SGF (37 °C, 60 min) showed a better survival for L. plantarum 299v, L. plantarum CIP A159 and L. plantarum 800 (respectively 7.76 ± 0.12, 6.67 ± 0.08 and 5.81 ± 0.25 log 10 CFU g − 1 ) when compared to uncoated beads (2.19 ± 0.09, 1.89 ± 0.09 and 1.65 ± 0.10 log 10 CFU g − 1 ) ( p < 0.05). Only bacteria in the coated beads survived in the SIF medium (37 °C, 180 min) after SGF treatment. This preliminary work showed that whey proteins are a convenient, cheap and efficient material for coating alginate beads loaded with bacteria.

Toxicity of CdSe Nanoparticles in Caco-2 Cell Cultures

BackgroundPotential routes of nanomaterial exposure include inhalation, dermal contact, and ingestion. Toxicology of inhalation of ultra-fine particles has been extensively studied; however, risks of nanomaterial exposure via ingestion are currently almost unknown. Using enterocyte-like Caco-2 cells as a small intestine epithelial model, the possible toxicity of CdSe quantum dot (QD) exposure via ingestion was investigated. Effect of simulated gastric fluid treatment on CdSe QD cytotoxicity was also studied.ResultsCommercially available CdSe QDs, which have a ZnS shell and poly-ethylene glycol (PEG) coating, and in-house prepared surfactant coated CdSe QDs were dosed to Caco-2 cells. Cell viability and attachment were studied after 24 hours of incubation. It was found that cytotoxicity of CdSe QDs was modulated by surface coating, as PEG coated CdSe QDs had less of an effect on Caco-2 cell viability and attachment. Acid treatment increased the toxicity of PEG coated QDs, most likely due to damage or removal of the surface coating and exposure of CdSe core material. Incubation with un-dialyzed in-house prepared CdSe QD preparations, which contained an excess amount of free Cd2+, resulted in dramatically reduced cell viability.ConclusionExposure to CdSe QDs resulted in cultured intestinal cell detachment and death; cytotoxicity depended largely, however, on the QD coating and treatment (e.g. acid treatment, dialysis). Experimental results generally indicated that Caco-2 cell viability correlated with concentration of free Cd2+ ions present in cell culture medium. Exposure to low (gastric) pH affected cytotoxicity of CdSe QDs, indicating that route of exposure may be an important factor in QD cytotoxicity.

Allergenic reactivity of the melon profilin Cuc m 2 and its identification as major allergen

Melon allergy is commonly associated with oral allergy syndrome (OAS) and with hypersensitivity to pollens and other plant foods. No melon allergen responsible for these clinical characteristics has yet been isolated, although profilin has been proposed as a potential target. To isolate natural and recombinant melon profilin, to evaluate its in vivo and in vitro reactivity, and to analyse its behaviour in simulated gastric fluid (SGF) and heat treatments. A pool or individual sera from 23 patients, and an additional group of 10 patients, all of them with melon allergy, were analysed by in vitro and in vivo tests, respectively. Natural melon profilin (nCuc m 2) and its recombinant counterpart (rCuc m 2) were isolated by poly-l-proline affinity chromatography, and characterized by N-terminal amino acid sequencing, matrix-assisted laser desorption/ionization analysis, DNA sequencing of cDNAs encoding rCuc m 2, and immunodetection with anti-profilin antibodies. In vitro analysis included IgE immunodetection, specific IgE determination, ELISA-inhibition assays, and histamine release (HR) tests. In vivo activity of nCuc m 2 was established by skin prick testing (SPT). The effect of SGF and heat treatment on rCuc m 2 was followed by immunodetection, ELISA inhibition, and HR assays. Both purified forms of melon profilin were recognized by rabbit anti-profilin antibodies and IgE of sera from allergic patients, and showed molecular sizes typical of the profilin family. nCuc m 2 had a blocked N-terminus, whereas rCuc m 2 rendered the expected N-terminal amino acid sequence, its full protein sequence being highly similar (98--71% identity) to those of profilins from plant foods and pollens. The natural allergen displayed a slightly higher IgE-binding capacity than its recombinant counterpart. Specific IgE to nCuc m 2 and rCuc m 2 was found in 100% and 78% of the 23 individual sera analysed, respectively. nCuc m 2 evoked positive SPT responses in all (10/10) patients tested, and rCuc m 2 induced HR in two out of three sera assayed. SGF treatment readily inactivated rCuc m 2, as shown by its loss of recognition by anti-profilin antibodies, lack of IgE binding, and inability to induce HR. In contrast, heat treatment did not affect the IgE-binding capacity of rCuc m 2. Profilin is highly prevalent in melon-allergic patients, and promptly inactivated by SGF, as expected for an allergen mainly linked to OAS.

Survival of Listeria monocytogenes Strain H7762 and Resistance to Simulated Gastric Fluid following Exposure to Frankfurter Exudate

Listeria monocytogenes strain H7762, a frankfurter isolate, was tested to determine whether it was able to survive at 4°C in frankfurter pack fluid (exudate) and to determine whether food exposure affects its acid sensitivity. Cultures were sampled and tested for acid sensitivity by challenge with simulated gastric fluid (SGF). SGF challenges performed immediately after inoculation revealed that between 20 and 26% of the cells survived the full 30 min of SGF challenge regardless of whether the cells were inoculated into brain heart infusion broth (BHI) or exudate. After 2 days of incubation, cells exposed to both exudate and BHI had significantly decreased SGF resistance; however, the cells exposed to exudate were significantly more SGF resistant than cells exposed to BHI (after 15 min of SGF treatment, 33% of the exudate-exposed cells survived and 12% of the BHI-exposed cells survived). L. monocytogenes exposed to exudate had greater SGF resistance at all challenge times compared with BHI-exposed cells from day 2 through day 4. From days 8 to 15, exudate-exposed cells continued to have greater SGF resistance than BHI-exposed cells up to 10 min of SGF challenge but were as sensitive as the BHI-exposed cells at 20 to 30 min of challenge. By day 25, cells exposed to exudate were significantly more sensitive to SGF challenge than BHI-exposed cells. The survivor data generated from SGF challenges were modeled by a nonlinear regression analysis to calculate the underlying distribution of SGF resistance found in the challenged populations. These analyses indicated that L. monocytogenes exposed to exudate at 48C had a broader distribution of resistance to SGF compared with cells exposed to BHI at 4°C. In addition, the mean time of death during SGF treatment was greater after exposure to exudate, indicating that cells exposed to exudate were more resistant to killing by SGF. These data suggest that exposure to frankfurter exudate might render L. monocytogenes more able to survive the stomach environment during the initial stages of infection.

Safety Assessment by in Vitro Digestibility and Allergenicity of Genetically Modified Maize with an Amaranth 11S Globulin

Prospective testing for allergenicity of proteins obtained from sources with no prior history of causing allergy has been difficult to perform. Thus, the objective of this work was to assess the food safety of genetically modified maize with an amaranth globulin protein termed amarantin. Transgenic maize lines evaluated showed, in relation to nontransgenic, 4-35% more protein and 0-44% higher contents of specific essential amino acids. Individual sequence analysis with known amino acid sequences, reported as allergens, showed that none of these IgE elicitors were identified in amarantin. Amarantin was digested within the first 15 min by Simulated Gastric Fluid treatment as observed by Western blot. Expressed amarantin did not induce important levels of specific IgE antibodies in BALB/c mice, as analyzed by ELISA. We conclude that the transgenic maize with amarantin is not an important allergenicity inducer, just as nontransgenic maize.

Cellulose Acetate Succinate as an Enteric Coating for Some Compressed Tablets

The preparation and testing of compressed tablets of barium sulfate and sodium chloride coated with cellulose acetate succinate (CAS) are described, and the statistical evaluation of the data obtained is presented. The most satisfactory coatings were obtained when the test material, in an acetone-ethyl acetate solution, was applied by a modified “pan” method using talc as a dusting powder. Generally, CAS coatings which were stable for 3 1/2 hours in Simulated Gastric Fluid USP XVI dissolved sooner than control coatings of CAP applied and treated in the same manner. Disintegration times were slightly lower for most tablets placed directly into Simulated Intestinal Fluid, USP XVI than they were for tablets first treated with Simulated Gastric Fluid USP XVI for 31/2 hours and then tested in the former solution. None of the tablet coatings showed signs of cracks as a result of storage at −2 ± 2° for 21 days or 45 ± 2° for 10 days; however, some of these showed statistically significant differences in average disintegration time when compared with samples stored at room temperature. When test and control tablets were stored at 40 ± 2° and approximately 81% humidity, the majority of coatings were unsatisfactory. Tablets lost less than 3.56% tablet weight as a result of the simulated gastric fluid treatment. Preliminary in vivo tests with human subjects indicate that CAS has merit as an enteric coating. Pancreatin appears to have little effect on the disintegration times of the test coating.