Process optimization for microencapsulation of probiotic yeasts

Background

Microencapsulation is a technique which improves the survival and viability of probiotics. We demonstrate encapsulation of five potential probiotic yeasts with alginate and gum as encapsulation matrices to improve their gastrointestinal transit.

Methods

Gum extracted from various cereals viz. rice, oats, barley, finger millet and pearl millet along with alginate have been used to encapsulate five potential probiotic yeasts. Screening was carried out by measuring swelling index, encapsulation efficiency and nutritional value of microcapsules encapsulated with alginate and gum. The concentration of OBG, sodium alginate and inoculum dosage of probiotic yeasts was optimized using response surface methodology (RSM). Efficiency of alginate OBG microcapsules with or without coating materials viz. whey protein and chitosan also tested. The mucoadhesion ability and storage stability of alginate OBG microcapsules with coating materials were tested.

Results

Highest encapsulation efficiency of probiotic yeasts was noted using oats bran gum (OBG) microcapsules along with alginate in all the five probiotic yeasts. Notably whey protein coated microcapsules showed maximum GIT tolerance (95%) and mucoadhesion (90%) for L. starkeyi VIT-MN03. The minimum loss of viability was observed in L. starkeyi VITMN03 microcapsules on 60th day of storage.

Conclusions

This is the first report on optimization and survival of microencapsulated probiotic yeasts under simulated GIT conditions using natural gum and alginate as encapsulation matrices and whey protein as coating material.

     

Investigation of the impact of feeding Lactobacillus plantarum CRL 1815 encapsulated in microbially derived polymers on the rat faecal microbiota

Aims

The aim of this study was to evaluate the impact of the administration of microencapsulated Lactobacillus plantarum CRL 1815 with two combinations of microbially derived polysaccharides, xanthan : gellan gum (1%:0·75%) and jamilan : gellan gum (1%:1%), on the rat faecal microbiota.

Methods and Results

A 10‐day feeding study was performed for each polymer combination in groups of 16 rats fed either with placebo capsules, free or encapsulated Lact. plantarum or water. The composition of the faecal microbiota was analysed by fluorescence in situ hybridization and temporal temperature gradient gel electrophoresis. Degradation of placebo capsules was detected, with increased levels of polysaccharide‐degrading bacteria. Xanthan : gellan gum capsules were shown to reduce the Bifidobacterium population and increase the Clostridium histolyticum group levels, but not jamilan : gellan gum capsules. Only after administration of jamilan : gellan gum‐probiotic capsules was detected a significant increase in Lactobacillus‐Enterococcus group levels compared to controls (capsules and probiotic) as well as two bands were identified as Lact. plantarum in two profiles of ileum samples.

Conclusions

Exopolysaccharides constitute an interesting approach for colon‐targeted delivery of probiotics, where jamilan : gellan gum capsules present better biocompatibility and promising results as a probiotic carrier.

Significance and Impact of Study

This study introduces and highlights the importance of biological compatibility in the encapsulating material election, as they can modulate the gut microbiota by themselves, and the use of bacterial exopolysaccharides as a powerful source of new targeted‐delivery coating material.     

Effects of Potassium Chloride and Sodium Chloride on the Thermal Properties of Gellan Gum Gels

The exothermic and endothermic peaks in cooling and heating curves of differential scanning calorimetry (DSC) for gellan gum gels without and with potassium chloride and sodium chloride were analyzed. The gelling and melting temperatures shifted to higher temperatures with increasing gellan and salt concentration in the concentration range of gellan from 0.3 to 2.0% (w/w). The exothermic and endothermic enthalpy increased with increasing gellan and salt concentrations. Cooling DSC curves showed one exothermic peak for samples with salts and at low gellan concentration. Heating DSC curves showed many peaks for all samples except 0.3% (w/w) gellan gum gels. The sol-gel transition of samples was examined numerically by using a zipper model approach. The introduction of cations increases the number of junction zones or zippers and decreases the rotational freedom of parallel links. This makes the structure of junction zones more heat resistant, and increases the elastic modulus of the gel.     

Micro-encapsulation of <Emphasis Type="Italic"> Bifidobacterium lactis</Emphasis> for incorporation into soft foods

Summary Micro-encapsulation of the probiotic micro-organism Bifidobacterium lactis isolated from a bio-yoghurt starter culture, was carried out using a mixture of hydrated gellan and xanthan gums. Rheological studies showed that the gum mix was suitable for encapsulation of B. lactis, for incorporation into soft foods/beverages. The gel behaved as a non-Newtonian material, and the flow curve fitted well to the Herschel–Bulkley model. The average yield stress of the gum was 1.515 Pa, indicating gum stability, and the yield stress range was 1 Pa over a temperature range of 35–50 °C. Almost constant minimum gum viscosity occurred between 46 and 61 °C. Oval/round capsules were synthesized manually using a monoaxial gum flow through a 27.5 G bevelled needle, together with a superposed air stream (air knife technique). The diameter of the capsules, measured using laser diffractometry, varied from 20 to 2200μm, with 50% of the capsules having a diameter of ≤637 μm. Numbers of viable B. lactis in the capsules were estimated using high power ultrasound (20 kHz). By using a concentrated inoculum of B. lactis, microcapsules containing log₁₀ 11–12 c.f.u. g−1 were synthesized. Apart from the anaerobic culturing of B. lactis, all other work was done in the presence of atmospheric oxygen. The organism exhibited a high degree of oxygen tolerance. A 21-day survival study of immobilized cells in 1 M sodium phosphate buffer (pH 7) stored at either 4 or 22 °C indicated that B. lactis survived in excess of log₁₀ 11 c.f.u. g−1 microcapsule. This technique represents a suitable means of supplying viable probiotics to the food and/or pharmaceutical industries.     

Irradiation depolymerized guar gum as partial replacement of gum Arabic for microencapsulation of mint oil

Spray dried microcapsules of mint oil were prepared using gum Arabic alone and its blends with radiation or enzymatically depolymerized guar gum as wall materials. Microcapsules were evaluated for retention of mint oil during 8-week storage during which qualitative changes in encapsulated mint oil was monitored using principal component analysis. The microcapsules with radiation depolymerized guar gum as wall material component could better retain major mint oil compounds such as menthol and isomenthol. The t(1/2) calculated for mint oil in microcapsules of gum Arabic, gum Arabic:radiation depolymerized guar gum (90:10), gum Arabic:enzyme depolymerized guar gum (90:10) was 25.66, 38.50, and 17.11 weeks, respectively. The results suggested a combination of radiation depolymerized guar gum and gum Arabic to show better retention of encapsulated flavour than gum Arabic alone as wall material.     

Design and Evaluation of the Release Characteristics of Caffeine-Loaded Microcapsules in a Medicated Chewing Gum Formulation

The aim of this study was to microencapsulate caffeine by the emulsion technique, trying to control its release from a medicated chewing gum. Three formulations were prepared using alginate, alginate-starch, and alginate-starch with chitosan coating as the wall materials. These microcapsules were characterized with regard to the morphology studied by using an optical microscope and scanning electron microscopy (SEM), particle size, and encapsulation efficiency. The microcapsules were then incorporated into the chewing gums. The chewing gums were characterized by thermal behavior (by differential scanning calorimetry [DSC]), texture profile analysis [TPA], and sensory evaluation. Furthermore, the release of caffeine from the chewing gum was studied in vitro using the masticatory simulator and in vivo by a chew-out study. The microcapsules revealed a spherical form and high encapsulation efficiency, representing the success of the technique. The outcomes indicated that it is possible to encapsulate caffeine with the techniques employed and the microcapsules prolonged the release of caffeine throughout mastication. The chewing gum containing alginate-starch with chitosan-coated microcapsules showed the great potential of the microcapsule in controlling the release of the caffeine from the chewing gum, thereby delaying its bitterness.     

Co-encapsulation of lactic acid bacteria and prebiotic with alginate-fenugreek gum-locust bean gum matrix: Viability of encapsulated bacteria under simulated gastrointestinal condition and during storage time

The aim of this study was to enhance the viability of probiotic strains Pediococcus pentosaceus KID7, Lactobacillus plantarum KII2, Lactobacillus fermentum KLAB6 and Lactobacillus helveticus KII13 in gastrointestinal transit, freeze-drying condition and during storage time by microencapsulation using a combination of alginate, fenugreek gum and locust bean gum. The microcapsules were prepared using various ratio of alginate to fenugreek gum to locust bean gum and tested for its dissolution in colonic fluid. The combination that efficiently dissolved in colonic fluid was selected for co-encapsulation of the probiotic strains and prebiotics to produce synbiotic microcapsules. Further, we observed that the bacteria encapsulated with alginate-fenugreek gum-locust bean gum (AFL) matrix tolerated gastrointestinal condition efficiently compared to non-encapsulated bacteria. The encapsulated bacterial cells retained higher viability than non-encapsulated cells during freeze-drying condition and subsequent storage for 3 months at 4°C. These results show the utility of AFL matrix in microencapsulation of probiotics for use in food industry.     

The role of hemagglutination and effect of exopolysaccharide production on bifidobacteria adhesion to Caco-2 cells in vitro

It is believed that an important criterion for a potential probiotic strain is that it is capable of adhering to mucosal surfaces in the human gastrointestinal tract. The purpose of this study was to investigate a possible relationship between exopolysaccharide production and adhesion to Caco-2 cells by Bifidobacterium breve A28 and Bifidobacterium bifidum A10. In a preselection process, the hemagglutination abilities of these bacteria were determined prior to undertaking adhesion studies. B. breve A28, which produces large amounts of EPS (97.00 ± 2.00 mg/l) and has good hemagglutination abilities (+3) was found to adhere strongly to Caco-2 cells. Under gastrointestinal conditions, the high EPS producing- B. breve A28 was found to have better viability and adhesion to Caco-2 cells than the low EPS producing- B. bifidum A10. Also, B. breve A28 was found to be more effective at inhibiting Escherichia coli ATCC 11229 than B. bifidum A10. This investigation showed that high EPS production and adhesion ability may be important in the selection of bifidobacteria as probiotic strains.     

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.     

Thermotolerance induced by heat, sodium arsenite, or puromycin: its inhibition and differences between 43 degrees C and 45 degrees C

When CHO cells were treated either for 10 min at 45-45.5 degrees C or for 1 hr with 100 microM sodium arsenite (ARS) or for 2 hr with 20 micrograms/ml puromycin (PUR-20), they became thermotolerant to a heat treatment at 45-45.5 degrees C administered 4-14 hr later, with thermotolerance ratios at 10(-3) isosurvival of 4-6, 2-3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR-20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP-70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 micrograms/ml) or PUR (100 micrograms/ml) after the initial treatments greatly inhibited thermotolerance to 45-45.5 degrees C in all cases. However, for a challenge at 43 degrees C, thermotolerance was inhibited only for the ARS and PUR-20 treatments. CHM did not suppress heat-induced thermotolerance to 43 degrees C, which was the same as heat protection observed when CHM was added before and during heating at 43 degrees C without the initial heat treatment. These differences between the initial treatments and between 43 and 45 degrees C may possibly be explained by reports that show that heat causes more redistribution of HSP-70 to the nucleus and nucleolus than ARS and that redistribution of HSP-70 can occur during heating at 42 degrees C with or without the presence of CHM. Heating cells at 43 degrees C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45 degrees C immediately after heating at 43 degrees C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 micrograms/ml) or PUR (100 micrograms/ml) to inhibit protein synthesis during heating at 43 degrees C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43 degrees C, protein synthesis was required during 43 degrees C for the development of additional thermotolerance to 45 degrees C.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plant regeneration from cell suspension-derived protoplasts of Phalaenopsis

Protoplasts isolated from cell suspension culture of Phalaenopsis “Wataboushi” were cultured by (a) embedding in gellan gum-solidified hormone-free 1/2 New Dogashima medium (1/2 NDM) containing 0.44 M sorbitol, 0.06 M sucrose and 0.1 g/l l-glutamine (standard method) and (b) beads method using beads of gellan gum or sodium alginate as the gelling agents which were surrounded by liquid NDM. Although, the two beads methods gave less frequency of initial protoplast division than the standard method, the former finally resulted in higher frequency of microcolony formation than the latter. The highest frequency of microcolony formation (23%) was obtained when protoplasts were embedded in 1% Ca-alginate beads and subcultured every two weeks by replacing the surrounding liquid culture medium with a decrease in sorbitol concentration by 0.1 M. Colonies visible to the naked eyes were observed within 2 months of culture and the regenerated calluses were transferred onto hormone-free NDM supplemented with 10 g/l maltose and 0.3% (w/v) gellan gum, on which PLBs were formed and proliferated profusely. The PLBs were regenerated into plantlets after changing the carbon source to 10 g/l sorbitol and successfully acclimatized to greenhouse conditions.     

Improved gellan gum production by a newly-isolated Sphingomonas azotifigens GL-1 in a cheese whey and molasses based medium

Gellan gum is a water-soluble exopolysaccharide, it has applications in the food, pharmaceutical and chemical industries. In this study, a gellan gum producing strain was isolated from rice root, and this strain was identified be the species of Sphingomonas azotifigens. The Plackett-Burman design was applied to investigate the main factors affecting gellan gum production by S. azotifigens GL-1 in a molasses and cheese whey based medium; the medium compositions were optimized by response surface methodology. The optimum cheese whey based medium consisted of cheese whey 68.34 g/L, Na₂HPO₄ 14.58 g/L and KH₂PO₄ 7.66 g/L, and the maximum gellan gum production that using this medium was 33.75 ± 1.55 g/L. 14.75 ± 0.65 g/L gellan gum was obtained with an optimized molasses medium, which consisted of molasses 50 g/L, Na₂HPO₄ 9.71 g/L and KH₂PO₄ 5.92 g/L. The molecular weight of gellan gum obtained from two medias were 1.06 × 10⁶ and 0.89 × 10⁶ Da, respectively. The cheese whey-derived gellan gum showed a higher rhamnose, lower glucuronic acid and higher glycerate content compared to the molasses-derived gellan gum. S. azotifigens GL-1 has a high gellan gum production capacity in a cheap medium suggesting it has great potential as an industrial gellan gum producer.     

Solution properties of gellan gum: change in chain stiffness between single- and double-stranded chains

Nine samples of gellan gum in the sodium form, ranging in weight-average molar mass from 3.47 x 10(4) to 1.15 x 10(5) at 40 degrees C, were investigated by static and dynamic light scattering and viscometry in 25 mM aqueous NaCl both at 40 and at 25 degrees C. The ratios of the molar mass at 25 degrees C (in the ordered state) to that at 40 degrees C (in the disordered state) were in the range of 1.99 to 2.07, supporting the scheme of the conformational transition of gellan gum between a disassociated single chain and an associated chain composed of two molecules. Focusing on the effects of polydispersity, the intrinsic viscosities, radii of gyration, and hydrodynamic radii were analyzed on the basis of unperturbed wormlike chain models. The persistence lengths were evaluated as 9.4 nm at 40 degrees C and 98 nm at 25 degrees C.     

Influence of microencapsulation and spray drying on the viability of Lactobacillus and Bifidobacterium strains

Improved production methods of starter cultures, which constitute the most important element of probiotic preparations, were investigated. The aim of the presented research was to analyse changes in the viability of Lactobacillus. acidophilus and Bifidobacterium bifidum after stabilization (spray drying, liophilization, fluidization drying) and storage in refrigerated conditions for 4 months. The highest numbers of live cells, up to the fourth month of storage in refrigerated conditions, of the order of 10(7) cfu/g preparation were recorded for the B. bifidum DSM 20239 bacteria in which the N-Tack starch for spray drying was applied. Fluidization drying of encapsulated bacteria allowed obtaining a preparation of the comparable number of live bacterial cells up to the fourth month of storage with those encapsulated bacteria, which were subjected to freeze-drying but the former process was much shorter. The highest survivability of the encapsulated L. acidophilus DSM 20079 and B. bifidum DSM 20239 cells subjected to freeze-drying was obtained using skimmed milk as the cryoprotective substance. Stabilization of bacteria by microencapsulation can give a product easy to store and apply to produce dried food composition.     

Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment

Since Robert Koch and colleagues found agar to be an effective gelling agent over a century ago, the pure culture method using agar plates has long been a standard of microbiology. Agar is undoubtedly easy to handle and useful for culture of microorganisms, but recent discovery of the ubiquity of microorganisms that cannot be cultured on agar raises a question: is agar really the best agent? In this study, we investigated the effect of two gelling agents, agar and gellan gum, on colony formation of a diverse array of microorganisms (total 108 strains) newly isolated from freshwater sediments and a representative microorganism as a slow grower on agar medium, Gemmatimonas aurantiaca, to clarify (i) whether they can grow on both agar and gellan gum plates, and (ii) the difference in time required for colony formation between the two gelling agents. Interestingly, 22 of 108 isolates showed no ability to form any visible colonies on the agar medium but did so on the gellan gum medium, and showed low 16S rRNA gene sequence similarities to their closest species. The remaining 86 isolates grew on both agar and gellan gum, but 52 of them grew much faster on gellan gum than on agar. Moreover, gellan gum also significantly stimulated the colony formation of the representative slow‐growing microorganism G. aurantiaca. Our results demonstrate that the gelling agent is a crucial factor for the growth of bacteria on plate media, and that alternatives to agar will be very important for increasing the culturability of yet‐to‐be cultured microorganisms.     

Rapid identification of potentially probiotic Bifidobacterium species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA

This study aimed at developing a novel multiplex polymerase chain reaction (PCR) primer set for identification of the potentially probiotic Bifidobacterium species B. adolescentis, B. animalis subsp. animalis (B. animalis), B. bifidum, B. breve, B. longum biovar infantis (B. infantis), B. animalis subsp. lactis B. lactis, B. longum biovar longum (B. longum) and B. pseudolongum. The primer set comprised specific and conserved primers and was derived from the integrated sequences of 16S and 23S rRNA genes and the rRNA intergenic spacer region (ISR) of each species. It could detect and identify type strains and isolates from pharmaceuticals or dairy products corresponding to the eight Bifidobacterium species with high specificity. It was also useful for screening of the related strains from natural sources such as the gastro-intestinal tract and feces. We suggest that the assay system from this study is an efficient tool for simple, rapid and reliable identification of Bifidobacterium species for which probiotic strains are known.     

Combination of Rolling Vibration and Serpentinite Induces the Formation of Penetration-Intermediates

Bacterial cells with nano-sized acicular materials were placed in a sliding friction field between a hydrogel and an interface-forming material, after which cells with surrounding materials, namely penetration-intermediates, were formed. This phenomenon is defined as the Yoshida effect. The Yoshida effect requires the following conditions: a nano-sized acicular material, a hydrogel, an interface-forming material, and a power source for inducing sliding friction. The penetration-intermediate in the Yoshida effect has been demonstrated to readily incorporate exogenous genes and to be transformed genetically based on the acquired genes. To predict the occurrence of the Yoshida effect in the natural environment, transformation efficiency of the penetration-intermediate with plasmid DNA was investigated, and chrysotile, gellan gum, serpentinite, and artificial rolling vibration were selected as the nano-sized acicular material, hydrogel, interface-forming material, and power source for inducing sliding friction, respectively. Colloidal solution consisting of Pseudomonas sp. or Bacillus subtilis as recipient cells, donor plasmid, and chrysotile was placed on the interface between gellan gum and the serpentinite plate. To generate a sliding friction field, the serpentinite plate in contact with gellan gum was subjected to rolling vibration at 30 to 520 Gal. The sliding friction allowed recipient bacteria to become antibiotic resistant by taking up donor plasmids. Incorporation of plasmids into bacteria at the interface of gellan gum required both rolling vibration and nano-sized acicular materials such as chrysotile. Chrysotile-bound plasmid DNA was protected from catalytic reactions by DNase I and Kpn I.     

Synthesis and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum with potential biomedical applications

N-(2-aminoethyl)-2-acetamidyl gellan gum (GCM-EDA) was prepared by carboxymethylation (via nucleophilic substitution of primary hydroxyl groups of the β-d-glucose unit of gellan gum, in the presence of alkali and chloroacetic acid) and reaction with tert-butyl N-(2-aminoethyl) carbamate (N-Boc-EDA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as an activator, followed by deprotection with trifluoroacetic acid. The structural confirmation and characterization of N-(2-aminoethyl)-2-acetamidyl gellan gum was performed by spectroscopic, rheological and thermogravimetric analysis, and in vitro tests showed a lack of cytotoxicity which is indicative of the potential of this material to be used in biomedical applications.     

The Effect of Sodium Azide on Basic and Induced Thermotolerance in Saccharomyces cerevisiae

The action mechanism of the mitochondrial inhibitor sodium azide on thermotolerance in Saccharomyces cerevisiae was studied. At ambient growth temperature, pretreatment with sodium azide was shown to improve the thermotolerance of parent cells and the hsp104 mutant. Treating with the inhibitor during a mild heat shock suppressed the development of induced thermotolerance due to the inhibition of heat shock protein (Hsp104) synthesis. Treating with the inhibitor immediately before lethal heat shock produced a variety of effects on thermotolerance depending on whether the yeast metabolism was oxidative or fermentative. The conclusions are: (1) the protective effect of sodium azide on the thermotolerance of S. cerevisiae cells grown on glucose-containing medium is not related to Hsp104 functioning, and (2) the mechanisms of basic and induced thermotolerance differ considerably.