Evaluation of pea protein–polysaccharide matrices for encapsulation of acid-sensitive bacteria

Protein–polysaccharide capsules containing Bifidobacterium adolescentis were produced and tested in a series of in vitro survival experiments to evaluate capsule protection of the bacterium to simulated stomach conditions, as well as their ability to release the encapsulated bacteria under conditions similar to those found in the lower gut. A protein fraction isolated from peas (pea protein isolate: PPI; 2.0%; w/v) was mixed with each of three different polysaccharides (0.5% (w/v) of either sodium alginate, iota-carrageenan and gellan gum) to produce capsules ranging in size from 2 to 3 mm diameter. All capsule formulations provided significant protection for cells exposed to synthetic stomach juice at 37 °C relative to non-encapsulated bacteria. In addition, PPI-alginate and PPI-iota-carrageenan capsules were found to dissolve in simulated intestinal fluid at 37 °C, releasing 70–79% of their bacteria “payload” within 3 h, with higher cell numbers being released from the freeze-dried capsules. PPI-gellan gum capsules did not dissolve to the same extent and the number of released cells was ~ 26–30% lower. Following a temporal rat feeding study with the test bacterium encapsulated in PPI-alginate, B. adolescentis-specific PCR and qPCR analyses confirmed the presence of DNA from this species in rat feces, but only during the period of capsule intake.     

Stability of probiotic Lactobacillus plantarum in dry microcapsules under accelerated storage conditions

This study investigated the stability of freeze dried and fluid bed dried alginate microcapsules coated with chitosan containing model probiotic bacteria, Lactobacillus plantarum, during storage for up to 45 days at different water activities (0.11, 0.23, 0.40 and 0.70) and temperatures (4, 30 and 37 °C). The loss in cell viability was around 0.8 log in the case of fluid bed drying and around 1.3 in the case of freeze drying, with the former method resulting in dried capsules of smaller size (~ 1 mm vs 1.3 mm), more irregular shape, and with a rougher surface. In both cases, the water activity and water content were less than 0.25 and 10% w/w, respectively, which favours high storage stability. The storage stability studies demonstrated that as the water activity and temperature decreased the survival of the dried encapsulated cells increased. Considerably better survival was observed for fluid bed dried encapsulated cells compared to freeze dried encapsulated cells and freeze dried free cells with 10% sucrose (control), and in some cases, e.g. at 4 and 30 °C at water activities of 0.11, 0.23 and 0.40, there was more than 1 log difference after 45 days, with concentrations higher than 10⁸ CFU/g after 45 days of storage. The results indicate that fluid bed drying is an effective and efficient manufacturing method to produce probiotic containing capsules with enhanced storage stability.     

Physical and antibacte rial properties of alginate-based edible film incorporated with garlic oil

Antibacterial alginate-based edible film has been studied by incorporation of garlic oil as a natural antibacterial agent. Initially, 0.1% v/v garlic oil was tested in in vitro experiments against some food pathogenic bacteria. The presence of 0.1% v/v garlic oil in the nutrient broth decreased viable cell counts for Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus cereus by 2.28, 1.24, 4.31 and 5.61 log cycles, respectively after 24 h incubation. Meanwhile, an increased cell population occurred on all accompanying controls. Antimicrobial alginate films were prepared by incorporating garlic oil up to 0.4% v/v. They were characterized for antibacterial activity, mechanical and physical properties. The edible film exhibited antibacterial activity against Staphylococcus aureus and B. cereus among bacteria tested by using agar diffusion assay. Tensile strength and elongation at break were significantly (p < 0.05) changed by incorporation of garlic oil at 0.3% and 0.4% v/v, respectively. Water vapor permeability decreased significantly (p < 0.05) with 0.4% v/v garlic oil incorporation, whereas total color difference remained same until 0.4% v/v. These results revealed that garlic oil has a good potential to be incorporated into alginate to make antimicrobial edible film or coating for various food applications.     

The influence of multi stage alginate coating on survivability of potential probiotic bacteria in simulated gastric and intestinal juice

The probiotics, Lactobacillus acidophilus PTCC1643 and Lactobacillus rhamnosus PTCC1637, were encapsulated into uncoated calcium alginate beads and the same beads were coated with one or two layers of sodium alginate with the objective of enhancing survival during exposure to the adverse conditions of the gastro-intestinal tract. The survivability of the strains, was expressed as the destructive value (decimal reduction time). Particle size distribution was measured using laser diffraction technique. The thickness of the alginate beads increased with the addition of coating layers. No differences were detectable in the bead appearance by scanning electron microscopy (SEM). The alginate coat prevented acid-induced reduction of the strains in simulated gastric juice (pH 1.5, 2 h), resulting in significantly (P < 0.05) higher numbers of survivors. After incubation in simulated gastric (60 min) and intestinal juices (pH 7.25, 2 h), number of surviving cells were 6.5 log cfu mL^?1 for L. acidophilus and 7.6 log cfu mL^?1 for L. rhamnosus by double layer coated alginate microspheres, respectively, while 2.3 and 2.0 log cfu mL^?1 were obtained for free cells, respectively.     

Physical stability of emulsion encapsulated in alginate microgel particles by the impinging aerosol technique

Emulsion filled alginate microgel particles can be applied as carrier systems for lipophilic actives in pharmaceutical and food formulations. In this study, the effects of oil concentration, emulsifier type and oil droplet size on the physical stability of emulsions encapsulated in calcium alginate microgel particles (20–80 μm) produced by a continuous impinging aerosol technique were studied. Oil emulsions emulsified by using either sodium caseinate (SCN) or Tween 80 were encapsulated at different oil concentrations (32.55, 66.66 and 76.68% w/w of total solids content). The emulsions were analysed before and after encapsulation for changes in emulsion size distribution during storage, and compared to unencapsulated emulsions. The size distribution of encapsulated fine emulsion (mean size ~ 0.20 μm) shifted to a larger size distribution range during encapsulation possibly due to the contraction effect of the microgel particles. Coarse emulsion droplets (mean size ~ 18 μm) underwent a size reduction during encapsulation due to the shearing effect of the atomizing nozzle. However, no further size changes in the encapsulated emulsion were detected over four weeks. The type of emulsifier used and emulsion concentration did not significantly affect the emulsion stability. The results suggest that the rigid gel matrix is an effective method for stabilising lipid emulsions and can be used as a carrier for functional ingredients.     

In vitro study of anti-oxidative effects of β-carotene and α-lipoic acid for nanocapsulated lipids

In this study we attempted to assess the efficacy of the in vitro activities of β-carotene and α-lipoic acid in conjugation with an encapsulated lipid. Nanoemulsion was prepared and freeze-dried to get nanocapsules. Highest encapsulation efficiency 84.32 ± 1.08% was achieved for α-lipoic acid nanocapsules, whereas for β-carotene nanocapsules it was 79.63 ± 1.41%. Morphology study showed that the dried capsules had an irregular, rough surface. Both anti-oxidants were equally effective in releasing core materials. Complete release of the total lipid occurred within 210 min from both β-carotene and α-lipoic acid bearing nanocapsules. Stability study revealed that β-carotene nanocapsules showed decrease in oil content from 79.63 g lipid to 72.8 g lipid for every 100 g lipid encapsulated, in 3 months, whereas α-lipoic acid nanocapsules retained oil much better, the oil retention being from 84.32 g lipid to 78.08 g lipid per 100 g lipid encapsulated. DPPH radical scavenging activity of β-carotene and α-lipoic acid ranged from 62.75% and 44.13% to 32.94% and 36.95% (after 90 days) respectively. Reducing activity of β-carotene was higher (absorbance 0.492 to 0.291 at 90 days) compared to α-lipoic acid (absorbance 0.243 to 0.109 after 90 days). Again β-carotene and α-lipoic acid metal-chelation activity ranged from 47.65% and 48.59% to 32.315% and 39.29% after 90 days.     

Coating papers with soy protein isolates as inclusion matrix of carvacrol

Antimicrobial papers were prepared by coating paper with soy protein isolate (SPI) solution as inclusion matrix of carvacrol, an antimicrobial agent. Addition of carvacrol (30% w/w of SPI) to SPI solution (10% w/v) prepared at 25 °C induced soy protein aggregates and viscosity decrease. Heat treatment (50, 70, 90 °C) of SPI solutions and carvacrol addition improved homogeneity reduced particles size and increased viscosity of solutions. The aggregated structure of SPI in the presence of carvacrol at 25 °C may play the role of a trapping structure leading to low carvacrol losses during coating and drying process of paper (9.6% against 37% after heat treatment at 90 °C) and to lower release rates specially the first three days (0.04 g/m²/day and 0.31 g/m²/day when SPI coating solutions were prepared at 25 and 90 °C, respectively). Regardless of the heat treatments received by the SPI solutions, residual carvacrol quantities in the coated papers after 50 days ranged between 0.6 and 0.7 g/m².     

Encapsulation of α-tocopherol in protein-based delivery particles

Two types of proteins, including β-lactoglobulin (BLG) and hen egg white protein (HEW), were examined for their ability to encapsulate α-tocopherol (α-TOC) after salt-induced gelation of the proteins. Parameters affecting encapsulation efficiency were investigated including the type of salt, as well as concentrations of salt, protein, and α-TOC. Concentrations of protein and α-TOC revealed to have an influence on encapsulation efficiency. The optimum preparation condition of BLG-encapsulated α-TOC was as follows: BLG of 0.5% (w/v); α-TOC of 100 mM; and CaCl₂ of 25 mM. The optimum preparation condition of HEW-encapsulated α-TOC was as follows: HEW of 4.0% (w/v); α-TOC of 50 mM; and ZnCl₂ of 25 mM. With the selected preparation conditions, encapsulation efficiency by BLG aggregates was approx. 20% and that by HEW was approx. 32%. From the in vitro estimation, the release of α-TOC was nearly 100% in simulated gastric condition. Alginate was therefore used for coating of these encapsulated particles to prolong the release of α-TOC till simulated intestinal condition. The α-TOC of approx. 55% and 38% were retained and released in the simulated intestinal condition from BLG- and HEW-encapsulated particles, respectively.     

Effect of alginate concentrations on survival of microencapsulated Lactobacillus casei NCDC-298

This study reports the tolerance of Lactobacillus casei NCDC-298 encapsulated in different alginate concentrations (2%, 3% or 4%), to low pH (1.5), high bile salt concentration (1% or 2%) and heat processing (55, 60 or 65 °C for 20 min). The release of encapsulated cells in simulated aqueous solution of colonic pH was also assessed. The survival of encapsulated L. casei was better at low pH, high bile salt concentration and during heat treatment as compared to free cells. The survival increased proportionately with increasing alginate concentrations without affecting the release of entrapped cells in solution of colonic pH.     

Characterization of emulsion stabilization properties of quince seed extract as a new source of hydrocolloid

The capability of seed extracts in stabilizing emulsions has particularly received interest in recent years. Upon soaking quince seeds into water, biopolymers inside the seeds are extracted to water, forming mucilage. This study investigates the physical stability, rheology and microstructure of oil (sunflower oil) in water emulsions, stabilized by 2% (w/v) whey protein isolate with varying concentrations of xanthan and quince seed gum. Quince seed gum resulted in emulsions with smaller low-shear viscosities and shear thinning capabilities compared to the same concentrations of xanthan. Quince seed gum emulsions with concentrations ≤ 0.1 (w/v), displayed rapid creaming due to bridging flocculation. Despite the difference in apparent viscosities, for gum concentrations < 0.2 (w/v), both gums demonstrated comparable stability with xanthan gum in general yielding marginally more stable emulsions. Gum concentrations > 0.3 (w/v) resulted in physically stable emulsions even after 5 months. Overall, quince seed gum displayed significant emulsification and stabilization properties.     

Effect of cryoprotectants,prebiotics and microencapsulation on survival of probiotic organisms in yoghurt and freeze-dried yoghurt

The survival of probiotic microorganisms including Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus rhamnosus and Bifidobacterium spp. was evaluated in yoghurt and freeze-dried yoghurt after processing and storage. The effectiveness of microencapsulating probiotic organisms as well as adding cryoprotectants and prebiotics in improving their viability was also investigated. The viability of Bifidobacterium infantis 17930 and L. rhamnosus GG was reduced by 0.07 log, while that of L. casei 1520 and Bifidobacterium longum 1941 was reduced by 0.28 and 0.39 log, respectively. There was a 7% improvement in the viability of L. casei 1520 when cryoprotectant ‘Unipectine™ RS 150’ was added at 2.5% (w/v). The prebiotic ‘Raftilose^®P95’ when added at 1.5% w/v to yoghurt improved the viability of the combined selected probiotic organisms by 1.42 log during four weeks of storage at 4 °C. Microencapsulation with alginate improved viability of combined selected probiotic organisms by 0.31 log in freeze-dried yoghurt stored at 21 °C.     

Rheology of emulsion-filled alginate microgel suspensions

Emulsion filled polysaccharide gels can be used as carrier systems of lipophilic bioactives in the food, pharmaceutical and cosmetics industry. This carrier system can exist either as bulk or discrete gel systems. In this study the rheological properties of discrete emulsion filled alginate microgel suspension was examined as a function of volume fraction (ϕ) and oil content. Fine emulsion (220 nm) was encapsulated within alginate microgels (mean size 36.2–57.8 μm) by using the impinging aerosol technique. The microgels (containing 0–77% w/w oil total solids basis) produced were estimated to have particle modulus in the range of 150–212 Pa. An increase in oil content in the microgels led to more deformable microgels due to the reduction in gel density. The deformability of microgels influenced the bulk modulus and apparent viscosity of the concentrated suspension. At the same suspension volume fraction (ϕ), suspensions with more deformable microgels exhibited a lower bulk modulus. We also showed that the Carreau and Cross models were adequate in predicting the flow behaviour of the concentrated emulsion filled microgel suspension.     

Diet-induced changes in alginate- and laminaran-fermenting bacterial levels in the caecal contents of rats

Brown algae contain soluble polysaccharides, such as alginic acid, fucoidan and laminaran. To assess the induction of dietary fiber-fermenting bacteria in the intestine, rats were fed diet containing no dietary fiber (control) or 2% w/w of the polysaccharides for 2 weeks. The levels of dietary fiber-fermenting bacteria in caecal contents were determined using decimal dilution culture containing 1% w/v of the fibers. Caecal microbiota in the rats was analyzed using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). In the culture method, 4–9 log viable cells/g caecal content of alginate-fermenting bacteria was detected in rats fed alginate, while this was not detected in rats fed the control diet. Although laminaran-fermenting bacteria were detected in control rats (4–9 log viable cells/g), the level observed in rats fed laminaran was 8 or 9 log viable cells/g. On the other hand, fucoidan-fermenting bacteria were not detected in rats fed fucoidan. DGGE analysis showed laminaran administration increased the diversity of bacterial bands. Clostridium spp. and Parabacteroides distasonis were detected as typical species in rats fed alginate and laminaran. The results indicate that the intake of soluble fermentable fibers in edible brown algae can alter the intestinal microbiota and its fermentation capacity.     

Extrusion of mixtures of starch and d-limonene encapsulated with β-cyclodextrin: Flavour retention and physical properties

d-Limonene was encapsulated with β-cyclodextrin to improve its retention during pre-added flavour starch extrusion. The objective of this work was to determine the effect of processing condition on the flavour retention and extrudate properties. Corn starch containing five levels of β-cyclodextrin-d-limonene capsules (0–5%) were extruded at five different maximum barrel temperatures (133–167 °C) and screw speeds (158–242 rpm) using a twin screw extruder. The effect of these parameters on the flavour retention, expansion, texture, colour difference (ΔE), Water Absorption Index, Water Solubility Index, and residence time distribution (RTD) were investigated. Barrel temperature and capsule level predominantly influenced flavour retention and extrudate properties, while screw speed primarily affected extruder performances such as torque, die pressure, specific mechanical energy and RTD.     

Electrostatic spraying of organic acids on biofilms formed by E. coli O157:H7 and Salmonella Typhimurium on fresh produce

Electrostatic spraying which has an even and retained surface coverage could be an effective novel technique to completely cover the surface of fresh produce to disrupt biofilm formation by pathogenic bacteria. Spinach leaves and cantaloupe rind were spot-inoculated with a bacterial culture and stored at 8 °C for 72 h to allow biofilm formation. Among various green fluorescent protein-labeled strains, ED 14 strain of E. coli O157:H7 and SD 10 strain of Salmonella Typhimurium had the best attachment based on colony counts. The produce samples were electrostatically sprayed with malic (MA) and lactic (LA) acid solutions alone (1.0/2.0/3.0/4.0% w/v) or in combination (0.5 + 0.5/1.0 + 1.0/1.5 + 1.5/2.0 + 2.0% w/v) to test for a reduction in the attached bacteria. A combined treatment of LA 2.0% w/v + MA 2.0% w/v had the highest log reduction (CFU/disk) of 4.14 and 3.6 on the attached E. coli strain ED 14 (spinach) and Salmonella strain SD 10 (cantaloupe), respectively. Crystal violet assay demonstrated the disruptive effect of organic acids on biofilms formed by the pathogenic bacteria. Application of electrostatic spray with a combination of malic and lactic acids resulting in a log reduction (CFU/disk) of 3.6 or higher can improve the microbial safety of spinach and cantaloupe by preventing the pathogenic biofilm formation and bacterial growth.     

Determination of the efficacy of ultrasound in combination with essential oil of oregano for the decontamination of Escherichia coli on inoculated lettuce leaves

Recent outbreaks related to fresh produce have shown that there is a need to develop new efficient and effective decontamination technologies that protect consumers' safety. Ultrasound and assisted ultrasound, i.e., the combination of ultrasound with another decontamination technology has been shown to be effective against bacteria. In this study equal surfaces of free-bacteria of Romaine lettuce were inoculated with Escherichia coli O157:H7 NCTC 12900. Leaves were then treated with an ultrasound probe system (26 kHz, 90 μm, 200 W, 14 mm Ø). Same processing time (300 s) was applied with continuous and pulsed modes of: (i) 10 s on/6 s off, (ii) 5 s on/5 s off and (iii) 2 s on/8 s off. Thus, total treatment durations were 5, 8, 10 and 25 min. Moreover, concentrations of essential oil (i.e. 0.010% v/v, 0.014% v/v. 0.018% v/v, 0.022% v/v and 0.025% v/v) of oregano were tested, operating with continuous and 2 s on/8 s off pulsed mode. The structure of treated samples was also analysed by SEM and light microscopy. Continuous ultrasound treatments led to a reduction of 2.65 ± 0.23 log CFU/cm² of E. coli on the lettuce. No significant differences were found among the tested pulsed and continuous modes. In addition no significant differences were found between injured and no-injured cells among all the applied treatments on the leaves of the lettuce. Application of the different concentrations of EO alone, at the levels mentioned previously reduced the bacteria on the surface of the lettuce. Less than 2 logs of total inactivation (on the lettuce and the treated water) were achieved when different concentrations of EO were tested alone. Assisted ultrasound enhanced the levels of microbial reduction on the surface of the lettuce at concentrations higher than 0.010% (v/v) of oregano EO. Significant differences were found at 0.018%, 0.022% and 0.025% (v/v) both in continuous and pulsed US modes when compared with samples treated only with US. Same concentrations of EO combined with continuous or pulsed mode of US reduced the level of E. coli in water below the limit of detection. When continuous or pulse mode of US was combined with 0.025% (v/v) of EO of oregano the result was synergetic. Images reported neither structural differences nor damage of the leaves among treated samples and controls. Concluding, the effectiveness of the assisted ultrasound process was dependent on the processing time rather than on the different configurations (continuous or on/off) or the total treatment duration. Furthermore, EO combined with US could work synergistically on enhancing the inactivation of the tested bacteria.     

Monitoring nisin desorption from a multi-layer polyethylene-based film coated with nisin loaded HPMC film and diffusion in agarose gel by an immunoassay (ELISA) method and a numerical modeling

Polyethylene-based films coated with nisin loaded HPMC films were put in contact with food simulants, i.e. agarose gels with 5 or 30% (w/w) fat. Nisin desorption from the multi-layer films and diffusion in agarose gels were monitored by ELISA (Enzyme Linked ImmunoSorbent Assay). The data obtained after 2 or 6 days of contact between antimicrobial films and agarose gels were employed to determine nisin mass transfer by numerical modeling following Fick's second law. The values were in the range from 0.87 × 10^? 3 m s^? 1 to 4.30 × 10^? 3 m s^? 1 and 6.5 × 10^? 11 m² s^? 1 to 3.3 × 10^? 10 m² s^? 1, for nisin apparent desorption and diffusion coefficients, respectively. The diffusion process was governed by interactions between food matrix simulant and nisin. Moreover, it was observed that the polymer in the coating did not modify plastic film initial mechanical resistance and water vapor permeability.Industrial relevanceThis paper concerns active packaging, considered as a new approach to preserve food shelf life. Active packaging is a real gain for plastic and Food industrials. The paper deals with coating as a manner to activate packaging. The impact of coating on film properties is investigated.Also, predictive models are proposed to determine antimicrobial agent desorption and diffusion during some storage conditions.     

Probiotic-loaded microcapsule system for human in situ folate production: Encapsulation and system validation

This study focused on the use of a new system, an alginate | Ɛ-poly-l-lysine | alginate | chitosan microcapsule (APACM), able to immobilize a folate-producing probiotic, Lactococcus lactis ssp. cremoris (LLC), which provides a new approach to the utilization of capsules and probiotics for in situ production of vitamins. LLC is able to produce 95.25 ± 26 μg·L^− 1 of folate, during 10 h, and was encapsulated in the APACM. APACM proved its capacity to protect LLC against the harsh conditions of a simulated digestion maintaining a viable concentration of 6 log CFU·mL^− 1of LLC. A nutrients exchange capacity test, was performed using Lactobacillus plantarum UM7, a high lactic acid producer was used here to avoid false negative results. The production and release of 2 g·L^− 1 of lactic acid was achieved through encapsulation of L. plantarum, after 20 h. The adhesion of APACM to epithelial cells was also quantified, yielding 38% and 33% of capsules adhered to HT-29 cells and Caco-2 cells, respectively.     

Assessment of agave fructans as lyoprotectants of bovine plasma proteins concentrated by ultrafiltration

The effectiveness of fructans as protectant agent to prevent protein denaturation was evaluated during freeze-drying and storage. Native agave fructans and two fractions obtained by ultrafiltration were assessed as protective agents. Since the protein denaturation during freeze-drying can alter the functional properties of proteins, the evaluation of bovine plasma protein properties was performed with different concentration of fructans, pH and freezing temperatures. The results showed that the incorporation of fructans as lyoprotectants improved functional properties, due to the reduction of protein denaturation with maximum stabilization of plasma bovine protein at a fructan concentration between 5% and 10% (w/v) for native fructans and 10% (w/v) for the fractions of fructans at − 40 °C. Moreover, when freezing at − 4 °C, fructans fraction at 15% (w/v) prevented protein denaturation while native fructans exerted no acceptable protection (P < 0.001). This behavior could be explained by a microstructure study of fructans, where the higher heterogeneity in size of native fructans may be the reason for the lower protective effect. Furthermore, the higher heterogeneity of the samples affected the extent of non-enzymatic browning in the range of the temperatures assessed. The shelf life of freeze-dried proteins was improved from 1.7 months for the control sample to 7–11, depending on the saccharide considered. Taking into account the health benefits of fructans, since they are categorized as prebiotic, the protein–saccharide mixture may be valuable functional ingredients for food formulations.     

Development of probiotic beads similar to fish eggs

Probiotic foods are mainly restricted to dairy and soy products. This study aimed to develop a new probiotic beads similar to fish eggs, commonly used in oriental cuisine. Beads were produced by the extrusion encapsulation technique with calcium alginate, added to one of the following cultures: Lactobacillus rhamnosus GG ATCC 53103 and Bifidobacterium animalis DN-173 010 and stored for 30 days at 4 °C. The beads were characterized by the size, weight, morphology and viability of the probiotic strains in different storage temperatures and in simulated gastric juice adjusted to different pH values. The beads were also evaluated by a sensorial affective hedonic scale. The beads present a 2.8 mm diameter and a weight of 0.01 g (p > 0.05). Free and encapsulated cells were tolerant to pH 3.0. At pH 2.5 only of the encapsulated cells presented counts above 6 Log colony-forming units per gram (CFU/g). Beads containing L. rhamnosus showed higher viability 10⁷ CFU/g in storage for 30 days under refrigeration. The beads may be stored at abusive temperature for 5 h without loss of viability cells. The probiotic product developed showed an 82.2% acceptability index of overall characteristics and good market potential as a new probiotic product.