Supercritical CO2 interpolymer complex encapsulation improves heat stability of probiotic bifidobacteria

The probiotic industry faces the challenge of retention of probiotic culture viability as numbers of these cells within their products inevitably decrease over time. In order to retain probiotic viability levels above the therapeutic minimum over the duration of the product’s shelf life, various methods have been employed, among which encapsulation has received much interest. In line with exploitation of encapsulation for protection of probiotics against adverse conditions, we have previously encapsulated bifidobacteria in poly-(vinylpyrrolidone)-poly-(vinylacetate-co-crotonic acid) (PVP:PVAc-CA) interpolymer complex microparticles under supercritical conditions. The microparticles produced had suitable characteristics for food applications and also protected the bacteria in simulated gastrointestinal fluids. The current study reports on accelerated shelf life studies of PVP:PVAc-CA encapsulated Bifidobacterium lactis Bb12 and Bifidobacterium longum Bb46. Samples were stored as free powders in glass vials at 30 °C for 12 weeks and then analysed for viable counts and water activity levels weekly or fortnightly. Water activities of the samples were within the range of 0.25–0.43, with an average a _w  = 0.34, throughout the storage period. PVP:PVAc-CA interpolymer complex encapsulation retained viable levels above the recommended minimum for 10 and 12 weeks, for B. longum Bb46 and B. lactis Bb12, respectively, thereby extending their shelf lives under high storage temperature by between 4 and 7 weeks. These results reveal the possibility for manufacture of encapsulated probiotic powders with increased stability at ambient temperatures. This would potentially allow the supply of a stable probiotic formulation to impoverished communities without proper storage facilities recommended for most of the currently available commercial probiotic products.     

Influence of encapsulation on the survival of probiotics in food matrix under simulated stress conditions

The main objective of the present study was to evaluate the influence of encapsulation by extrusion technique using two hydrogels, namely; sodium alginate (Na-ALG) and whey protein isolate (WPI) on Bifidobacterium bifidium viability and stability of yoghurt under simulated gastrointestinal conditions. Probiotic bacteria (free or encapsulated) were added to yogurt for four weeks to test their viability and stability. Physicochemical and sensory analysis of yoghurt were conducted. Viability of B. bifidium in the simulated gastrointestinal conditions pH 2 and pH 7.5 was determined. Also, the efficiency of encapsulated final yield of the microcapsules was determined. With storage time, the pH of yoghurt containing encapsulated bacteria increased more than that of yoghurt containing free probiotic bacteria, resulting in a decrease in acidity. When compared to yoghurt containing encapsulated bacteria, the lactose level of yoghurt containing free probiotic bacteria decreased over time. The viscosity of yoghurt containing encapsulated WPI remained stable over the storage period, with syneresis remaining stable. The sensory properties of yoghurt containing free probiotics deteriorated over time. Cell viability was significantly reduced in yoghurt-containing free probiotics compared to other treated yoghurts. Cell viability in free probiotics yoghurt was lower than in encapsulated ones when exposed to simulated gastric and intestinal juice. In conclusion, WPI- encapsulated probiotics showed better stability over 28 days of storage in both yoghurt and gastrointestinal conditions, followed by sodium alginate.     

Effect of Encapsulation on Viability of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> CFR815j and Physiochemical Properties of Ice Cream

The health beneficial attributes of bifidobacteria and its safe association with the host gut has increased its significance as a probiotic. However delivering probiotic bifidobacteria with Minimum Biological Value (MBV) through product has always been a challenge. In the present study, an attempt was made to maintain the viability of native isolate of Bifidobacterium longum CFR 815j and deliver through ice-cream. B. longum CFR815j was microencapsulated in alginate starch capsules by emulsification followed by evaluation of bead stability in simulated gastrointestinal conditions. After incorporation in ice-cream, the effect on chemical properties, sensory parameters and meltdown characteristics of the product were also evaluated. Survival studies of B. longum revealed higher counts than 10⁷ in the product which is essential for probiotic bacteria to exhibit beneficial effect. Further, all the properties of this ice-cream were comparable to the regular ice-cream. Our studies conclude that encapsulation was able to maintain the requisite MBV of bifidobacteria in ice-cream without affecting the sensory characteristics.     

Role ofBifidobacterium longum in the induction of apoptotic deletion in the human enterocyte-like Caco-2 cell line

Bifidobacterium longum is a probiotic, known for its beneficial effects to the human gut and even for its immunomodulatory and antitumor activities. Recently, many studies have stressed out the intimate relation between probiotic bacteria and the GIT mucosa and their influence on human cellular homeostasis. We focused on the apoptotic deletion of cancer cells induced byB. longum. This has been valuedin vitro, performing the incubation of threeB. longum strains with enterocyte-like Caco-2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains tested were defined for their adhesion properties using adhesion and autoaggregation assays. These features are considered necessary to select a probiotic strain. The three strains named B12, B18 and B2990 resulted respectively: “strong adherent”, “adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to investigate apoptotic deletion. Cocultures of Caco-2 cells withB. longum resulted positive in DNA fragmentation test, only when adherent strains were used (B12 and B18). These results indicate that the interaction with adherentB. longum can induce apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the gastrointestinal tract and in restoring the ecology of damaged colon tissues.     

Use of viability staining in combination with flow cytometry for rapid viability assessment of Lactobacillus rhamnosus GG in complex protein matrices

The aim of this study was to demonstrate that flow cytometry (FACS) could potentially be employed for rapid viability assessment of probiotic bacteria immobilized or encapsulated in complex matrices. Lactobacillus rhamnosus GG was immobilized within six different protein environments using whey protein isolate (WPI) and yoghurt matrices and encapsulated within protein micro-beads, all of which ranged in structural complexity. Following a series of environmental-stress trials, survival of the strain was examined using FACS compared to traditional plate count techniques. Cell extraction and digestive pre-treatments were designed to release cells and reduce the protein background, respectively, which represent compositional obstacles for efficient FACS analysis. Physico-chemical properties of protein-probiotic components revealed the mechanism necessary for efficient cell delivery during FACS analysis. This assay required 40 min sample preparation and distinct functional populations were discriminated based on fluorescent properties of thiazole orange (TO) and propidium iodide (PI). This assay yielded 45-50 samples/h, a detection range of 10²-10¹⁰ cfu/ml of homogenate and generated correlation coefficients (r) of 0.95, 0.92 and 0.93 in relation to standard plate counts during heat, acid and storage trials, respectively. In conclusion, this methodology provides impetus for dynamic progression of FACS for rapid viability assessment of live bacteria immobilized/encapsulated within complex protein systems.     

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures.The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.     

Inhibitory effect of essential oils of Allium sativum and Piper longum on spontaneous muscular activity of liver fluke, Fasciola gigantica

Effects of essential oil of Allium sativum (garlic) and Piper longum (Indian long pepper) were evaluated on muscular activity of whole Fasciola gigantica and its strip preparation. The whole flukes and longitudinal strip preparations of the flukes were isometrically mounted to record the spontaneous muscular activity (SMA) and to evaluate effects of cumulative doses (0.1, 0.3, 1.0 and 3.0 mg/ml) of the plant essential oils. Whole flukes and the strip preparations exhibited continuous SMA without any significant difference in its baseline tension, frequency and amplitude for 2 h. Essential oil of A. sativum produced significant reduction in the frequency and the amplitude of the SMA of whole fluke at 1 and 3 mg/ml concentrations. It caused complete paralysis of the fluke after 15 min of administration of 3 mg/ml concentration. Similar to whole fluke, essential oil of A. sativum (3 mg/ml) also produced flaccid paralysis in the strip preparations of the flukes. Essential oil of P. longum firstly induced marked excitatory effect and then there was flaccid paralysis of the whole fluke following 15 min exposure at 3 mg/ml concentration. Complete flaccid paralysis of the strip preparation was also ensued after 15 min of administration of 3 mg/ml concentration of P. longum. In both the essential oils, the whole fluke and strip preparations did not recover from paralysis following 2-3 washes. In conclusion, the observations demonstrated irreversible paralytic effect of essential oils of A. sativum and P. longum on F. giganticain vitro which might possibly help to developing herbal-based anthelmintic.     

Synthesis and characterization of chitosan-based pH-sensitive semi-interpenetrating network microspheres for controlled release of diclofenac sodium

pH-Sensitive semi-interpenetrating networks (IPNs) based on chitosan (Cs) and acrylamide-grafted hydroxyethylcellulose (AAm-g-HEC) were prepared in the form of microspheres (MPs) by emulsion-crosslinking technique using glutaraldehyde (GA) as a crosslinker. Diclofenac sodium (DS) drug was successfully encapsulated into IPN microspheres by varying the ratio of Cs and AAm-g-HEC, % drug loading, and amount of GA. DS encapsulation of up to 83% was obtained as measured by UV spectroscopy. MPs with average particle sizes in the range of 188-310 μm were obtained. MPs were characterized by Fourier transform infrared spectroscopy (FTIR), Scanning electron microscopy (SEM), and Differential scanning calorimetry (DSC). Diffusion coefficients (D) of water transport through the microspheres were determined using an empirical equation. In vitro release of DS from these matrices has been investigated in pH 1.2 and 7.4 media.     

Quantitative in vitro assay to evaluate the capability of yeast cell wall fractions from Trichosporon mycotoxinivorans to selectively bind gram negative pathogens

Yeast cell wall fractions have been proposed to bind enteropathogenic bacteria. The aim of this study was to develop a quantitative assay by measuring the optical density as growth parameter of adhering bacteria. The exponential growth phase of adhering bacteria was determined by optical density reading and compared with the colony count (CFU/mL). A linear regression was compiled and the bacterial number bound to the yeast cell wall product could be determined. Further focus was the investigation of a yeast cell wall from strain Trichosporon mycotoxinivorans (MTV) for its ability to bind gram negative Salmonella, E. coli and Campylobacter strains and gram positive probiotic bacteria of the genera lactobacilli and bifidobacteria as well as gram positive Clostridium perfringens quantitatively. The gram negative probiotic strain E. coli Nissle 1917 was also investigated. Seven out of 10 S. Typhimurium and S. Enteritidis strains adhered to the cell wall product with an amount between 10³ and 10⁴ CFU/10 μg. Four out of 7 E. coli strains showed an average binding capability (10² CFU/10 µg) whereas 4 × 10³E. coli F4 cells bound per 10 μg yeast cell wall. E. coli 0149 K91, E. coli 0147 K89, C. jejuni and C. perfringens as well the genera lactobacilli and bifidobacteria did not bind to the yeast cell wall. E. coli Nissle 1917 was bound with 2 × 10² CFU/10 μg. These results demonstrate that cell wall from MTV can be used to differentially bind E. coli spp. and Salmonella spp. up to 8 × 10⁴ CFU/10 μg. Thus certain yeast cell walls may prevent enteric infections caused by selective bacteria. This methodical approach would be an accurate tool in the feed industry for quality control of yeast cell wall products.     

Sequence Analysis of Two Cryptic Plasmids from Bifidobacterium longum DJO10A and Construction of a Shuttle Cloning Vector

Bifidobacterium longum DJO10A is a recent human isolate with probiotic characteristics and contains two plasmids, designated pDOJH10L and pDOJH10S. The complete sequences of both these plasmids have now been determined and consist of two circular DNA molecules of 10,073 and 3,661 bp, with G+C contents of 62.2% and 66.2%, respectively. Plasmid pDOJH10L is a cointegrate plasmid consisting of DNA regions exhibiting very high sequence identity to two other B. longum plasmids, pNAC2 (98%) and pKJ50 (96%), together with another region. Interestingly, the rolling circular replication (RCR) regions of both the pNAC2- and pKJ50-like plasmids were disrupted during the recombination event leading to a further recombination event to acquire a functional replicon. This consists of a new fused rep gene and an RCR-type ori consisting of a conserved DnaA box in an AT-rich region followed by four contiguous repeated sequences consistent with an iteron structure and an inverted repeat. The smaller pDOJH10S had no sequence similarity to any other characterized plasmid from bifidobacteria. In addition, it did not contain any features consistent with RCR, which is the replication mechanism proposed for all the bifidobacteria plasmids characterized to date. It did exhibit sequence similarity with several theta replication-related replication proteins from other gram-positive, high-G+C bacteria, with the closest match from a Rhodococcus rhodochrous plasmid, suggesting a theta mechanism of replication. S1 nuclease analysis of both plasmids in B. longum DJO10A revealed single-strand DNA intermediates for pDOJH10L, which is consistent for RCR, but none were detected for pDOJH10S. As the G+C content of pDOJH10S is similar to that of Rhodococcus rhodochrous (67%) and significantly higher than that of B. longum (60.1%), it may have been acquired through horizontal gene transfer from a Rhodococcus species, as both genera are members of the Actinomycetes and are intestinal inhabitants. An Escherichia coli-B. longum shuttle cloning vector was constructed from pDOJH10S and the E. coli ori region of p15A, a lacZ gene with a multiple cloning site of pUC18, and a chloramphenicol resistance gene (CAT) of pCI372 and was transformed successfully into E. coli and B. longum. It could not be introduced into lactic acid bacteria (Lactococcus and Lactobacillus), showing it was not very promiscuous. It was stably maintained in B. longum in the absence of antibiotic pressure for 92 generations, which is consistent with the segregational stability of theta-replicating plasmids in gram-positive bacteria. This is the first cloning vector for bifidobacteria that does not utilize RCR and should be useful for the stable introduction of heterologous genes into these dominant inhabitants of the large intestine.     

The complete genome sequence of Bifidobacterium longum LTBL16, a potential probiotic strain from healthy centenarians with strong antioxidant activity

B. longum LTBL16 is a potential probiotic strain that was isolated from healthy centenarians in Bama, China. In vitro experiments show that B. longum LTBL16 has a strong antioxidant activity and the complete genome of B. longum LTBL16 was sequenced in this work. The genome consists of one 2,430,682 bp circular chromosome that is plasmid free. The circular chromosome has a GC content of 61.23% and contains 2071 coding sequences (CDSs), 4 rRNA manipulators and 55 tRNA coding genes. Genetic analysis showed that at least five protein-coding genes were associated with antioxidant activity, and the abundance of these genes may be related to free radical scavenging rates and oxygen tolerance. In addition, the safety of B. longum LTBL16 was evaluated using a virulence factor database and antibiotic resistance gene database. The results indicate that B. longum LTBL16 has the good potential for the development and utilization as a probiotic.     

Bifidobacterium longum supplementation improves age-related delays in fracture repair

Age-related delays in bone repair remains an important clinical issue that can prolong pain and suffering. It is now well established that inflammation increases with aging and that this exacerbated inflammatory response can influence skeletal regeneration. Recently, simple dietary supplementation with beneficial probiotic bacteria has been shown to influence fracture repair in young mice. However, the contribution of the gut microbiota to age-related impairments in fracture healing remains unknown. Here, we sought to determine whether supplementation with a single beneficial probiotic species, Bifidobacterium longum (B. longum), would promote fracture repair in aged (18-month-old) female mice. We found that B. longum supplementation accelerated bony callus formation which improved mechanical properties of the fractured limb. We attribute these pro-regenerative effects of B. longum to preservation of intestinal barrier, dampened systemic inflammation, and maintenance of the microbiota community structure. Moreover, B. longum attenuated many of the fracture-induced systemic pathologies. Our study provides evidence that targeting the gut microbiota using simple dietary approaches can improve fracture healing outcomes and minimize systemic pathologies in the context of aging.     

Improvement of catalytic activity of lipase from Candida rugosa via sol-gel encapsulation in the presence of calix(aza)crown

Lipase from Candida rugosa (CRL) was encapsulated within a chemically inert sol-gel support in the presence of calix(aza)crowns as the new additives. The catalytic activity of the encapsulated lipases was evaluated both in the hydrolysis of p-nitrophenyl palmitate (p-NPP) and the enantioselective hydrolysis of racemic Naproxen methyl ester. It has been observed that the percent activity yields of the calix(aza)crown based encapsulated lipases were higher than that of the free lipase. Improved enantioselectivity was observed with the calix(aza)crown-based encapsulated lipases as compared to encapsulated free lipase. The reaction of Naproxen methyl ester resulted in 48.4% conversion for 24 h and 98% enantiomeric excess for the S-acid, corresponding to an E value of >300 (E = 166 for the encapsulated free enzyme). Moreover, the encapsulated lipases were still retained about 18% of their conversion ratios after the sixth reuse in the enantioselective reaction.     

Complete Genome Sequence of Bifidobacterium longum JDM301

Bifidobacteria, known as probiotic bacteria, are high-G+C Gram-positive bacteria which naturally inhabit the human gastrointestinal tract and vagina. Recently, we completely sequenced Bifidobacterium longum JDM301, which is a widely used Chinese commercial strain with several probiotic properties.Bifidobacterium spp., which are considered model probiotic bacteria like Lactobacillus spp., play an important role in the stability of the intestinal microflora, the modulation of the immune response, and so on (6, 8). We determined the complete genome sequence of B. longum JDM301, which is commercially used in China as a probiotic strain, using the GS 20 system (454 Life Science Corporation) (7). A total of 192,888 reads with an average length of 210 bp were assembled into 112 contigs by the 454 assembly tool. Among these, 92 large contigs were larger than 500 bp. We determined the order of the largest contigs through BLAST analysis with the reference strain B. longum ATCC 15697 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP001095","term_id":"213522389","term_text":"CP001095"}}CP001095) (10) and arranged the others by multiplex PCR. Gaps were closed by sequencing gap-spanning PCR products or clones using ABI 3730 xl DNA sequencers. Primer design and sequence assembly were performed with the Phred/Phrap/Consed software package (2, 3).The complete genome of B. longum JDM301 is composed of a 59.8% G+C circular chromosome of 2,477,838 bp without any plasmid. The genome of JDM301 (2.48 Mb) is smaller than that of B. longum ATCC 15697 (2.83 Mb) and slightly larger than the complete genomes of B. longum NCC2705 (2.26 Mb; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AE014295","term_id":"58012118","term_text":"AE014295"}}AE014295) and DJO10A (2.38 Mb; GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"CP000605","term_id":"189427298","term_text":"CP000605"}}CP000605). The JDM301 genome contains 1,959 protein-coding genes, three rRNA operons, and 55 tRNA genes. There are four rRNA operons with two cascaded in the three reference B. longum strains, while there are three in JDM301. We resequenced the three regions containing rRNA operons in the genome of JDM301 and found no cascaded rRNA operon. However, the locations of the three rRNA operons in JDM301 are the same as in the other three B. longum strains. No complete prophages were found in the genome sequence, but 12 phage-related fragments were identified. The genome also contains 15 pseudogenes, which is evidence of the recent and ongoing genome reduction of lactic acid bacilli (9). In addition, 15 complete or disrupted insertion sequence (IS) elements were found in the entire genome, which were identified as 5 derivatives of ISBlo5, 3 complete copies of ISBlo3, and 7 other ISs or derivatives belonging to the IS256, IS3, IS21, and IS30 family elements, respectively. One of them, named ISBad1, was 98.08% identical to that of B. adolescentis, and the others have been reported in the B. longum NCC2705 genome (8).Genome analysis revealed 14 response regulators and 14 sensor histidine kinases throughout the JDM301 chromosome, which may suggest a less complex regulatory network in JDM301 than that in ATCC 15697 (10). JDM301 has been grown as a commercial strain in stable and rich nutritional medium for such a long time that the regulatory networks in the genome may degenerate slowly, which is required for responses to dynamic environmental cues in the gastrointestinal tract.A gene (for BLJ_1359) encoding a serpin (serine protease inhibitor) with 92.69% identity to that of NCC2705 (4) was found in the genome sequence. The serpin is a potential probiotic effector molecule, and it may contribute to the immunomodulation of this B. longum strain. As we know, modulation of the immune system is one of probiotic functions of lactic acid bacteria (5).As model probiotic bacteria, Bifidobacterium spp. attract more and more interest since the molecular mechanisms of their probiotic activities remain unclear to a large degree (11). On the other hand, the biosafety of probiotics has attracted more attention with their enlarged and wide applications in food and drug products (1). We believe that this work will bring us deeper insight into the probiotic activities and safety of the strain widely used in China.     

Contribution of PTHrP to mechanical strain-induced fibrochondrogenic differentiation in entheses of Achilles tendon of miniature pigs

Background

Fibrochondrocytes are involved in entheses repair, but their response to mechanical strain (MS) is ill known.

Objective

To determine if parathyroid hormone-related protein (PTHrP) expression in fibrochondrocytes from fibrocartilaginous entheses is modulated by MS, and to further observe the regulatory effects of human (h) PTHrP on fibrochondrocyte differentiation.

Methods

Fibrochondrocytes from fibrocartilaginous entheses of Guizhou miniature pig?s Achilles tendon were submitted or not to MS (4%, 8% or 12% cyclic tensile strain; 1 Hz). Fibrochondrocytes were also exposed to: cyclopamine (Indian hedgehog (Ihh) inhibitor) (10 μM), hPTHrP (10 nM) or cyclopamine/hPTHrP (cyclopamine 10uM+hPTHrP 10 nM). Types I, II and X collagen and PTHrP expressions were measured by real-time RT-PCR and Western blot.

Result

Under 4% strain load for 12 h, types I and II collagen mRNA expressions were increased (+324% and +659%, P<0.001), while type X collagen was decreased (−89%, P<0.001). At 12%, types I and II collagen mRNA expressions were decreased (−62% and −62%, P<0.001), while type X collagen was increased (+375%, P<0.05). Under 4% strain load, PTHrP mRNA expression was increased in relation with strain duration (from 3 to 12 h: +168%, P<0.001), while at 12%, PTHrP expression decreased with time (from 3 to 12 h: −81%, P<0.001). Using cyclopamine for 24 h, PTHrP mRNA expression was significantly decreased (−88%, P<0.05), types I and II collagen were decreased (−90% and −82%, P<0.001), and type X collagen was increased (+261%, P<0.001).

Conclusions

Dynamic MS modulate PTHrP expressions. Thus, PTHrP might play an important role in fibrochondrocyte differentiation, indirectly revealing a role in entheses? formation and repair.     

Large catalase based bioelectrode for biosensor application

A large catalase (CAT) (Mr ~ 90 kDa), immobilized on multiwalled carbon nanotubes—Nafion^® (MWCNT-NF) matrix and encapsulated with polyethylenimine (PEI) on glassy carbon electrode (GCE), showed a pair of nearly reversible cyclic voltammetric peaks for Fe^(III)/Fe^(II) couple with formal potential of about −0.45 V (vs. Ag/AgCl electrode at pH 7.5). PEI significantly reduced the charge transfer resistance and stabilized the bioelectrode through electrostatic interaction. The electron transfer rate constant and surface coverage of the immobilized CAT were 1.05 ± 0.2 s⁻¹ and 2.1 × 10⁻¹⁰ mol cm⁻², respectively. Studies on electrocatalytic activity and kinetics of GCE/MWCNT-NF/CAT/PEI for hydrogen peroxide (H₂O₂) showed the apparent Michaelis-Menten constant of 3 mM, linear response in the range of 10 μM to 5 mM, response time of ~ 2 s for steady state current, and detection limit of ~ 1 μM. A high operational and storage stability was also demonstrated for the bioelectrode. Hence, the direct electrochemistry of the large catalase and its potential biosensor application have been established through this investigation.     

Magnetic resonance microscopy of collagen mineralization

A model mineralizing system was subjected to magnetic resonance microscopy to investigate how water proton transverse (T₂) relaxation times and magnetization transfer ratios can be applied to monitor collagen mineralization. In our model system, a collagen sponge was mineralized with polymer-stabilized amorphous calcium carbonate. The lower hydration and water proton T₂ values of collagen sponges during the initial mineralization phase were attributed to the replacement of the water within the collagen fibrils by amorphous calcium carbonate. The significant reduction in T₂ values by day 6 (p < 0.001) was attributed to the appearance of mineral crystallites, which were also detected by x-ray diffraction and scanning electron microscopy. In the second phase, between days 6 and 13, magnetic resonance microscopy properties appear to plateau as amorphous calcium carbonate droplets began to coalesce within the intrafibrillar space of collagen. In the third phase, after day 15, the amorphous mineral phase crystallized, resulting in a reduction in the absolute intensity of the collagen diffraction pattern. We speculate that magnetization transfer ratio values for collagen sponges, with similar collagen contents, increased from 0.25 ± 0.02 for control strips to a maximum value of 0.31 ± 0.04 at day 15 (p = 0.03) because mineral crystals greatly reduce the mobility of the collagen fibrils.     

Adsorption immobilization of Escherichia coli phytase on probiotic Bacillus polyfermenticus spores

The immobilization of enzymes on edible matrix supports is of great importance for developing stabilized feed enzymes. In this study, probiotic Bacillus spores were explored as a matrix for immobilizing Escherichia coli phytase, a feed enzyme releasing phosphate from phytate. Because Bacillus spore is inherently resistant to heat, solvents and drying, they were expected to be a unique matrix for enzyme immobilization. When mixed with food-grade Bacillus polyfermenticus spores, phytases were adsorbed to their surface and became immobilized. The amount of phytase attached was 28.2 ± 0.7 mg/g spores, corresponding to a calculated activity of 63,960 U/g spores; however, the measured activity was 41,120 ± 990.1 U/g spores, reflecting a loss of activity upon adsorption. Immobilization increased the half life (t_1/2) of the enzyme three- to ten-fold at different temperatures ranging from 60 to 90 °C. Phytase was bound to the spore surface to the extent that ultrasonication treatment was not able to detach phytases from spores. Desorption of spore-immobilized phytase was only achieved by treatment with 1 M NaCl, 10% formic acid in 45% acetonitrile, SDS, or urea, suggesting that adsorption of phytase to the spore might be via hydrophobic and electrostatic interactions. We propose here that Bacillus spore is a novel immobilization matrix for enzymes that displays high binding capacity and provides food-grade safety.     

Lateral packing of mineral crystals in bone collagen fibrils

Combined small-angle x-ray scattering and transmission electron microscopy studies of intramuscular fish bone (shad and herring) indicate that the lateral packing of nanoscale calcium-phosphate crystals in collagen fibrils can be represented by irregular stacks of platelet-shaped crystals, intercalated with organic layers of collagen molecules. The scattering intensity distribution in this system can be described by a modified Zernike-Prins model, taking preferred orientation effects into account. Using the model, the diffuse fan-shaped small-angle x-ray scattering intensity profile, dominating the equatorial region of the scattering pattern, could be quantitatively analyzed as a function of the degree of mineralization. The mineral platelets were found to be very thin (1.5 nm ∼ 2.0 nm), having a narrow thickness distribution. The thickness of the organic layers between adjacent mineral platelets within a stack is more broadly distributed with the average value varying from 6 nm to 10 nm, depending on the extent of mineralization. The two-dimensional analytical scheme also leads to quantitative information about the preferred orientation of mineral stacks and the average height of crystals along the crystallographic c axis.     

Mucoadhesive chitosan/gelatin films for buccal delivery of propranolol hydrochloride

The aim of this work was to develop and characterize chitosan/gelatin films as innovative mucoadhesive system for buccal delivery of propranolol hydrochloride. FT-IR and TGA analysis confirmed the interaction between chitosan and gelatin. The presence of higher chitosan amounts in chitosan/gelatin films allowed the lowest percent water-uptake ability (235.1 ± 5.3%) and the highest in vivo residence time in the buccal cavity (240 ± 13 min). Moreover, the presence of mannitol in the formulation allowed 80% drug permeation through porcine buccal mucosa in 5 h. This behaviour suggests that the application of four and two films containing 5 mg of propranolol hydrochloride could be suitable for achieving the proposed daily dose for hypertension and atrial fibrillation treatment, respectively. Another interesting aspect of chitosan/gelatin films was their compatibility with buccal microflora in the absence of drug and their ability to determine growth inhibition for pathogen bacteria, but not for probiotic species, when loaded with drug.