Survival of freeze dried Lactobacillus plantarum in instant fruit powders and reconstituted fruit juices

The aim of this study was to investigate the survival of freeze dried Lactobacillus plantarum cells mixed with several freeze dried instant fruit powders (strawberry, pomegranate, blackcurrant and cranberry) during storage for 12 months as well as after reconstitution with water each month. Inulin and gum arabic were also added to the instant fruit powders at two levels (10% and 20% w/w of dry weight) to improve the cell survival and functional properties of the product. The best cell survival over the 12 months of storage was observed for the blackcurrant powder (almost no decrease) followed by strawberry (~ 0.3 log decrease), pomegranate (~ 0.9 log decrease), whereas the worst survival was obtained in cranberry powder (~ 4.5 logs). To explain these results multiple regression analysis was conducted with the log decrease [log10N0 month − log10N12 months] as the dependent variable and water activity, pH, citric acid, dietary fibre and total phenol as the independent variables. The results indicated that among all the examined factors, the [log10N0 month − log10N12 months] depended only on the water activity (P < 0.05). Inulin and gum arabic demonstrated a substantial protective effect on cell survival (1–1.5 log) in the case of cranberry, which was likely due to a physical interaction between the cells and the carbohydrates. After reconstituting the dried fruit powders at room temperature and measuring cell viability for up to 4 h, it was shown that in the case of strawberry juice there was no decrease, and very little in the case of pomegranate and blackcurrant juices (< 0.5 log). On the other hand, a significant decrease was observed for cranberry juice (P < 0.05), which increased as the storage time of the dried cranberry powder increased, indicating that the cells became more susceptible with prolonged storage. Multiple regression analysis indicated that the main factors influencing cell survival were water activity and pH, while citric acid, dietary fibre and total phenol did not have an effect. Furthermore, inulin and gum arabic addition did not have a significant (P > 0.05) effect upon reconstitution of the dried fruit powder. This study showed that instant juice powders are very good carriers of probiotic cells and constitute good alternatives to highly acidic fruit juices.

Protection of dried probiotic bacteria from bile using bile adsorbent resins

Enteric coated oral tablets or capsules can deliver dried live cells directly into the intestine. Previously, we found that a live attenuated bacterial vaccine acquired sensitivity to intestinal bile when dried, raising the possibility that although gastric acid can be bypassed, significant loss of viability might occur on release from an enteric coated oral formulations. Here we demonstrate that some food-grade lyophilised preparations of Lactobacillus casei and Lactobacillus salivarius also show temporary bile sensitivity that can be rapidly reversed by rehydration. To protect dried bacterial cells from temporary bile sensitivity, we propose using bile acid adsorbing resins, such as cholestyramine, which are bile acid binding agents, historically used to lower cholesterol levels. Vcaps™ HPMC capsules alone provided up to 830-fold protection from bile. The inclusion of 50% w/w cholestyramine in Vcaps™ HPMC capsules resulted in release of up to 1700-fold more live Lactobacillus casei into simulated intestinal fluid containing 1% bile, when compared to dried cells added directly to bile. We conclude that delivery of dried live probiotic organisms to the intestine may be improved by providing protection from bile by addition of bile adsorbing resins and the use of HPMC capsules.

Biorefinery strategies for upgrading Distillers’ Dried Grains with Solubles (DDGS)

Distillers’ Dried Grains with Solubles (DDGS) is the major by-product of bioethanol and distillery plants. Due to its high content of proteins, water-soluble vitamins and minerals, DDGS has been long marketed as animal feed for livestock. EU legislation on liquid biofuels could raise the demand on bioethanol production in Europe, with a resulting increase in DDGS availability. DDGS contains a spectrum of complex organic macromolecules, particularly polysaccharides, in addition to proteins and vitamins, and its use as a starting raw material within a biomass-based biorefining strategy could lead to the development of multi-stream processes for the production of commodities, platform molecules or speciality chemicals, with concomitant economic benefits and waste reduction for bioethanol plants. The present review aims to outline the compositional characteristics of DDGS and evaluate its potential utilisation as a starting material for the production of added-value products. Parameters of influence on the chemical and physical characteristics of DDGS are discussed. Moreover, various pre-treatment strategies are outlined in terms of efficient DDGS fractionation into several added value streams. Additional processing steps for the production of medium and high added value compounds from DDGS are evaluated and their potential applications in the food and chemical industry sector are identified.

Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project

An efficient and robust method to measure vitamin D (25-hydroxy vitamin D3 (25(OH)D3) and 25-hydroxy vitamin D2 in dried blood spots (DBS) has been developed and applied in the pan-European multi-centre, internet-based, personalised nutrition intervention study Food4Me. The method includes calibration with blood containing endogenous 25(OH)D3, spotted as DBS and corrected for haematocrit content. The methodology was validated following international standards. The performance characteristics did not reach those of the current gold standard liquid chromatography-MS/MS in plasma for all parameters, but were found to be very suitable for status-level determination under field conditions. DBS sample quality was very high, and 3778 measurements of 25(OH)D3 were obtained from 1465 participants. The study centre and the season within the study centre were very good predictors of 25(OH)D3 levels (P<0·001 for each case). Seasonal effects were modelled by fitting a sine function with a minimum 25(OH)D3 level on 20 January and a maximum on 21 July. The seasonal amplitude varied from centre to centre. The largest difference between winter and summer levels was found in Germany and the smallest in Poland. The model was cross-validated to determine the consistency of the predictions and the performance of the DBS method. The Pearson's correlation between the measured values and the predicted values was r 0·65, and the sd of their differences was 21·2 nmol/l. This includes the analytical variation and the biological variation within subjects. Overall, DBS obtained by unsupervised sampling of the participants at home was a viable methodology for obtaining vitamin D status information in a large nutritional study.

Exploring the association of diary product intake with the fatty acids C15:0 and C17:0 measured from dried blood spots in a multi-population cohort: findings from the Food4Me study

Scope: The use of biomarkers in the objective assessment of dietary intake is a high priority in nutrition research. The aim of this study was to examine pentadecanoic acid (C15:0) and heptadecanoic acid (C17:0) as biomarkers of dairy foods intake. Methods and results: The data used in the present study were obtained as part of the Food4me Study. Estimates of C15:0 and C17:0 from dried blood spots and intakes of dairy from an FFQ were obtained from participants (n=1,180) across 7 countries. Regression analyses were used to explore associations of biomarkers with dairy intake levels and receiver operating characteristic (ROC) analyses were used to evaluate the fatty acids. Significant positive associations were found between C15:0 and total intakes of high-fat dairy products. C15:0 showed good ability to distinguish between low and high consumers of high-fat dairy products. Conclusion: C15:0 can be used as a biomarker of high-fat dairy intake and of specific high-fat dairy products. Both C15:0 and C17:0 performed poorly for total dairy intake highlighting the need for caution when using these in epidemiological studies.

The variation in transparency of amniotic membrane used in ocular surface regeneration

Background/aims: Scant consideration has been given to the variation in structure of the human amniotic membrane (AM) at source or to the significance such differences might have on its clinical transparency. Therefore, we applied our experience of quantifying corneal transparency to AM. Methods: Following elective caesarean, AM from areas of the fetal sac distal and proximal (ie, adjacent) to the placenta was compared with freeze-dried AM. The transmission of light through the AM samples was quantified spectrophotometrically; also, tissue thickness was measured by light microscopy and refractive index by refractometry. Results: Freeze-dried and freeze-thawed AM samples distal and proximal to the placenta differed significantly in thickness, percentage transmission of visible light and refractive index. The thinnest tissue (freeze-dried AM) had the highest transmission spectra. The thickest tissue (freeze-thawed AM proximal to the placenta) had the highest refractive index. Using the direct summation of fields method to predict transparency from an equivalent thickness of corneal tissue, AM was found to be up to 85% as transparent as human cornea. Conclusion: When preparing AM for ocular surface reconstruction within the visual field, consideration should be given to its original location from within the fetal sac and its method of preservation, as either can influence corneal transparency.

Effect of growth time on the surface and adhesion properties of Lactobacillus rhamnosus GG

Aims: To investigate the changes in the surface properties of Lactobacillus rhamnosus GG during growth, and relate them with the ability of the Lactobacillus cells to adhere to Caco-2 cells. Methods and Results: Lactobacillus rhamnosus GG was grown in complex medium, and cell samples taken at four time points and freeze dried. Untreated and trypsin treated freeze dried samples were analysed for their composition using SDS-PAGE analysis and Fourier transform infrared spectroscopy (FTIR), hydrophobicity and zeta potential, and for their ability to adhere to Caco-2 cells. The results suggested that in the case of early exponential phase samples (4 and 8 h), the net surface properties, i.e. hydrophobicity and charge, were determined to a large extent by anionic hydrophilic components, whereas in the case of stationary phase samples (13 and 26 h), hydrophobic proteins seemed to play the biggest role. Considerable differences were also observed between the ability of the different samples to adhere to Caco-2 cells; maximum adhesion was observed for the early stationary phase sample (13 h). The results suggested that the adhesion to Caco-2 cells was influenced by both proteins and non-proteinaceous compounds present on the surface of the Lactobacillus cells. Conclusion: The surface properties of Lact. rhamnosus GG changed during growth, which in return affected the ability of the Lactobacillus cells to adhere to Caco-2 cells. Significance and Impact of the Study: The levels of adhesion of Lactobacillus cells to Caco-2 cells were influenced by the growth time and reflected changes on the bacterial surface. This study provides critical information on the physicochemical factors that influence bacterial adhesion to intestinal cells.

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutics

Post-abscission, pre-dispersal seeds of Digitalis purpurea remain in a developmental state that is not terminated by desiccation ex planta

Seed quality may be compromised if seeds are harvested before natural dispersal (shedding). It has been shown previously that slow or delayed drying can increase potential quality compared with immediate rapid drying. This study set out to investigate whether or not there is a critical moisture content, below which drying terminates maturation events for seeds harvested after mass maturity but before dispersal. Seeds of foxglove (Digitalis purpurea) in the post-abscission pre-dispersal phase were held at between 15 and 95 % RH for 4 or 8 d, with or without re-hydration to 95 % RH for a further 4 d, before drying to equilibrium at 15 % RH. In addition, dry seeds were primed for 48 h at -1 MPa. Subsequent seed longevity was assessed at 60 % RH and 45 degrees C. Rate of germination and longevity were improved by holding seeds at a wide range of humidities after harvest. Longevity was further improved by re-hydration at 95 % RH. Priming improved the longevity of the seeds dried immediately after harvest, but not of those first held at 95 % RH for 8 d prior to drying. Maturation continued ex planta in these post-abscission, pre-dispersal seeds of D. purpurea dried at 15-80 % RH at a rate correlated positively with RH (cf. ageing of mature seeds). Subsequent re-hydration at 95 % RH enabled a further improvement in quality. Priming seeds initially stored air-dry for 3 months also allowed maturation events to resume. However, once individual seeds within the population had reached maximum longevity, priming had a negative impact on their subsequent survival.

Priming and re-drying improve the survival of mature seeds of Digitalis purpurea during storage

Most priming studies have been conducted on commercial seed lots of unspecified uniformity and maturity, and subsequent seed longevity has been reported to both increase and decrease. Here a seed lot of Digitalis purpurea L. with relatively uniform maturity and known history was used to analyse the effects of priming on seed longevity in air-dry storage. Seeds collected close to natural dispersal and dried at 15 % relative humidity (RH), 15 degrees C, were placed into experimental storage (60 % RH, 45 degrees C) for 14 or 28 d, primed for 48 h at 0, -1, -2, -5, -10 or -15 MPa, re-equilibrated (47 % RH, 20 degrees C) and then returned to storage. Further seed samples were primed for 2 or 48 h at -1 MPa and either dried at 15 % RH, 15 degrees C or immediately re-equilibrated for experimental storage. Finally, some seeds were given up to three cycles of experimental storage and priming (48 h at -1 MPa). Priming at -1 MPa had a variable effect on subsequent survival during experimental storage. The shortest lived seeds in the control population showed slightly increased life spans; the longer lived seeds showed reduced life spans. In contrast, seeds first stored for 14 or 28 d before priming had substantially increased life spans. The increase tended to be greatest in the shortest lived fraction of the seed population. Both the period of rehydration and the subsequent drying conditions had significant effects on longevity. Interrupting air-dry storage with additional cycles of priming also increased longevity. The extent of prior deterioration and the post-priming desiccation environment affect the benefits of priming to the subsequent survival of mature seeds. Rehydration-dehydration treatments may have potential as an adjunct or alternative to the regeneration of seed accessions maintained in gene banks for plant biodiversity conservation or plant breeding.

The potential of combining Pasteuria penetrans and neem (Azadirachta indica) formulations as a management system for root-knot nematodes on tomato

Initial applications of 10(4) spores g(-1) of Pasteuria penetrans, and dried neem cake and leaves at 3 and 2% w:w, respectively, were applied to soil in pots. Juveniles of Meloidogyne javanica were added immediately to the pots (500, 5,000 or 10,000) before planting 6-week-old tomato seedlings. The tomatoes were sampled after 64 days; subsequently a second crop was grown for 59 days and a third crop for 67 days without further applications of P. penetrans and neem. There was significantly less root-galling in the P. penetrans combined with neem cake treatment at the end of the third crop and this treatment also had the greatest effect on the growth of the tomato plants. At the end of the third crop, 30% of the females were infected with P. penetrans in those treatments where spores had been applied at the start of the experiment. The effects of neem leaves and neem cake on the nematode population did not persist through the crop sequences but the potential for combining the amendments with a biological control agent such as P. penetrans is worthy of further evaluation.

Response of free eggs on infective juvenile root-knot nematode Meloidogyne javanica through neem (Azadirachta indica A. Juss) amended soil

Air-dried and 3 mm pore size sieved soil was amended with neem crude formulations (leaves and cake) @ 3% w/w and a refined product, aza @ 0.05 and 0.1 w/w. Three days after treatment, 500 eggs of M. javanica held in 2 ml water were added in each dish. In another experiment, soil was amended with neem crude formulations @ 10. 5, 2.5 and 1% w/w and refined formulation aza @ 0.025, 0.05, 0.1 and 0.5% w/w. Three days after amendment 1000 plus minus 21 freshly hatched J2 held in 3 ml water were added to the amended soil. Untreated soil was kept as control. Comparison of treatments means showed that all the neem formulations caused significant reduction of hatching. Neem crude formulations were more effective in reducing hatching as compared to commercial product aza. Among the crude formulations, neem leaves were most effective in reducing hatching. In other experiment all the doses of neem crude and refined formulations differed significantly with control in reducing the mobility of juveniles. It was observed that by increasing the dose of the formulations the mobility was reduced accordingly.

Drying method influences the development of germinability, dessication tolerance and subsequent longevity of immature seeds of sumauma (Ceiba pentandra (L.) Gaertn. [Bombacaceae])

The ability to germinate, tolerate desiccation and survive in air-dry storage was investigated during early seed development in planta and subsequent ex planta maturation of sumauma (Ceiba pentandra). Immature fruits were collected on three different dates (i.e. from about 5 days before until 7 days after mass maturity). Immature fresh seeds were not able to germinate. Fruits or seeds were subjected immediately after each collection to three different drying treatments with progressively slower rates of dessication: (i) seeds were extracted from the fruits and dried immediately; (ii) fruits were dried in a thin layer; (iii) fruits were dried in a tied polyethylene bag (with 10 holes of 1cm diameter). Drying was in a room maintained at 25 degrees C +/- 3 degrees C and 65%+/- 5% r.h. For treatment (i) the seeds were dried for 6 days in order to reduce moisture content to around 13% ( +/- 2%) moisture content. For treatments (ii) and (iii) the fruits were subjected to different periods of drying depending upon collection date. The results of these post-collection treatments showed generally that the more immature the seeds the slower the rate of drying that is required to improve ability to germinate, ability to tolerate desiccation and potential longevity, but at the third harvest, 7 days after mass maturity, the intermediate drying rate treatment was the most beneficial. Thus post fruit collection treatments can be modified depending upon the stage of seed development in order to provide good to high quality seeds of sumauma when collection has to be made at a site with difficult access at less than ideal times. The results are relevant to seed collection practices for both forestry and ex situ plant biodiversity conservation.